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BIOCHEMICAL INVESTIGATION OF TAU AND MAP2 POLYMERIZATION:
IMPLICATIONS FOR NEUROPATHIC FILAMENT ASSEMBLY IN ALZHEIMER'S
DISEASE
By
LUCA DI NOTO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2002
This dissertation is dedicated to my family for their endless support and encouragement
over the years and at times, for their supernatural patience
ACKNOWLEDGMENTS
I would like to thank my mentor and guide over these years, Daniel Purich. He is
an inspiration as a scientist and as a man and I have the privilege to consider him a good
friend. I would also like to thank the former members of the lab, especially Michael
DeTure, Yuri Bukhtiyarov, Will Zeile and Edy Zhang for their friendship, and the
interesting discussions (scientific and nonscientific) shared with them over the years.
Finally I would like to thank the members of my committee and the Department.
iii
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ................................................................................................. iii
LIST OF ABBREVIATIONS........................................................................................... vii
LIST OF FIGURES ........................................................................................................... ix
ABSTRACT....................................................................................................................... xi
CHAPTER
1 INTRODUCTION ...........................................................................................................1
The Cell Cytoskeleton: an Overview.............................................................................. 1
Tubulin............................................................................................................................ 5
Identification and General Properties....................................................................... 5
Structural Properties................................................................................................. 6
Nucleotide Binding Sites. ........................................................................................ 7
Microtubules ................................................................................................................... 8
Structural Properties................................................................................................. 8
Microtubule Assembly. .......................................................................................... 10
Microtubule-Associated Proteins.................................................................................. 11
Motor-driven MAPs. .............................................................................................. 12
Structural or Fibrous MAPs .................................................................................. 13
MAP2 ..................................................................................................................... 14
Tau ......................................................................................................................... 18
Tau Phosphorylation .............................................................................................. 19
Tau in Alzheimer’s Disease and Other Neurodegenerative Diseases........................... 22
Tau Phosphorylation in Alzheimer’s Disease............................................................... 24
Other Neurodegenerative Diseases Exhibiting Neurofibrillary Tangles ...................... 25
In Vitro Studies of Tau Polymerization ........................................................................ 28
Methods Available to Measure Tau Assembly............................................................. 32
Aims of This Research Project ..................................................................................... 34
2 CHARACTERIZATION OF PAIRED HELICAL FILAMENT-PRODUCING
CONSTRUCTS OF MAP2............................................................................................37
Introduction................................................................................................................... 37
Materials and Methods.................................................................................................. 42
iv
Expression and Purification of Wild Type Tau123 and Module-B Mutants ......... 44
In Vitro Assembly of Filaments with tRNA or Heparin ........................................ 46
Electron Microscopy .............................................................................................. 47
Morphometry ......................................................................................................... 47
Fluorescence Spectroscopy .................................................................................... 48
Results........................................................................................................................... 48
Image Analysis of the MAP2 MTBR Mutants Generated by the Laboratory. ...... 49
Investigation of the Role of Charges in ModuleB ................................................. 60
Discussion ..................................................................................................................... 61
3 RETENTION OF MICROTUBULE ASSEMBLY-PROMOTING CAPACITY OF
TAU AND MAP2 UPON CONVERSION TO DISULFIDE-CROSSLINKED
HOMODIMERS ............................................................................................................67
Introduction................................................................................................................... 67
Material and Methods ................................................................................................... 69
Expression and Purification of Wild Type Tau123 and Module-B Mutants ......... 70
Tau and MAP2 MTBR Dimer Purification............................................................ 72
Tubulin Preparation and Polymerization ............................................................... 73
Results........................................................................................................................... 75
Discussion ..................................................................................................................... 80
4 REDOX PROPERTIES OF CYSTEINE RESIDUES IN THE MICROTUBULEBINDING REGION OF TAU PROTEIN .....................................................................85
Introduction................................................................................................................... 85
Material and Methods ................................................................................................... 87
Expression and Purification of Wild Type Tau123 ............................................... 89
Tau-123 MTBR Dimer Purification....................................................................... 90
HPLC Analysis of Tau Dimer and Monomer After Reaction with Thiols ............ 91
Tau-123-GSH Conjugation .................................................................................... 91
Electron Microscopy .............................................................................................. 93
Time-course of Monomer Formation and Tau-thiols Reactivity Experiments...... 93
Results........................................................................................................................... 94
Discussion ................................................................................................................... 102
5 A FLUORIMETRIC ASSAY OF TAU POLYMERIZATION ..................................106
Introduction................................................................................................................. 106
Matherials and Methods.............................................................................................. 107
Expression and Purification T-123 with N-terminal Serine................................. 109
Oxidation of N-terminal Serine............................................................................ 111
Fluorescent Labeling of Tau123 .......................................................................... 113
In Vitro Assembly of Filaments with tRNA or Heparin ...................................... 114
Electron Microscopy ............................................................................................ 114
Results......................................................................................................................... 116
Discussion ................................................................................................................... 119
v
6 CONCLUSIONS AND FUTURE DIRECTIONS.......................................................127
APPENDIX
ELECTRON MICROSCOPY..........................................................................................135
Transmission Electron Microscopy ............................................................................ 135
Resolution and Contrast. ...................................................................................... 136
Shadow Casting.................................................................................................... 137
Positive Staining. ................................................................................................. 138
Negative Staining. ................................................................................................ 138
LIST OF REFERENCES.................................................................................................140
BIOGRAPHICAL SKETCH ...........................................................................................162
vi
LIST OF ABBREVIATIONS
AD
Alzheimer’s disease
AGE
advanced glycation end product
APP
amyloid precursor protein
ADP
adenosine diphosphate
ATP
adenosine triphosphate
BSA
bovine serum albumine
C
centigrades
cAMP
cyclic adenosine monophosphate
cDNA
complementary deoxyribonucleic acid
DNA
deoxyribonucleic acid
DNase
deoxyribonuclease
DTT
dithiothreitol
EGTA
ethyleneglycol-bis-(b-amino-ethyl ether) N,N’-tetraacetic acid
GDP
guanosine diphosphate
GTP
guanosine triphosphate
GSK
glycogen synthase kinase
GSH
glutathione
HPLC
high performance liquid chromatography
IPTG
isopropyl thiogalactopyranoside
KDa
kilo Daltons
M
molar
MALDI-TOF matrix-assisted laser desorption/ionization – time of flight
MAP
microtubule-associated protein
MBP
Module-B point mutant
MES
morpholino ethanesulfonic acid
MOPS
morpholino propanesulfonic acid
vii
MT
microtubule
MTBR
microtubule-binding region
NF
neurofilament
NFT
neurofibrillary tangles
PAGE
polyacrylamide gel electrophoresis
PCR
polymerase chain reaction
PHF
paired helical filament
PMSF
phenylmethylsulfonyl fluoride
RNA
ribonucleic acid
SDS
sodium dodecyl sulfate
SF
straight filament
Tris
tris (hydroxymethyl) aminomethane
tRNA
transfer ribonucleic acid
viii
LIST OF FIGURES
Figure
page
1-1. Dynamic microtubule filament....................................................................................9
1-2. Schematic representation of a MAP interaction with a microtubule.........................15
1-3. Schematic representation of the six isoforms of tau expressed in the human
adult brain ..............................................................................................................20
2-1. Sequence comparison of MAP2 and tau microtubule-binding regions and
identification of Module-A and Module-B. ...........................................................40
2-2A. Montage of electron micrographs illustrating the SF morphology of
MAP2-123 [Module-A] polymers. ........................................................................51
2-2B. Montage of electron micrographs illustrating the PHF-like morphology of
MAP2-123 [Module-B] polymers..........................................................................51
2-3. Frequency and relative abundance of PHF formation ...............................................52
2-4. Length distribution of the MAP2 mutants containing the entire Module-A or
Module-B from tau or the single-residue mutations from Module-B....................53
2-5. Comparison of amino acid sequence between MAP2-123 [Module-B] and
tau-123 [Module-A] ...............................................................................................54
2-6. Montage of electron micrographs illustrating the SF morphology of tau-123
[Module-A] polymers. ...........................................................................................55
2-7. Fluorescent emission spectrum of thioflavin-S reacted in the presence of
polymers from different tau or MAP2 mutants......................................................56
2-8. Sequence of the microtubule-binding region of MAP2-123[Module-B] mutant. .....57
2-9. Fluorescent emission spectra of thioflavin-S reacted in the presence of
polymers from different tau or MAP2 mutants......................................................62
3-1. General features of three-repeat tau and MAP2, including sequences of the
microtubule-binding regions used in this study. ....................................................76
3-2. HPLC and SDS gel electrophoresis of tau and MAP2 monomer and dimer.............77
ix
3-3. Promotion of tubulin polymerization by tau and MAP2 monomer and
disulfide-liked dimer..............................................................................................79
3-4. Electron micrographs of microtubules assembled with MAP2 and tau . ..................81
3-5. Affinity of tau and MAP2 monomers and dimers for microtubules..........................82
4.1. Complete sequence of human adult tau.. ...................................................................95
4-2. Time-course experiments of tau mixed to GSH or DTT ...........................................98
4-3. HPLC analysis of tau reacted with glutathione or dithiothreitol. ..............................99
4-4. MALDI-TOF mass spectrometry of peak isolated from HPLC ..............................100
4-5. Amount of tau monomer generated by reaction with increasing amounts of
glutathione or dithiothreitol. ................................................................................101
4-6. Montage of electron micrographs illustrating different filaments assembled
from tau-GSH conjugate.. ....................................................................................103
5-1a. Periodate oxidation reaction. .................................................................................112
5-1b. Conjugation of fluorescent molecule to the protein ..............................................112
5-2. HPLC profiles of the products of conjugation reaction...........................................115
5-3. The fluorescent molecule does not interfere with polymerization of tau or
MAP2 [Module-B]...............................................................................................118
5-4. Emission spectra of the Alexa350-conjugated tau-123 protein, at different
incubation times. ..................................................................................................120
5-5. Electron micrographs of the solution containing polymerized tau-123
conjugated to Alexa350. ......................................................................................123
5-6. Fluorescent measurements of solution containing polymerized tau-123
conjugated to Alexa350. ......................................................................................124
5-7. Release of fluorescent molecules from tau-123 upon its polymerization ...............125
x
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
BIOCHEMICAL INVESTIGATION OF TAU AND MAP2 POLYMERIZATION:
IMPLICATIONS FOR NEUROPATHIC FILAMENT ASSEMBLY IN ALZHEIMER'S
DISEASE
By
Luca Di Noto
May 2002
Chair: Daniel L. Purich
Department: Biochemistry and Molecular Biology
Neurodegenerative diseases of the brain accompanied by dementia affect 5 to 10 %
of individuals over the age of 65 in the western world and represent one of the main
health-related economic problems of our society. Alzheimer’s disease (AD) is the most
frequent form of dementia. Many other forms of progressive neuropathological
degenerations have been characterized that share similar pathological features. Among
them are intracellular aggregates known as neurofibrofibrillary tangles. These inclusions
consist of hyperphosphorylated microtubule-associated protein tau that accumulates
inside affected neuronal cells, disrupting their microtubule network.
Tau protein in healthy cells promotes tubulin polymerization, reduces microtubule
instability and plays a role in maintaining neuronal integrity, axonal transport and axonal
polarity. Tau protein is abundant in both the central and peripheral nervous systems. In
brain it is predominantly found in neurons concentrated in axons.
xi
Since its correlation to neurodegenerative diseases, tau tendency to aggregate into
filaments has been the focus of many studies. Nevertheless, the phenomenon remains a
complex and challenging model to study. The mechanism by which the protein stops
acting as a microtubule-stabilizing factor and starts building up into the fibrillary
structure is still largely unknown.
This dissertation investigates how two particular stretches of amino acids inside tau
sequence, previously identified by our laboratory and named Module-A and Module-B,
play a role in driving the protein toward aggregation and influence Tau filament
morphology. My results show that other residues outside of the modules are also likely
to be involved in filament assembly and morphology.
This work also investigates the importance and biochemical characteristics of the
only cysteine residue present in three-repeat isoforms of tau. This cysteine is believed to
be important in nucleating the polymerization of Tau, by generating tau-tau homodimers
that act as a building block for filaments. This work shows that tau homodimers are still
able to bind to microtubules and to stabilize them. My results also show that tau has a
high reactivity with the intracellular reducing agent, glutathione. A specific protective
role is proposed for glutathione against the oxidation of tau.
xii
CHAPTER 1
INTRODUCTION
The Cell Cytoskeleton: an Overview
The cytoskeleton is an important and complex component of nearly all eukaryotic
cells. Despite a name that suggests a rigid and long-lived structure, the cytoskeleton is a
surprisingly dynamic, three-dimensional organelle, one that is characterized by its highly
interactive collection of protein fibers that serve as molecular scaffolds for other signaland energy-transducing elements of the cell. The cytoskeleton is comprised of three
different fibers – actin filaments, intermediate filaments (including the cytokeratins), and
microtubules – as well as fiber-specific proteins that presently number in the hundreds.
By associating with one or several of these polymeric networks, the various
cytoskeleton-associated proteins modify the properties of these fibers in a cell- and
tissue-specific manner. The cytoskeleton effectively partitions the cytoplasmic
compartment into various subcompartments, each of which contributes to the overall
structure, stability, and motility of muscle and nonmuscle cells. Beyond its role in
maintaining cell shape, the cytoskeleton possesses molecular motors that generate the
forces needed to continuously reshape resting cells as well as other migrating and/or
dividing cells. Proximity to the nuclear envelope and the peripheral membrane facilitates
cytoskeletal involvement in numerous functions, including cellular trafficking, anchoring
of membrane-bound components to both intra- and extracellular components,
chromosome segregation, and cytokinesis. The vital importance of the cytoskeletal
1
2
organelle in modulating cellular dynamics is evident from its extremely high evolutionary
stability and by the lethality arising from certain mutations and/or gene deletions.
Microfilaments are 7 nm diameter, rod-like filaments composed of actin monomers
that self-assemble in an ATP-dependent polymerization reaction (Pollard 1990).
Filamentous actin is a two-chain helical structure, in which the head-to-tail self-assembly
mechanism leads to the intrinsic structural polarity obvious in electron micrographs.
Eukaryotic cells have six actin isoforms: two in striated muscle, two in smooth muscle
and two in nonmuscle cells. Three isoelectric variants, α-, β- and γ-actin have been
isolated, but despite the differences all three variants share the same apparent
polymerization properties (Whalen et al. 1976). In nonmuscle cells, actin filaments line
the periphery of the cell to propagate cell motility and this localization facilitates rapid
responses to chemotaxis components. When ATP is present, the two ends of each
filament are energetically disparate, such that the macroscopic critical actin concentration
[i.e., Actinmacro = (koff+ + koff-)/(kon+ + kon-)] has an intermediate value lying between the
more stable plus-end critical actin concentration (i.e., Actinplus = koff+/kon+) and the less
stable minus-end critical actin concentration (i.e., Actinminus = koff-/kon-). For this reason,
the minus-end is intrinsically less stable than the plus-end, a condition that immediately
results in treadmilling, which is the steady-state, opposite end assembly/disassembly
process where (+)end monomer addition is fueled by (-)end monomer loss. In nonmuscle
cells, actin polymerization is also responsible for actin-based motility, and Dickinson and
Purich (2002) recently offered a clamped-filament elongation reaction scheme, called the
“Lock, Load & Fire” mechanism, for the actoclampin motor.
3
Intermediate filaments (IFs) are divergent structures that are cell type-specific and
are generated from a variety of protein subunits (Steinert and Roop 1988). The various
IF proteins differ considerably in molecular weight and isoelectric charge, but all share a
common conserved molecular arrangement from the amino to the carboxyl termini: head,
rod and tail domains. Neurofilament proteins, which are localized almost exclusively in
axon-containing neurons, include 68-, 102-, and 112-kD polypeptides that are
respectively designated as low- (NF-L), medium- (NF-M), and high-molecular-weight
(NF-H) proteins. Only NF-L can self-assemble into a homopolymer; NF-H and NF-M
form what are obligate heteropolymers that coassemble with NF-L, which acts as a
scaffold. Neurofilaments have 10 nm diameters in electron micrographs – hence the
usage of “intermediate” to distinguish them from 5 to 7 nm actin filaments and 25 nm
microtubules. NFs are thought to play a role in establishing and maintaining caliber (i.e.,
diameter) of axonal processes. Larger caliber axons are characterized by faster rates of
neuronal signal transmission via membrane polarization-depolarization. Except for their
capacity to interact with certain microtubule-associated proteins (see below), there are no
other activities are currently known for the stable filaments formed from intermediate
filament proteins.
Reaching 25-28 nm in diameter and 50 µm in length, microtubules (MT) are by far
the largest of the cytoskeletal components. These hollow cylindrical fibers are formed by
the indefinite polymerization of tubulin, a heterodimer composed of a α- and a
β-polypeptide. Just prior to self-assembly, GTP is bound at a single site located on each
of the subunits; the α-subunit has a non-exchangeable GTP binding site, and the
β-subunit has a nonexchangeable GTP binding site (Purich and Kristofferson 1984). In a
4
manner reminiscent of actin filaments, head-to-tail polymerization of tubulin is
responsible for the MT’s structural polarity. The assembly reactions are also
characterized by fast- and slow-growing ends in vitro, but tubulin adds exclusively to the
(+)end in vivo. Because tubulin-GTP addition induces GTP hydrolysis in the
immediately underlying dimer, a stabilizing, one-dimer deep boundary of
GTP-containing tubulin stabilizes the ends of microtubules (Karr et al. 1979). Stochastic
release of these tubulin-GTP dimers results in loss of this stabilizing boundary layer,
leading to the rapid and extensive loss of numerous tubulin-GDP dimers. This
phenomenon was later termed dynamic instability by Mitchison and Kirschner (1984)
who first directly observed the behavior in vitro. Later experiments confirmed that this
phenomenon occurred in living cells. In 1990, a third member of the tubulin protein
family, now known as γ-tubulin, was identified (Oakley et al. 1990). Found almost
exclusively in the gel-like pericentriolar cloud of the centrosome, γ-tubulin is thought to
nucleate the assembly of MTs from αβ-tubulin (Joshi et al. 1992), leading to the ability
of the centrosome to serve as a microtubule-organizing center (MTOC). The details of
this nucleation process have remained elusive.
In addition to the aforementioned fiber subunits, the cytoskeleton possesses
numerous, less abundant associated proteins. Through biospecific binding interactions
with tissue-specific/selective MF-, IF-, and MT-associated proteins, the fibers interact
with other fibers of like kind to generate the cytoskeletal organelles required within
specific cells. These structures also act as supramolecular scaffolds for such molecular
motors as dynein, kinesin, or myosin to propel intracellular organelles and/or
chromosomes. In the case of the actin cytoskeleton, there are at least 140 such proteins
5
that can be classified functionally as monomer-sequestering proteins (e.g., thymosin-β4,
profilin and actobindin), filament-capping proteins (e.g., CapG, CapZ, and profilin),
filament cross-linking proteins (e.g., α-actinin, fimbrin, and ABP-280),
membrane-anchoring proteins (e.g., zyxin, vinculin, talin, and ponticulin), and
severing/depolymerizing proteins (e.g., gelsolin, cofilin and villin). Likewise,
intermediate proteins appear to interact with certain micro as well as many protein
kinases and phosphoprotein phosphatases that comprise various signal-transduction
pathways. Microtubules-associated proteins (MAPS) are thought to influence cell
dynamics via microtubules binding. MAPs have either structural (e.g., MAP-2 and Tau)
or enzymatic activities (e.g., kinesin and MAP-1c). The remainder of this chapter is
devoted to a discussion of the roles of microtubules and their microtubule-associated
proteins (abbreviated MAPs) in the neuronal cytoskeleton.
Tubulin
Identification and General Properties.
The first tubulin was identified as a protein with a Mr of 100,000 daltons able to
bind to the anti-mitotic drug colchicine (Borisy and Taylor 1967, Wilson and Friedkin
1967). It was then purified from rat brain homogenates using radiolabeled colchicine
(Weisenberg et al. 1968). The α- and β−subunits were visualized by polyacrylamide gel
electrophoresis, but researchers were unable to reassemble tubulin in vitro until 1972 as
shown in electron micrographs (Weisenberg 1972). Polymerization studies conducted
with partially purified tubulin showed the requirement of GTP, magnesium, and EGTA;
on the contrary, calcium was shown to inhibit microtubule assembly (Olmsted and Borisy
1975, Rosenfeld et al. 1976). The two-step polymerization process has an initial lag
6
phase for nucleation to occur, followed by a period of growth or elongation (Barton and
Riazi 1980, Bryan 1976, Karr et al. 1979). Assembly and disassembly of the polymer
depends on the concentration of the tubulin (Barton and Riazi 1980, Gaskin et al. 1974,
Karr et al. 1979). The kinetics of both stages depends also on the temperature of the
reaction. Currently, microtubule protein (MTP) preparations use repeated cycles of
warm-induced assembly and cold-induced disassembly in a glycerol-containing buffer
(Karr et al. 1979, Shelanski et al. 1973). In conjunction with electron microscopy,
microtubule assembly and disassembly properties are routinely measured as turbidity
measurements in a spectrophotometer (Gaskin et al. 1974).
Structural Properties.
In solution, αβ tubulin remains predominantly associated in its dimeric form, and
each subunit has an approximate molecular weight of 50,000 daltons. The dimeric form
was demonstrated by crosslinking solubilized chick brain tubulin via
dimethyl-3,3’-(tetramethylenedioxy)dipropionimidate followed by nondenaturing gel
electrophoresis (Luduena et al. 1977). Circular dichroism studies revealed purified
tubulin contains nearly 50 % random coil, 20 % alpha-helical structure and about 30 %
beta sheet. (Ventilla et al. 1972). Amino acid sequence determination reveals both
subunits are rich with glutamic acid residues (Ponstingl et al. 1981, Valenzuela et al.
1981) sharing 36-42 % homology (Little and Seehaus 1988). Despite their similarities, in
mammals there are multiple α- and β-tubulin genes, four and six, respectively, and
greater than 20 variant isoforms have been identified (Field et al. 1984, Silflow and
Rosenbaum 1981). The C-terminus region is where α- and β-tubulin are the most
divergent. This region of the two subunits is modified post translationally, with α-tubulin
7
undergoing acetylation (L'Hernault and Rosenbaum 1985), glutamylation (Edde et al.
1990) and tyrosinylation (Raybin and Flavin 1977) while β-tubulin is phosphorylated
(Gard and Kirschner 1985). The significance of these modifications is still unknown, but
it has been suggested that microtubules are rendered more or less stable by a particular
modification. Indeed, codfish microtubules, which are particularly stable in cold
temperature, are enriched with acetylated and tyrosinylated tubulin (Detrich et al. 1987),
although stability has not been directly demonstrated with these covalent modifications.
In 1998 an atomic model of the alpha-beta tubulin dimer, fitted to a 3.7 Å density map,
was obtained by electron crystallography of zinc-induced tubulin sheets. From the model
it is evident that the structures of α- and β-tubulin are basically identical: a core of two
beta-sheets surrounded by alpha-helices forms each monomer. Furthermore the
monomer structure is very compact, but can be divided into three functional domains: the
amino-terminal domain containing the nucleotide-binding region, an intermediate domain
containing the Taxol-binding site, and the carboxy-terminal domain, which probably
constitutes the binding surface for motor proteins (Nogales et al. 1998).
Nucleotide Binding Sites.
The alpha-beta tubulin dimer is the building unit of microtubules. Both the α- and
the β-tubulin monomers contain a binding site for guanine nucleotide, but the sites are
nonidentical. Early experiments demonstrated distinguished abilities to exchange
radiolabeled GTP from the medium. The exchangeable site (E-site) readily replaces GTP
for GDP within 15 minutes, whereas the nonexchangeable site (N-site) does not
exchange, even over several hours (Berry and Shelanski 1972, Weisenberg et al. 1968).
Use of 8-azido-GTP as a photo-affinity probe indicated that hydrolysis occurred on
8
β-tubulin concurrently with polymerization (Geahlen and Haley 1977, Geahlen and
Haley 1979). Additionally, David-Pfeuty et al. (1977) showed the participation of a
GTPase activity in microtubule polymerization, and MacNeal and Purich (1978) showed
that tubulin incorporation into MTs is correlated to the hydrolysis of GTP on the
exchangeable site in a one-to-one stoichiometry. Karr et al. (1979) used nonhydrolysable
GTP analogs to demonstrate that GTP is involved early in nucleation, while GDP
stabilizes microtubules during elongation. Later Angelastro and Purich (1992)
discovered deoxy-GTP in the N-site of nerve growth factor-treated PC12
pheochromocytoma cells as well as embryonic chick dorsal root ganglion neurons.
Microtubules
Structural Properties.
Microtubules are hollow cylinders composed of longitudinally arranged
protofilament arrays of αβ tubulin (see Figure 1-1). They have a fast growing
(conventionally defined the plus end) and a slow growing (defined as minus) end. The
extremes of the microtubule can be differentiated by the direction of movement of motor
proteins (i.e., kinesin and dynein) that use them as vectorially defined scaffolds for
intracellular trafficking. The detailed packing of the globular subunits has been
investigated by diffraction analysis of negatively stained flagellar and brain microtubules
(Amos and Klug 1974, Erickson 1974). The data, from both populations, revealed a
4 nm axial periodicity and 8 nm wall and shows a heterologous interaction between
protofilaments. In axonemes a double ringlet is observed with two single filaments
surrounded by nine outer doublet microtubules composed of an A and B tubule. In
general in vitro electron microscopy studies unusual microtubule structures are observed
9
Figure 1-1. Dynamic microtubule filament. Microtubules are composed of α- and βtubulin subunits in a linear array. Both the plus and minus ends are dynamic and have a
GTP cap which can be subsequently lost over time. The α-subunit has GTP bound at all
times (non-exchangeable), while the β-subunit has either GTP or GDP bound
(exchangeable). The drawing shows how one end (plus or growing end) is more dynamic
than the other
10
from ribbons, sheets and rings (Matsumura and Hayashi 1976, McEwen and Edelstein
1980). These structures depend on buffer and pH conditions, and it is not clear if they
occur in nature. Microtubules are highly acidic because of numerous negatively charged
residues present in the C-terminal part of tubulin. This acidic nature of microtubules
facilitates their association with over 350 drugs (e.g., taxol, colchicine and vinblastine).
Microtubule Assembly.
The assembly of tubulin into microtubules is a polymerization reaction that can be
divided into three different phases: nucleation, elongation and length redistribution. The
nucleation and elongation steps can be followed spectrophotometrically with a distinct
separation between the initial rate and steady-state kinetic properties. Length
redistribution refers to a property of the microtubule ends observed both in vitro
(Kristofferson et al. 1986, Mitchison and Kirschner 1984) and in vivo (Sammak and
Borisy 1988), known as dynamic instability. This property of microtubules is described
as a continuous flux of shrinking and growing microtubule populations. Some
microtubules continue to grow while others are lost by depolymerization at the distal
ends. Growing and shrinking microtubules rarely interconvert. By means of electron and
direct fluorescent microscopy techniques it has been demonstrated that the kinetics at
each end is independent of the other as evidenced under in vivo conditions where minus
ends are held at MTOC and thus are not free to undergo any exchange. The sudden loss
of GTP cap followed by a rapid shortening of the microtubule is termed a “catastrophe”.
Subsequently, the filaments can be “rescued” by the addition of tubulin dimers or the
association with fibrous MAPs which blocks further depolymerization (Kowalski and
Williams 1993, Simon and Salmon 1990).
11
The affinity constants for both ends of the microtubule have been measured using
electron microscopy and spectrophotometric assays and the dissociation constants for the
plus and the minus ends have been determined to be 17 s-1 and 7 s-1, respectively (Bergen
and Borisy 1980, Carlier and Pantaloni 1981). Several investigators have shown a direct
correlation between GTP hydrolysis and microtubule polymerization (Davis et al. 1994,
Karr et al. 1979, O'Brien et al. 1987, Stewart et al. 1990). Additionally the carboxyl
terminus of each tubulin subunit, where post-translational modifications are present, has a
control of MT assembly. This part of the tubulin molecule has a conserved GTP binding
motif (G-X-X-X-X-G-K) as well as a proposed site for MAP-MT interactions, and the
region may also regulate interactions with drugs such as colchicine (Avila et al. 1987). In
fact the selective removal of 4 amino acids from the C-terminal domain by subtilisin
treatment results in a loss of MAP binding and assembly regulation (Dingus et al. 1991).
Microtubule-Associated Proteins
Many proteins interact with microtubules. They can be divided in three main
classes. The “microtubule-associated proteins”, or MAPs, are sometimes called more
specifically “structural” or “fibrous” MAPs because they can bind to microtubules
stabilize them and promote their assembly, and because they can be copurified with
tubulin through several cycles of microtubule assembly and disassembly (Shelanski et al.
1973). Another broad class comprises the motor proteins, so called because they
generate movement along microtubules using the chemical energy of ATP hydrolysis.
The motors do not copurify through cycles of assembly and disassembly, although they
can bind tightly to microtubules. A third and more general class includes heterogeneous
proteins not normally defined as MAPs but often found associated with microtubules.
The interactions exerted by this third group of proteins are not always well defined, but
12
contribute to the idea for a cytoskeleton as a temporary docking site for many
cytoplasmic proteins.
Motor-driven MAPs.
Motor proteins recognize the asymmetry of microtubules and move along MTs as
though they are tracks with a defined polarity (Gibbons 1965). Axonemal dynein is a
two-headed, minus-end directed motor protein, which propagates with a sliding motion
by forming crossbridges between A- and B-tubules of adjacent doublets (Gibbons 1988).
This motor is composed of a heavy chain, two or three intermediate chains and four light
chain polypeptides. Cytoplasmic dynein is morphologically indistinguishable from
axonemal dynein (Vallee et al. 1988), although composed of nine similar subunits
(Paschal et al. 1987). Cytoplasmic dynein is widely distributed and plays a role in
retrograde organelle transport with rates of 1.3-2.0 mm/sec (Paschal et al. 1987). In 1990
two independent observations both showed dynein association with kinetochore
microtubules of the mitotic spindle, and the data suggest a direct involvement in
chromosomal separation (Pfarr et al. 1990, Steuer et al. 1990).
The kinesin super family of proteins is perhaps the best characterized of the motor
proteins. Structurally, each consists of three domains: a globular, two-headed catalytic
region; a coil-coil polypeptide stalk, and a fan-like tail (Amos 1987, Scholey et al. 1989).
The catalytic region or head domain of kinesin binds to β-tubulin with a stoichiometry of
one kinesin head per tubulin heterodimer, generating an axial periodicity of 8 nm (the
height of the dimer) and a 'B'-lattice in which adjacent protofilaments are staggered by
about 0.9 nm.(Harrison et al. 1993). The head domain is conserved among the members
of this family with 40 % homology, whereas other regions are highly divergent. Kinesins
13
are directed toward the plus-end of microtubules and are ATP-dependent. They were
first isolated as an activity that moved microtubules along latex beads at 2-4 mm/sec
(Vale et al. 1985). Immunofluorescence microscopy experiments indicate that kinesin is
associated with organelles but unlike dynein, can transport vesicles in vitro without added
cellular factors (Pfister et al. 1989).
Structural or Fibrous MAPs
The use of brain extracts to assemble microtubules in vitro (Weisenberg 1972) laid
the foundation for the identification of cytoplasmic protein components that associate
with the MT lattice. Fractions enriched in fibrous MAPs promoted MT assembly at
lower critical concentration than did tubulin alone (Sloboda et al. 1976, Weingarten et al.
1975). Among the best-characterized structural MAPs are MAP2, MAP4 and tau
proteins. Additionally, MAP-1a and -1b were identified in both axonal and dendritic
processes as a major neuronal component (Bloom et al. 1985). However, the inability to
quantitatively purify this protein has hindered its biochemical analysis.
Tau proteins were first purified from porcine brain homogenates under nonstringent
conditions (Weingarten et al. 1975), while Herzog and Weber (1978) and Kim et al.
(1979), heat-treated brain microtubule proteins as a mean to isolate the thermostable
proteins, MAP2 and tau. Figure 1-2 shows the current working model of a MAP
structure and binding to the microtubule lattice. MAP2 is a multifunctional domain
protein with two major regions: an acidic (pI 4.8) N-terminal projection arm domain that
extends away from the microtubule and a highly basic (pI 10.5) MT-binding domain
(Flynn and Purich 1987). The latter only is necessary for MT polymerization, as
demonstrated by experiments in vitro where the projection arm was selectively removed
without altering the assembly properties of the MAP (Vallee and Borisy 1977).
14
Although the tissue and subcellular distribution of MAP2, tau and MAP4 is
distinctly different these proteins appear structurally related. MAP2 and Tau localize to
the dendrite and the axon, respectively, and they share nonidentical tandem repeats of a
sequence of 18 amino acids stretches, repeated three to four times (see Figure. 1-2)
(Lewis et al. 1988). These repeats are highly conserved in the MT-binding region of
these proteins as well as in MAP4. Independent reports by Joly et al. (1989) and Ennulat
et al. (1989) revealed that synthetic peptides corresponding to the second repeated
sequence in either MAP2 or tau, respectively, competed with native protein and promoted
MT assembly. Additionally in tau the first repeat was also shown to bind to
microtubules, implying that MAP2 and tau, although structurally quite similar, are
distinct proteins and this may account for their specific roles in dendrites and axons.
Likewise, MAP4, originally purified from adrenal cortex, is an ubiquitously expressed
protein which has been associated with spindle pole bodies and is a member of this MAP
superfamily (Olmsted 1986). Interestingly all three proteins are rich in proline, which
may explain their lack of defined structure and rod-like shapes (Hirokawa et al. 1988,
Murofushi et al. 1986, Voter and Erickson 1982).
MAP2
Microtubule-associated protein 2 (MAP2) is a family of heat-stable,
phosphoproteins expressed predominantly in the cell body and dendrites of neurons.
Three major MAP2 isoforms, (MAP2a, MAP2b, MAP2c) are differentially
expressed during the development of the nervous system and have an important role in
microtubule dynamics. A single gene on chromosome 2 codes for the proteins by
alternative splicing of a single transcript into 6 kb and 9 kb transcripts (Neve et al. 1986).
All MAP2 share a common structure with an amino-terminal the projection arm, a
15
Figure 1-2. Schematic representation of a MAP interaction with a microtubule. The
microtubule-binding region (MTBR) can be seen in close association with the MT while
the projection arm extends away from the microtubule acting as a spacer between
adjacent microtubules, or possibly crosslinker. A hinge region susceptible to protease
activity connects the two parts of MAP. The MTBR is shown magnified and three repeats
of 18 amino acids are represented separated by the 13 amino acid inter-repeat regions.
16
carboxy-terminal MTBR and a protease accessible hinge connecting the two domains
(Rubino et al. 1989, Vallee 1980)
Two proteins, MAP2a and MAP2b are the high-molecular-weight forms, with an
extended projection arm at the amino-terminus and three repeats in the carboxy-terminal
microtubule-binding region (Kalcheva et al. 1995). MAP2a, the larger isoform, is found
only in adults while MAP2b is present throughout life. The low-molecular-weight forms
are MAP2c and MAP2d, and they contain three and four repeats respectively (Doll et al.
1993, Garner et al. 1988). The juvenile three-repeat MAP2c and adult four-repeat
MAP2d each are missing 1372 amino acids of the projection arm. It appears that MAP2a
replaces the juvenile MAP2c as the transition from juvenile to adult occurs, although it is
not excluded that the adult low-molecular-weight form MAP2d could also be involved in
this developmentally regulated switch of forms (Tucker et al. 1988).
The microtubule-binding region of MAP2 was originally identified as a 28-36 kDa
protease cleavage product of the carboxyl-terminal tail that promoted MT assembly
(Flynn et al. 1987, Flynn and Purich 1987, Vallee 1980). This region was subsequently
cloned from rat, mouse, bovine and human and led to the elucidation of the repeats and
residues responsible for MT binding. Previous work from this lab demonstrated that the
second octadecapeptide repeat M2 is the major binding repeat of MAP2 (Coffey 1994,
Zhang 1997). Additionally, other experiments showed that synthetic octadecapeptides
corresponding to the second repeat only, were able to compete with MAP2 for MTs
binding and promoted MT assembly (Joly et al. 1989, Joly and Purich 1990, Zhang
1997).
17
All MAP2 proteins are highly phosphorylated with an amino terminal PKA RII
binding site on the projection arm (Rubino et al. 1989, Vallee 1980). There are nearly
200 serine and threonine residues that are potential phosphorylation sites in high
molecular weight MAP2 with consensus sequences for cAMP-dependent kinase,
calcium/phospholipid-dependent kinase, calcium/calmodulin-dependent kinase, glycogen
synthase kinase and several proline-directed kinases. At least two of these kinases, PKA
and MAPK-2, purify with cycled microtubules and are known to phosphorylate the
protein in vitro (Lopez and Sheetz 1995, Theurkauf and Vallee 1982). In neurons, MAP2
is heavily phosphorylated as observed when the protein is purified using focused
microwave irradiation to denature kinases and phosphatases in situ. It has been
determined that there are 46 moles of phosphoryl groups incorporated per mole of MAP2
in vivo (Tsuyama et al. 1987), but only one serine in position 136 has been identified as
being phosphorylated in vivo by a proline-directed kinase such as MAPK or GSK-3
(Berling et al. 1994). Recently MAP2c has been shown to interact with the nonreceptor
tyrosine kinase Fyn. An SH3-binding motif (R-T-P-P-K-S-P) adjacent to the first repeat
of the microtubule-binding domain is threonine/serine-phosphorylated by Fyn in vivo, as
shown by coimmunoprecipitation of Fyn and MAP2c from human fetal homogenates and
cotransfection of MAP2c and Fyn in COS7 cells (Zamora-Leon et al. 2001).
Identification of phosphorylation sites in MAP2 is a particularly important issue
since in vitro phosphorylation of MAP2 has been shown to affect the dynamics of
microtubule assembly and disassembly (Ainzstein and Purich 1994). More work is being
done to uncover the roles of other conserved amino acids and regions in the MTBR of
MAP2.
18
Tau
In 1975 Weingarten et al. described a heat-stable protein that copurified with
tubulin through repeated cycles of polymerization and facilitated the formation of
microtubules (Weingarten et al. 1975). Subsequently, the protein was purified to near
homogeneity and was shown to migrate on SDS-polyacrylamide gels as several closely
spaced bands with apparent molecular masses between 55 and 62 kD. The protein was
characterized as being highly phosphorylated with an average pI value of ~7.3 and highly
asymmetric with little defined secondary structure and no significant amounts of α-helix
or β-sheet conformation (Cleveland et al. 1977a, Cleveland et al. 1977b, Schweers et al.
1994). This extended conformation of tau may result from high levels of proline and
glycine in its primary structure (Goedert et al. 1989, Lee et al. 1988).
The lack of a defined structure probably contributes to the protein heat stability
(Weingarten et al. 1975), solubility in 2.5 % perchloric acid (Lindwall and Cole 1984a)
and retarded migration on SDS-polyacrylamide gels (Lee et al. 1988). Tau is
predominantly a neuronal protein located mostly in the axon, to a lesser extent in cell
bodies, and almost totally absent from dendrites (Binder et al. 1985). It is expressed in
six different isoforms in adult human brain from alternative splicing of a single gene
located in chromosome 17 (Goedert et al. 1991, Goedert et al. 1989, Himmler 1989, Lee
et al. 1988). Additional isoforms of tau have been identified including a higher molecular
weight form, largely localized to the peripheral nervous system (Andreadis et al. 1992,
Couchie et al. 1992).
The 6 tau isoforms differ from each other by the presence or absence of three
inserts encoded by exons 2, 3 and 10 (Figure. 1-3). Alternative splicing of exon 10
19
generates isoforms with three (exon 10 missing) or four (exon 10 included)
microtubule-binding domains (3R and 4R tau) (Goedert et al. 1991). In the adult human
brain the ratio of 3R and 4R isoforms is approximately 1:1; however, in fetal brain only
3R tau is present, demonstrating developmental regulation of exon 10 alternative
splicing. The repeats and some adjoining sequences constitute the microtubule-binding
domains of tau. The repeats traditionally have been divided into the first 18 amino acids,
which constitute the originally described repeated regions of tau (T1-T4) and the
following 13 or 14 amino acids that were termed the inter-repeats. Synthetic
octadecapeptides corresponding to T1 and T2 have been shown to promote the assembly
of MTs while synthetic peptides of T3, T4 and randomized T1 did not (Ennulat et al.
1989). In addition, the inter-repeat between T1 and T2 of adult tau was found to bind
MTs in a strong electrostatic interaction that required three lysine residues (Goode and
Feinstein 1994). This tightly binding region is thought to confer extra stability to MTs in
the axons of adults by providing an additional MT binding site that reduces microtubule
depolymerization.
Tau protein promotes microtubule assembly and binds to microtubules, which are
stabilized as a result.
Tau Phosphorylation
The tau repeats region has many potential phosphorylation sites, and many kinases
have been shown to phosphorylate tau within and around the microtubule-binding region.
Similar to MAP2, this phosphorylation is believed to alter microtubule binding,
proteolysis and intracellular trafficking of tau (Billingsley and Kincaid 1997, Mandelkow
et al. 1995). In vitro tau is a substrate for a multitude of protein kinases. These include,
but are not limited to, Ca++/calmodulin-dependent protein kinase II (CaMKII)
20
Figure 1-3. Schematic representation of the six isoforms of tau expressed in the human
adult brain, by alternative splicing of exons E2, E3, and E10. The isoforms range from
352 to 441 amino acids in length and they differ by the presence of either three (3R-tau)
or four (4R-tau) carboxy-terminal tandem repeat sequences of 31 or 32 amino acids each.
Additionally, the triplets of 3R-tau and 4R-tau isoforms differ as a result of alternative
splicing of E2 and E3 exons that code for two 29 amino acid long inserts in the aminoterminal part of the molecule, with unknown functions. (Spillantini and Goedert 1998)
21
(Johnson 1992, Steiner et al. 1990), casein kinase II (Greenwood et al. 1994),
cAMP-dependent protein kinase (cAMP-PK) (Litersky and Johnson 1992, Pierre and
Nunez 1983); MAP kinase (also known as ERK2) (Drewes et al. 1992), a neuronal
cdc2-like protein kinase (cdk5/p35) (Lew and Wang 1995, Paudel et al. 1993) and
glycogen synthase kinase 3 (GSK3) (Hanger et al. 1992, Mandelkow et al. 1992).
However, it is still unclear which protein kinases phosphorylate tau in vivo.
Data from various groups during the past years made it clear that the
phosphorylation of specific sites, rather than the overall extent of phosphorylation, was
the important factor in modulating the ability of tau to bind microtubules and promote
microtubule assembly.
Phosphorylation of tau at just a few sites within the microtubule-binding regions
(Ser 262, Ser 356, and to a lesser extent Ser 324 and Ser 293) by p110mark (MARK)
virtually abolishes the ability of tau to bind microtubules (Drewes et al. 1995). The
phosphorylation state of tau modulates parameters other than its interaction with
microtubules. For example, phosphorylation apparently plays a role in regulating the
conformation of the molecule as evidenced by the fact that dephosphorylated tau migrates
faster in SDS-polyacrylamide gels than phosphorylated tau (Lindwall and Cole, 1984a;
Lindwall and Cole, 1984b), and phosphorylation changes the paracrystal structure of tau
(Hagestedt et al. 1989).
Although the in vitro phosphorylation of tau has been studied extensively, the
phosphorylation of tau in situ is less characterized. However, several recent studies have
begun to elucidate the specific protein kinases that regulate the phosphorylation state of
tau in vivo. Lovestone et al. (1994) demonstrated that cotransfection of tau and GSK3
22
into COS cells resulted in a decrease in the electrophoretic mobility of tau and increased
reactivity with a panel of monoclonal antibodies against phosphoepitopes on tau. In
contrast, neither ERK1 nor ERK2 phosphorylated tau in COS cells transiently transfected
with tau and kinase cDNAs (Anderton et al. 1995, Lovestone et al. 1994).
Tau in Alzheimer’s Disease and Other Neurodegenerative Diseases
In 1906, Alzheimer using a silver impregnation technique observed abnormal
tangle-like structures in the brain of a patient with dementia, which he described as
neurofibrillary tangles (NFTs). In 1963, Kidd and Terry independently demonstrated that
the NFTs seen in Alzheimer's disease consist of dense perinuclear filamentous aggregates
of a unique nature (Kidd 1963, Terry 1963) prevalently located in the hippocampal
formation, the transentorhinal region and the entorhinal region. Kidd described these
filamentous structures as paired helical filaments (PHFs) based on their morphological
characteristics (Kidd 1963). These structures have a characteristic periodic modulation in
diameter, which varies from 10-20 nm with a crossover distance of 75-80 nm.
Early studies reported that in Alzheimer's disease there was a correlation between
the number of cortical NFTs and the degree of cognitive impairment (Roth et al. 1967).
Subsequent studies have also revealed that the extent of tau pathology (NFTs and
dystrophic processes) correlate with dementia severity (Arriagada et al. 1992). Once the
basic structure of filaments that composed the NFTs had been identified, investigators
analyzed the composition and organization of the PHFs (Crowther and Wischik 1985,
Wischik et al. 1985). One difficulty in studying these structures biochemically was the
fact that the PHFs of the NFTs were extremely insoluble (Selkoe et al. 1982). Ihara et al.
(1983) found that antisera raised against PHFs did not recognize normal cytoskeletal
proteins demonstrating that PHFs were unique pathological formations.
23
In 1986 several groups simultaneously discovered that the main component of the
PHFs found in the NFTs of Alzheimer's disease brain was the microtubule-associated
protein tau in an abnormally phosphorylated state (Brion et al. 1986, Grundke-Iqbal et al.
1986, Ihara et al. 1986, Kosik et al. 1986, Yang et al. 1993). All six isoforms of tau were
shown to be core components of the PHFs (Goedert et al. 1989). Furthermore when
observed by electron microscopy with negative staining, PHF filaments appeared
surrounded by a fuzzy coat of material. Treatment of the filaments with the proteases
cocktail Pronase was able to remove the fuzzy coat leaving a core filament that when
analyzed was found to contain only the C-terminal microtubule-binding region from tau
(Goedert et al. 1988, Wischik et al. 1988).
The finding that hyperphosphorylated tau was the predominant protein of PHFs and
NFTs launched a new era of research characterizing the functional properties of tau in
Alzheimer's disease brain. When sections from normal and Alzheimer's disease brain
were stained for tau localization the most intense tau staining in control tissue was found
in the white matter, consistent with tau being predominantly axonal (Papasozomenos and
Binder 1987). However, in Alzheimer's disease prominent, tau-immunoreactive fibers
without classical NFT formation were detected in the cell bodies of affected neurons. In
addition, NFTs consisted of perinuclear tau-reactive filaments that often extended into
proximal dendrites and sometimes terminated as fine dystrophic neurites (Kowall and
Kosik 1987). These data indicate that the localization of tau protein in Alzheimer's
disease brain is strikingly abnormal, and may contribute to neuronal dysfunction. NFTs
are found predominantly in the perinuclear region, closely encircling the nucleus. The
PHFs of these NFTs are located not only in the outer membrane of the nuclear envelope,
24
but also inside the nuclei. In addition, the intranuclear PHFs occasionally appear to be
attached to the inner membrane of the nuclear envelope (Metuzals et al. 1988).
Tau Phosphorylation in Alzheimer’s Disease.
Tau isolated from AD patients runs on an SDS-PAGE gel as three major bands of
60, 64 and 68 kDa with a minor band at 72 kDa. This reduced mobility on SDS-PAGE
gels can be restored by phosphatase treatment of the protein, showing a
hyperphosphorylation of PHF-tau (Greenberg and Davies 1990, Lee et al. 1991). This
hyperphosphorylation state changes in reactivity with phosphorylation-dependent
antibodies to tau. Tau isolated from soluble PHFs (PHF-tau) is extensively
phosphorylated compared to normal tau at both Ser/Thr-Pro and nonSer/Thr-Pro sites
(Morishima-Kawashima et al. 1995).
Use of monoclonal antibodies that recognize specific phosphoepitopes has been the
tool of choice for many investigations on PHF phosphorylation and in fact, much
information about which sites are phosphorylated in PHF-tau has been gained through
these molecules. Tau-1, which recognizes tau when it is dephosphorylated at Ser
199/202, Thr 205, was the first phosphate-dependent antibody to be developed (Binder et
al. 1985). In subsequent years many other antibodies that recognize specific
phosphoepitopes were made. Several kinases, which phosphorylate normal tau at the
same sites that are phosphorylated in AD-tau, have been found, and phosphopeptide
mapping of phosphorylation sites in AD-tau fragments has permitted the identification of
several candidate kinases that may play a role in Alzheimer disease. These include the
proline-directed kinases p34-cdc2 and GSK-3 that are known to phosphorylate tau and to
produce retarded mobility on SDS-PAGE gels (Lovestone et al. 1994). Whether these
kinases affect tau polymerization into paired helical filaments or whether they are
25
phosphorylated after filament assembly remains to be determined. Although several
early AD epitopes are phosphorylation-sensitive, more work is clearly needed to
determine how phosphorylation is altered during the course of Alzheimer filament
assembly.
Other Neurodegenerative Diseases Exhibiting Neurofibrillary Tangles
Neurodegenerative diseases of the brain accompanied by dementia affect 5-10% of
individuals over the age of 65 in the western world and represent one of the main health
related economical problem of our society. In the majority of cases, these patients suffer
from Alzheimer’s disease (AD), but many other forms of progressive neuropathological
degenerations have been characterized, that present NFT inclusions in neuronal cells of
affected areas of the brain. These include Down syndrome (Cork 1990, Olson and Shaw
1969, Wisniewski et al. 1976), Parkinson-dementia of Gaum (Hirano et al. 1968, Hirano
et al. 1961), diffuse Lewy body disease (Gibb et al. 1987, Pollanen et al. 1992),
Gerstmann-Strausller-Scheinker disease (Ghetti et al. 1994, Tagliavini et al. 1993), prion
protein amyloid angiopathy (Hsiao et al. 1992, Tranchant et al. 1997), familial presenile
dementia (Spillantini et al. 1996, Sumi et al. 1992), dementia pugilistica (Wisniewski et
al. 1976), Pick’s disease (Love et al. 1995, Murayama et al. 1990), corticobasal
degeneration (Feany and Dickson 1995, Ksiezak-Reding et al. 1994, Paulus and Selim
1990), progressive supranuclear palsy (Flament et al. 1991, Vermersch et al. 1994) and
familial multiple system tauopathy (Spillantini et al. 1997).
These diseases have different phenotypic manifestations, but they are all linked to
progressive accumulation of tau filament inclusions in the affected cells. The
circumstantial evidence of tau involvement in neurodegenerative disease was further
substantiated in 1998 when multiple tau gene mutations were discovered in
26
frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), a group
of clinically heterogeneous neurodegenerative disorders, sharing common pathological
features like neuronal loss in affected brain regions, with glial fibrillary deposits of
hyperphosphorylated tau protein, but without evidence of Aß deposits or other
disease-specific brain lesions. This finding provided unequivocal evidence that tau
abnormalities alone are sufficient to cause the disease (Hutton et al. 1998, Poorkaj et al.
1998, Spillantini et al. 1998), fueling a new and increased interest in tau as a major player
in neurodegenerative diseases.
Since 1998 several mutations in the tau gene have been found associated with
familial and sporadic forms of progressive neuronal degeneration of the brain. These
mutations in the tau gene lie within the C-terminal region of the protein where the
microtubule-binding domains are located. They are missense, deletion or silent
mutations in the coding region of exons 9, 10, 12 and 13 or intronic mutations located
close to the splice-donor site of the intron following exon 10, which codes for the fourth
repeat in the microtubule-binding region of tau. Some of the mutations have been shown
to unbalance the ratio of 3- to 4-repeat isoforms in favor of the 4-repeat forms.
Overproduction of 4-repeat tau is thus sufficient to lead to filamentous tau pathology,
resulting in neurodegeneration and dementia. Other point mutations affect all isoforms,
being in coding regions other than exon 10. Whatever is the effect of these mutations,
affected tau isoforms have a reduced ability to interact with microtubules as demonstrated
by in vitro studies using recombinant tau in cell-free assays or cells transfected with
wild-type and mutant tau cDNA constructs (Dayanandan et al. 1999, Hasegawa et al.
1998, Hong et al. 1998).
27
Despite all the observations, the mechanism by which most of these mutations
affect neuronal cells and result ultimately in the disease is still not completely
understood. In the past two years animal models have been developed to gain new
perspectives in the comprehension of these neurodegenerations. Transgenic mice
overexpressing the four-repeat form of human tau show numerous abnormal,
tau-immunoreactive nerve cell bodies and dendrites. Enlarged axons containing
neurofilament- and tau-immunoreactive spheroids are observed especially in spinal cord.
Furthermore, signs of Wallerian degeneration and neurogenic muscle atrophy are
observed, along with signs of muscular weakness although neurofibrillary tangles are not
observed (Probst et al. 2000). Overexpression of the most common tau mutation (P301L)
in FTDP-17 results in motor and behavioral deficits in transgenic mice, with age- and
gene-dose-dependent development of NFT (Lewis et al. 2000).
Another genetic model of tau-related neurodegenerative disease was obtained by
expressing wild-type and mutant forms of human tau in the fruit fly Drosophila
melanogaster. Transgenic flies showed key features of the human disorders: adult onset,
progressive neurodegeneration, early death, enhanced toxicity of mutant tau,
accumulation of abnormal tau, and relative anatomic selectivity. However,
neurodegeneration occurred without the neurofibrillary tangle formation that is seen in
human disease and some rodent tauopathy models (Wittmann et al. 2001). Although
these animal models may provide an interesting insight into the mechanism underlining
this group of diseases, a biochemical model that explains polymerization kinetics of
neurodegenerative tau is still missing. In order to obtain such information many studies
28
are being conducted utilizing the ability of tau to polymerize into paired helical or
straight filaments in vitro.
In Vitro Studies of Tau Polymerization
Paired helical filament, the principal component of the neurofibrillary tangles
characteristic of Alzheimer’s disease, consists of two structurally distinct parts, as
observed by electron microscopy: an external fuzzy region that can be removed by
Pronase treatment and a Pronase-resistant morphologically recognizable core. Analysis
of the Pronase resistant core has shown that the microtubule-binding domain of tau is
tightly and specifically bound in the core of the paired helical filament (Crowther et al.
1989). Furthermore PHF-like filaments can be assembled in vitro from bacterially
expressed, nonphosphorylated 3-repeat microtubule-binding region of tau (Crowther et
al. 1992, Wille et al. 1992). These observations along with the availability of large
quantities of recombinant human tau isoforms and the ease with which tau fragments can
be expressed have facilitated studies aimed at producing synthetic tau filaments. The
formation of these filaments lent strong support to the idea that the repeat region of tau is
the only component necessary for the morphological appearance of the PHF. However,
these studies failed to provide any insight into filament formation in vivo because the tau
filaments were obtained only with truncated tau under nonphysiological conditions, in
contrasts with PHFs from AD brain, which consist of full-length tau protein (Goedert et
al. 1992).
In a later study Goedert et al. obtained paired helical-like filaments under
physiological conditions in vitro, when nonphosphorylated recombinant tau isoforms
with three microtubule-binding repeats were incubated with sulphated
glycosaminoglycans such as heparin or heparan sulphate (Goedert et al. 1996). The
29
assembly into filaments of tau isoforms in the presence of sulphated glycosaminoglycans
occurs after a lag period and is heavily concentration dependent, consistent with a
nucleation-dependent process (Arrasate et al. 1997, Friedhoff et al. 1998, Goedert et al.
1996, Hasegawa et al. 1997, Perez et al. 1996). Glycosaminoglycans with a higher linear
charge density appear to be more efficient in facilitating polymerization. Subsequent to
this work, RNA (Hasegawa et al. 1997, Kampers et al. 1996) and arachidonic acid
(Wilson and Binder 1997) were also shown to induce the bulk assembly of full-length
recombinant tau into filaments. Incubation times in the presence of either heparin or
tRNA are reduced from 3 weeks to one day for filament assembly. The concentration of
three-repeat tau required for assembly was lowered ten-fold in respect to nonanionic
conditions, and four-repeat full-length gave similar results. Pathological colocalization of
sulphated glycosaminoglycans (Goedert et al. 1996, Snow et al. 1990, Verbeek et al.
1999) and RNA (Ginsberg et al. 1998) with hyperphosphorylated tau protein suggests
that these findings may also be relevant for the assembly of tau in AD.
Hyperphosphorylation is believed to be an early event in the pathway that leads to
the formation of insoluble and filamentous tau protein (Braak et al. 1994), however it is
unclear whether phosphorylation is sufficient for assembly into filaments, as there is no
experimental evidence that links hyperphosphorylation of tau to filament assembly.
Phosphorylation of tau at Ser/Thr-Pro sites does not significantly influence
heparin-induced assembly (Goedert et al. 1996). However, it has been reported that
phosphorylation at other sites, such as Ser214 and Ser262, is strongly inhibitory toward
assembly (Schneider et al. 1999).
30
Full length tau can polymerize in a number of settings (Crowther et al. 1994,
Dudek and Johnson 1993, Goedert et al. 1996, Kampers et al. 1996, Montejo de Garcini
and Avila 1987, Montejo de Garcini et al. 1988, Montejo de Garcini et al. 1986, Perez et
al. 1996, Troncoso et al. 1993, Wilson and Binder 1995, Wilson and Binder 1997), but
they do not assemble as well as small fragments containing only the microtubule-binding
region containing the repeat region (Crowther et al. 1992, Schweers et al. 1995, Wille et
al. 1992). Dimerization of tau through disulfide bond formation is believed to be an
essential step in the nucleation process that precedes the filament building. When
three-repeat tau fragments containing only one cysteine (Cys322) are used, homodimers
are formed that can be reverted to monomers by treatment with reducing agents.
Additionally, these tau polypeptides show a greatly reduced ability to form filaments if
reducing agents are added to the assembly buffer or if the cysteine is mutated to an
alanine (Schweers et al. 1995). The four-repeat tau isoforms contain two cysteine
residues with a second residue (Cys291) in the extra repeat. These tau isoforms behave
differently in the dimerization process, as they tend to form intramolecular disulfide
bonds as well as intermolecular ones. When intramolecular disulfide bonds form, the
protein remains monomeric, has an altered mobility on a native gel, and is poorly
incorporated into filaments (Schweers et al. 1995). When the second cysteine in the
four-repeat isoform is mutated to an alanine its ability to build PHF-like filaments is
comparable to the three-repeat form (Schweers et al. 1995). Despite its importance in the
nucleation of filament formation, dimerization of tau is not in itself an impairing process,
since tau dimers retain the ability to bind to microtubules and can still exert their
stabilizing effect, as demonstrated by our lab (Di Noto et al. 1999).
31
The use of recombinant tau constructs for polymerization in vitro studies can
provide useful hints on the kinetics of filament formation, although it may not reflect the
actual model in vivo. For example it is known that MAP2 can only form straight
filaments in vitro. Our lab was able to identify a stretch of 5 residues (R-V-K-I-E-S-V)
inside the microtubule binding region of MAP2 that can generate PHF-like filaments in
MAP2 fragments containing the microtubule-binding region if switched with the
corresponding residues of tau (Q-V-E-V-K-S-E), indicating that the information on the
polymer conformation resides in the protein sequence and is not dependent on
posttranslational modifications such as phosphorylation or glycosylation (DeTure 1998).
Even more interestingly, a change of a single residue in that motif (V-to-E) is enough to
generate the conformational switch from straight to paired helical filaments. More
recently von Bergen et al. (2000) were able to identify a short amino acid sequence
(V-Q-I-V-Y-K) inside the third repeat of tau that is thought to be essential for
heparin-induced filament assembly. This motif coincides with the highest predicted
β-structure potential in tau. A 43 amino acid long recombinant tau fragment containing
the hexapeptide was shown to readily assemble into thin filaments without a paired
helical appearance, and these filaments were highly competent to nucleate bona fide
PHFs from full-length tau (von Bergen et al. 2000).
Assembly of recombinant material offers several advantages to the researchers.
Recombinant tau can be made available in large amounts, all proteins are in the same
nonphosphorylated state, and they tend to assemble best under oxidizing conditions at
high salt and a variety of pH (Crowther et al. 1992, Crowther et al. 1994, Schweers et al.
1995, Wille et al. 1992).
32
Methods Available to Measure Tau Assembly
One impediment to studying tau in vitro has been the lack of a consensus method
for measuring this process. Several techniques are utilized to measure polymer assembly,
but they all have inherent advantages and limitations on their potential applicability.
Among the different techniques employed, are sedimentation assays (Arrasate et al. 1999,
Schneider et al. 1999), qualitative electron microscopy (Goedert et al. 1996, Hasegawa et
al. 1997, Nacharaju et al. 1999) and quantitative electron microscopy (King et al. 1999,
King et al. 2000, Wilson and Binder 1997). Quantitation from electron micrographs
holds some promise, but the technique is full of potential problems. The technique
implies the choice of six to eight random fields from which the number of tau polymers
are then counted and measured. But the amount of protein that polymerizes is only a
portion of the total amount of protein in the reaction. The polymerization solution is not
homogeneous because of the nature of the reaction and any clumping or aggregation of
filaments can skew the results. Furthermore, special care must be taken to ensure that the
adsorption rate is constant for all the different constructs and conditions tested. This is
not always feasible especially when polyanions are present, since they are known to
facilitate the aggregation of tau driving the reaction toward heterogeneity of the solution
thus making a statistical analysis of the quantitation more difficult. Nevertheless certain
systems may be amenable to quantitation and comparison by this method. Electron
microscopy may not be suitable for quantitation of tau polymers, but remains the
selective method for qualitative and structural studies of the fibrillary tangles (for a brief
introduction to electron microscopy see Appendix).
Spectrophotometric measurement of the turbidity of protein solutions following the
induction of polymerization can also be used (Gaskin et al. 1974). However,
33
turbidimetric analysis requires that enough filaments be formed to cause a decrease in the
amount of light transmitted through a protein solution, and this technique has not proven
sensitive enough to measure tau polymerization at physiological protein concentrations
(1-4 µM). Furthermore other type of aggregates could negatively influence the accuracy
of this method.
A standard method to assess the polymerization of filamentous macromolecular
compounds is the use of dyes, which fluoresce upon binding to aggregates. Among these
dyes, thioflavin-S and thioflavin-T had already been successfully employed to measure
amyloid plaques in samples from brains of patients affected by Alzheimer’s disease
(Naiki et al. 1989). Binding of thioflavin-S to paired helical filaments in Alzheimer
neurofibrillary tangles has been used as a diagnostic marker to confirm the disease on
post mortem brain samples. The dye is known to exhibit a characteristic fluorescence
emission at 480-490 nm when bound to Alzheimer paired helical filaments as well as
filaments formed in vitro from recombinant tau fragments (Schweers et al. 1995). The
method has been characterized for the measurement of tau polymers in vitro (Friedhoff et
al. 1998, King et al. 1999). Although the method is at present widely utilized to
determine the amount of tau polymer formed, it has some limitations. The mechanism by
which the fluorophore thioflavin binds to the polymer is not known; nor is known the
stoichiometry of the reaction, and the increase of fluorescence upon binding of the dye to
the polymer is linear only up to 4-5 µM final concentration of the protein. Furthermore
RNA cannot be used as an anionic facilitator in in vitro polymerization of tau, as the
intrinsic fluorescence-quenching property of the nucleic acid interferes with the emission
spectrum of thioflavin (Friedhoff et al. 1998).
34
Although many methods are currently available and are utilized to measure tau
polymerization in vitro, none of these methods is complete enough to be the ultimate
assay in the field.
Aims of This Research Project
MAP2 and tau are two microtubule-associated proteins abundantly present in
neuronal cells, where they locate in different cell compartments. MAP2 is mostly present
in the neuronal cell body and in the dendritic spines, while tau is mostly present at the
axonal level. Although the two proteins share many common features, tau is the only
component of aberrant filaments known as fibrillary tangles, which accumulate in
neuronal cells during neurodegenerative diseases. Since its correlation to
neurodegenerative diseases, tau has been well characterized and its ability to aggregate
into filaments has been the focus of many studies. Nevertheless, the phenomenon remains
a complex and challenging model to study. The mechanism by which the protein
interrupts its normal function and starts building up into the well-known fibrillary
structure is still largely unknown. Many questions remain unanswered.
Previous studies from our research group demonstrated that a small stretch of
residues in the microtubule-binding region is paramount to the morphology of the
filaments formed in tau. MAP2, which forms only straight filaments in vitro and has
never been found in the neurofibrillary tangles, could form paired helical filaments when
a small sequence from tau was substituted to the corresponding one in MAP2. Many
MAP2 mutants containing different tau residues alternatively substituted were generated.
The studies carried on some of those mutants lead us to believe that the information
concerning the ability of tau to form aberrant filaments and the morphology of the
filaments formed is indeed stored in the sequence of the protein itself or more precisely in
35
the distribution of charges in specific areas of the microtubule binding region (DeTure
1998).
The first part of this study characterizes various site-specific mutants of MAP2’s
MTBR previously generated by the Purich laboratory. To assess the significance of
certain residues contained within the stretches named Module-A and Module-B, the in
vitro polymerization data from the MAP2 mutants resembling tau behavior are analyzed.
Also other point mutations changing the distribution of charges in the Module-B are
generated, to investigate the importance of the residues contained in Module-B and their
role in filament morphology. Furthermore the first part of this work aims to address the
significance of the Modules by generating tau mutants that have the Module-A and B
motifs switched to the corresponding ones of MAP2.
A required first step in the formation of aberrant tau filaments is the dimerization of
the tau polypeptide, which serves as a nucleating step that increases the polymerization
rate (Schweers et al. 1995). Because oxidative stress is another pathological feature of
Alzheimer’s disease, it is reasonable to consider the likelihood that oxidative
stress-induced dimerization of tau, and possibly MAP2 as well, could impair the capacity
of these proteins to stabilize microtubules. To understand the significance of the dimer
formation, this work analyzed the ability of tau and MAP2 dimers to interact with
microtubules and to promote tubulin assembly and compared it with the monomers
interaction. Tau dimers are generated through formation of a covalent disulfide bridge
between cysteine residues of two separated proteins. The redox properties of the only
cysteine contained in the three-repeat forms of tau are also investigated in this work. The
reactivity of three-repeat tau MTBRs with different reducing agents, in particular
36
glutathione and dithiothreitol has also been investigated in this dissertation research
project.
One of the main problems encountered by researchers in studying tau
polymerization in vitro has been the lack of a consensus method to measure the process.
Different techniques have been developed and used for this purpose during the recent
years. The method most frequently used and widely accepted as a standard tool for
polymerization measurement involves the use of thioflavin-S and thioflavin-T dyes that
fluoresce upon binding to the polymeric structure (Friedhoff et al. 1998, Schweers et al.
1995). Although widely accepted as a standard method of tau polymer measurement, this
method has intrinsic limitations. The final part of this research project dealt with the
development of an alternative method to measure polymerization of tau in vitro using an
extrinsic fluorescent probe attached covalently to the N-terminal residue of tau-123
fragment. Although this method requires further development, the results presented in
this dissertation clearly suggest that this method could represent a valid alternative for
monitoring tau polymerization kinetics in vitro.
CHAPTER 2
CHARACTERIZATION OF PAIRED HELICAL FILAMENT-PRODUCING
CONSTRUCTS OF MAP2
Introduction
The notion that neurofibrillary tangles, a pathological feature observed in
Alzheimer’s disease and several other neurodegenerative diseases, are made of tau
protein aggregated into filaments has generated a great interest in this microtubuleassociated protein (Brion et al. 1986, Grundke-Iqbal et al. 1986, Ihara et al. 1986, Kosik
et al. 1986, Yang et al. 1993). Even greater excitement attended the recent discovery that
natural occurring mutations in tau are highly correlated to occurrence of familial forms of
frontotemporal dementias (Hutton et al. 1998, Poorkaj et al. 1998, Spillantini et al. 1998).
One of the questions that investigators have tried to answer is what type of mechanism
lies behind the polymerization of this microtubule-associated protein into filaments. In
order to generate a model that could fit the kinetics of this process, most of the research
conducted in the field has relied on in vitro experiments using recombinant tau complete
isoforms as well as tau fragments to assess the behavior of this protein under different
conditions. The use of recombinant tau has some advantages and some limitations. Clear
advantages are that recombinant tau can be produced in large amounts, all proteins are in
the same nonphosphorylated state, and they tend to assemble best under oxidizing
conditions at high salt and a variety of pH (Crowther et al. 1992, Crowther et al. 1994,
Schweers et al. 1995, Wille et al. 1992). The limitation on using recombinant material
and in general the limitation of in vitro studies resides in the fact that results do not
37
38
necessarily reflect the situation in vivo, especially the chronic multi-year progression of
the afflicted neurons. Nonetheless the use of recombinant tau and MAP2 proteins and
fragments are essential to the understanding of polymerization kinetics of MAPs.
Previous work from our lab demonstrated that MAP2 fragments containing the
microtubule-binding region are capable of polymerizing in vitro into straight filaments at
concentrations that are an order of magnitude lower than those needed with recombinant
tau (DeTure 1998). Like purified tau (Wilson and Binder 1997), MAP2 fragments can
assemble from the ends of Alzheimer paired helical filaments without forming paired
helical filaments, suggesting that MAP2 fragments and tau share similar, although not
necessarily identical, binding surfaces. The fact that thioflavin-S binds Alzheimer paired
helical filaments as well as filaments formed with tau-123 or MAP2-123 fragments
(fragments containing only the three repeats of the microtubule-binding region) suggests
that all of these filaments share a similar thioflavin S binding pocket. These experimental
results along with the fact that MAP2 is present in the cell body where Alzheimer
neurofibrillary tangles originate, point to the existence of factors that permit tau
filaments, but not MAP2, to form and accumulate in the cell.
Several studies during these years showed that polymerization of tau in vitro can be
greatly stimulated by incubation of the protein with certain classes of polyanions.
Sulphated glycosaminoglycans such as heparin and heparan sulfate (Goedert et al. 1996,
Perez et al. 1996) or other classes of polyanions, like polyglutamic acid (Perez et al.
1996) and RNA (Kampers et al. 1996) all accelerate the rate of filaments formation with
better efficiency for heparin than RNA and polyglutamate respectively. The
polymerization is a nucleation-dependent reaction, and dimerization of tau through a
39
disulfide bridge also appears to be a necessary step in the process (Schweers et al. 1995).
The current model for anionic interaction supports the idea that the anions facilitate the
polymerization of tau in two ways: first polyanions may act to concentrate tau allowing
for tau-tau interaction and polymerization at lower concentrations than in their absence;
and second, polyanions may bind tau in a way that exposes the repeat region that is
especially assembly-competent. Interestingly the polymerization enhancement of tau
constructs observed in the presence of polyanions is not seen for corresponding MAP2
constructs. Sulphated glycosaminoglycans drive MAP2 toward generic aggregation
rather than filament assembly; when filaments form they are straight filaments, and no
PHF is observed. RNA seems to inhibit assembly (DeTure 1998). These observations led
to the hypothesis that the conformation of the filaments formed from the different MAPs
is not dominated by external factors such as polyanions or post-translational
modifications; rather, morphology depends on the nature of the protein itself. Thus, there
may be regions in MAP2 protein that prevent paired helical filament formation, whereas
regions in tau drive PHF formation. Examination of the sequences of tau-123 and
MAP2-123 allowed our group to identify two regions, termed Module-A and Module-B,
which were thought to control filament morphology (Figure 2-1). These motifs in tau
MTRB sequence were introduced by cassette mutagenesis at the corresponding sites in
MAP2-123 (DeTure 1998). MAP2-123 [Module-A] formed mostly straight type of
filamentous structure but was able to polymerize in the presence of tRNA in contrast to
MAP2-123, which does not polymerize in the presence of polyanions. The MAP2-123
[Module-B] mutants generated mostly paired helical filament-like structures. These
40
Figure 2-1. Sequence comparison of MAP2 and tau microtubule-binding regions and
identification of Module-A and Module-B. Close examination of the sequences shows
that they share 70 % identity with most of the significant differences included in the
Modules. Underlined are the three repeats.
41
mutants actually formed a larger proportion of paired helical filament-like structures than
tau-123; and they resembled the tau-123 paired helical filaments made with heparin
(Kampers et al. 1996). These results indicated that individual amino acid residues in the
Module-B region of MAP2-123 may control paired helical filament formation (DeTure
1998).
To further investigate the motif named Module-B several MAP2 mutants were
generated from recombinant DNA carrying single-point mutations where residues of tau
inside Module-B sequence were substituted to the corresponding MAP2 ones (Figure
2-1). None of the individual point mutants in Module-B of MAP2-123 turned out to
behave like MAP2-123. (DeTure 1998). These experiments demonstrated that the in
vitro polymerization properties of MAP2-123 are exquisitely susceptible to small changes
in the primary sequence around the second repeat. This second repeat contains the only
cysteine in the microtubule-binding region of three-repeat tau isoforms, and it has been
demonstrated that the second repeat sequence stimulated microtubule assembly (Coffey
et al. 1994, Joly et al. 1989). Additionally, phosphorylation sites controlling
MAP2-stimulated microtubule assembly are localized in and around this repeat
(Ainzstein and Purich 1994), further emphasizing the importance of the second repeat in
MAP2 interactions with microtubules. Moreover, two natural occurring mutations found
in familial forms of frontotemporal dementias fall inside our Module-B motif (Lippa et
al. 2000, Spillantini et al. 1998) also underscore its possible contribution to filament
formation and filament morphology in aberrant tau.
The work in this chapter aims to extend the aforementioned study from our lab
(DeTure 1998). Polymerization data from the MAP2 mutants resembling tau behavior
42
in vitro are analyzed to detail the behavior of the different MAP2-123 Module-B mutants
in different polymerization conditions and determine the distribution of the different
filament morphologies generated. To further assess the importance of the residues
contained in Module-B and the role they play in determining the morphology of tau and
MAP2 polymeric filaments other point mutations changing the distribution of charges in
the Module-B are generated. Furthermore this work aim to address the significance of the
Modules by generating tau mutants that have the Module-A and B motifs switched to the
corresponding ones of MAP2.
Materials and Methods
Genemed Synthesis Incorporated made synthetic single-stranded oligonucleotides
for cloning and mutations. cDNA products were ligated into the pETh-3b vector for
cloning and expression. This vector was a derivative of pBR-322 and was a gift from Dr.
Donald McCarty at the University of Florida. Invitrogen One Shot competent cells and
DM1 competent cells from Gibco-BRL were used for cloning, and E. coli BL21 (DE3)
pLYS S competent cells from Novagen were used for protein expression. Agar, tryptone
and yeast extract were obtained from DIFCO Laboratories. Tris base, sodium acetate,
phenylmethylsulfonyl fluoride, dithiothreitol, sodium dodecylsulfate, ampicillin,
magnesium chloride, 2-(N-morpholino) ethanesulfonic acid, DNase, sodium chloride,
Folin reagent, thioflavin S, heparin, heparan sulfate, tRNA and
isopropyl-β-thiogalacto-pyranoside were from Sigma Chemical Company. DNA high
melt agarose, Wizard Minipreps, dNTPs, Lambda DNA markers and certain restriction
enzymes were obtained from the bioproducts division of Fisher Scientific. Most
restriction enzymes were purchased from New England Biolabs while T4 DNA ligase,
43
Taq DNA polymerase and chloramphenicol were purchased from Boehringer-Mannheim.
FPLC materials including HiTrap SP disposable columns and Sephaglass Bandprep kits
were obtained from Pharmacia. Amicon made Microcon, Centricon and Centriprep
devices for protein sample concentration. Whatman Corporation provided the P11
phosphocellulose, and Coomassie Brilliant Blue R 250 was from Crescent Chemicals.
Sitting bridge crystallization cells and ancillary material for polymerization experiments
were obtained from Hampton Research. Purified paired helical filament preparations
from Alzheimer brain tissue were a generous gift from Dr. Peter Davies at Albert
Einstein University. A previous member of the group generated the mutants MAP2ModuleB, MAP2-123-MBP1, MAP2-123-MBP2, MAP2-123-MBP3 and
MAP2-123-MBP4 (DeTure 1998).
Tau-123 cDNA was mutated so that the expressed protein contained residues that
were significantly different from the corresponding residue(s) in MAP2 contained inside
Module-A. This was accomplished by cassette mutagenesis. The mutations in Module-A
are at the end of the first repeat and the inter-repeat before the start of the second repeat.
A Bam HI site on the 5’ side of the mutations and an Afl II site on the 3’ side of the
mutations flank these mutations in tau-123. Oligonucleotides were designed that
annealed to one another, coded for the mutations of interest, created overhangs which
complemented the Bam HI and Afl II sticky ends and destroyed the Bam HI and Afl II
sites when inserted into the cDNA sequence. Cloning simply involved digesting tau-123
with Bam HI and Afl II, purifying the released fragment from the plasmid by gel
electrophoresis and ligating the annealed oligonucleotides into the plasmid. Transformed
Invitrogen One Shot competent cells were plated and colonies were collected for
44
screening of their plasmids. Plasmids that had lost both their Bam HI and Afl II sites
were grown in quantity and sequenced by the ICBR DNA sequencing core at the
University of Florida.
Tau-123 Module-A containing an extra mutation outside of the modules (G326K)
was generated using four primers PCR. Internal primers were designed that
complemented one another, contained the mutations and coded for a silent mutation that
created a Hind III site on the 3’ side of the mutations for screening. These primers were
used individually with primers for the ends of the clone that contained restriction sites for
cloning. The PCR products for the front and back of the molecule were purified by gel
electrophoresis and used as template in a second reaction with the primers for the 5’ and
3’ ends of the molecule. The product from this PCR reaction was digested with Nde I
and Eco RI and cloned into the pETh-3b plasmid that had been digested with the same
restriction enzymes and purified. Plasmids from colonies were screened for the addition
of the Hind III site, and positives were grown. The ICBR DNA sequencing core at the
University of Florida sequenced the positive colonies.
Individual point mutations in Module-B were also generated where positively
charged residues are substituted with negatively charged ones, or vice versa (E338K and
K340E: numeration refers to human tau longest isoform), were also generated using four
primers PCR.
Expression and Purification of Wild Type Tau123 and Module-B Mutants
MAP2-123[Module-B] and tau-123[Module-A] mutants, were expressed and
purified using a modified version of the protocol based on the heat stability and cationic
nature of the MAPs (Coffey et al. 1994). For expressing proteins, cDNAs in the pETh-3b
vector were transformed into E. coli BL21 (DE3) cells. Overnight cultures of 10 mL of
45
these cells were used to inoculate one-liter cultures of LB media containing 50 mg/mL of
ampicillin and 34 mg/mL of chloramphenicol. The cells were grown at 37° C until the
cultures reached an OD-600 of 0.4 to 0.5 at which time they were induced by bringing the
media to 0.5 mM IPTG. This agent allows the bacterial gene expression of T7 RNA
polymerase, which then binds the T7 promoter found upstream of the cloned cDNA.
This resulted in massive transcription and translation of the cDNA of interest. After 2
hours of protein expression the cells were harvested by centrifugation at 5,000 g for 5
minutes. The cells were then washed by resuspending them with ice-cold 1x MEM
buffer (100 mM MES, 1 mM EGTA and 1 mM MgCl2 at pH 6.8) and pelleting again at
5,000 g for 5 minutes. Bacteria from one liter of culture were resuspended in 20 mL of
cell lysis buffer (100 mM Tris, 500 mM NaCl, 1 mM MgCl2, 1 mM PMSF, 20 units/mL
DNase and 1 mM DTT) and frozen at –20° C overnight. The pellets were then thawed
and sonicated with a Branson Model 450 Sonifier at 20 watts output at 60 % for four twominute sessions on ice. This step lysed the cells and fragmented the DNA. The solution
with the lysated bacteria was then heated at 80° C for 10 minutes before being placed on
ice for 20 minutes. The solution was then freed of denatured, aggregated proteins by
centrifugation at 40,000 g for 20 minutes. The supernatant from this spin contained
mostly heat-stable MAP2 or tau fragments in high salt. The sample was diluted with
water to bring the salt concentration below 150 mM NaCl before loading it onto a 1 mL
phosphocellulose column equilibrated in 1x MEM. The sample was loaded by gravity,
and the column was washed extensively with 10 mL of 50 mM MEM. The sample was
eluted with 50 mM MEM containing 1 M NaCl and 1 mL fractions were collected. The
46
fractions with and OD-280 above 0.1 were pooled and then dialyzed into 2 x 200
volumes of 50 mM MEM at pH 6.8 for 16 hours.
The samples prepared as described were then processed directly in filament
assembly reactions or aliquoted and frozen at -20° C. Sample purity was confirmed by
SDS-PAGE, and the molecular weights of the polypeptides was verified by matrixassisted laser desorption mass spectroscopy. Some samples contained lower-molecularweight bands, presumably formed by incomplete translation or proteolysis (Zhang 1997).
The samples were finally isolated to purity on HPLC using a Jupiter C4 reverse-phase
column (Phenomenex).
In Vitro Assembly of Filaments with tRNA or Heparin
Studies demonstrating the efficiency of tau polymerization in the presence of
polyanions, such as sulfated glycosaminoglycans or RNA, prompted the use of similar
conditions to determine which mutants assembled more like tau do. If the sample
behaved like tau, then assembly with these agents permitted polymerization in a single
day using concentrations below 1 mg/mL. The samples were typically 2 mg/mL, as
determined using the method of Lowry (Lowry et al. 1951), standardized as described
elsewhere (Coffey and Purich 1994). Samples at 50-200 mM were placed in 1.5 mL
Eppendorf tubes with concentration of tRNA or heparin ranging between 0.01 and 0.50
mg/mL. The concentrations of polyanions that worked best were between 0.01 and 0.10
mg/mL, which were lower than those reportedly used for tau polymerization (Goedert et
al. 1996) (Perez et al. 1996) (Kampers et al. 1996). These samples were then incubated
overnight at 37° C and examined by electron microscope for filament assembly as
described below.
47
Electron Microscopy
Carbon-coated grids were prepared by coating 400-mesh copper grids with grid
glue (i.e., 1 inch Scotch tape washed into 10 mL chloroform) to ensure proper adhesion
of carbon films onto the grids. Carbon films created by vapor adsorption of elemental
carbon onto mica strips were floated on water and then picked up with copper grids.
These carbon-coated grids were prepared freshly for each microscope session. Sample
was adsorbed on to the grids by floating a grid on a drop of sample for 30 seconds. The
nonadsorbed sample was then wicked off and the grid was negatively stained by floating
it on a drop of 1 % uranyl acetate or 1 % phosphotungstic acid for 30 seconds. Grids
were examined after 15 minutes on a Hitachi H-7000 transmission electron microscope at
75 kV.
Morphometry
To determine the frequency and amount of SF and PHF formation, so called
negative-stained filaments were examined by electron microscopy. Six to eight random
fields were randomly chosen from the grids and digital images were taken of the different
filaments in the field. Based on the distinctive morphologies of SFs and PHFs, filaments
were classified as paired helical, only when a distinct periodicity was detectable from the
image. All the filaments without a defined periodicity and with a more uniform contour
were considered straight for the purpose of these measurements. For polymer length
measurements, digital pictures at 125,000x or 250,000x magnification were taken of the
filaments. The lengths of filaments only partially present in the picture (i.e., filaments
with at least one end outside of the image field) were doubled according to Johnson and
Borisy (1977). The digitally scanned images were analyzed using Image-1 software
(Universal Imaging Inc.). PHF mass distribution was estimated as the relative abundance
48
of protein in the SF and PHF structures by determining the total length of SFs and PHFs
in each set of electron micrographs and dividing the total PHF length by the total length
of filaments. This estimation was based on the image reconstruction study of Crowther
(1991), which demonstrated that SF and PHF structures have virtually identical masses
per unit length.
Fluorescence Spectroscopy
Binding of thioflavin S to paired helical filaments in Alzheimer neurofibrillary
tangles has traditionally been used as a diagnostic marker to confirm the disease on post
mortem brain samples. The fluorophore is known to exhibit a characteristic fluorescence
at 480-490 nm when bound to Alzheimer paired helical filaments as well as filaments
formed in vitro from recombinant tau fragments (Schweers et al. 1995). Binding of
thioflavin S to polymers of recombinant tau-123 or tau-123 Module-A mutants was
monitored by the change of extrinsic fluorescence of the dye using a Fluorolog-3 (JY
Horiba) photon-counting spectrofluorimeter. Samples were prepared at concentrations
from 1 to 10 µM final. Each sample was measured in the presence of 0.5 mM thioflavin
S, final concentration in MOPS buffer 20 mM at pH 6.8. The excitation wavelength was
set at 440 nm, and the emission spectrum was recorded from 460 to 550 nm. The
spectrum of thioflavin S alone was used as a control in each experiment as it gives little
or no emission peak in the 460 to 500 nm range.
Results
Several reports have documented that certain polyanions like, glycosaminoglycans
and RNA, enhance the in vitro polymerization of recombinant tau (Goedert et al. 1996,
Kampers et al. 1996, Perez et al. 1996). These substances lower the concentrations of
tau-123 needed for assembly to values below 100 mM, and these agents also accelerate
49
assembly so polymerization only takes one day, not one month. Full-length three- and
four-repeat tau that failed to assemble in vitro without polyanions, polymerized in one
day when 0.05 to 0.50 mg/mL tRNA or heparin was added. Tau assembled in the
presence of these agents still formed a variety of structures, including paired helical
filaments, straight filaments and other form of undefined aggregates. On the other hand,
our MAP2 MTBR mutants containing a defined stretch of charged residues in tau
Module-B region were able to polymerize in 1-2 days in the presence of polyanions and
the filaments formed were almost exclusively PHFs (DeTure 1998). MAP2 MTBRs with
single-residue substitutions from tau also showed interesting results producing different
type of filaments ranging from mostly straight for some of the mutants to mostly paired
helical filaments for other MAP2 [Module-B] mutants.
Image Analysis of the MAP2 MTBR Mutants Generated by the Laboratory.
In order to assess the importance of all the single-residue substitutions as well as
[Module-A] and [Module-B] mutants generated in this lab, the first part of this research
was aimed to quantify the type and frequency of filaments generated by the mutants of
interest. Digital micrographs of the polymerized samples of interest were obtained from
6 to 8 electron microscopy fields randomly chosen per sample. Image analysis was
carried on the micrograph and the filaments observed were classified according to their
morphology and length. Only when a distinct periodicity was detectable from images of
negatively stained filaments, these were classified as paired helical. Frequency of PHFs
and SFs was determined in this analysis, as well as mass and length distribution of PHFs.
MAP2 [Module-A] shows little or no ability to generate paired helical filaments
(see Figure 2-2A). The polymers formed after incubation in the presence of polyanions
were of a regular diameter without any evident periodicity in the contour of the
50
negatively stained filaments. Only about 3 % of the filaments gave any indication of
periodicity. When periodicity was observed it was not as well defined and certainly not
of the type found with mutants generated from substitutions in the Module-B motif. The
length distribution of the filaments was homogenous with most of the filaments, and
lengths ranged from 0.5 to 1.5 µm. Only a small number of filaments reached higher
lengths (Figure 2-4).
MAP2 [Module-B], on the other hand, showed clear evidence of PHFs production.
About 87 % of the filaments showed a regular periodicity and were classified as paired
helical (Figure 2-2B). These filaments exhibited a marked and distinct periodicity of 55
to 65 nm, which compares to a 80 nm periodicity for AD filaments and a 90 to 120 nm
periodicity typical of FTDs. Most of the filaments had an average length centered
between 0.5 and 1 µm, and no filaments over 2-2.5 µm were observed (Figure 2-4).
To determine the minimum number of changes needed for paired helical filament
formation, point mutations were introduced in MAP2-123. The four single-point
mutations differed in terms of PHF formation. MAP2-123 modB P1 showed little or no
ability to form PHF, similar to MAP2-123 [Module-A], with about 93 % of the filaments
in the straight form (Figure 2-3A). The average filament length was about 0.7 µm, with
most of the filaments measuring less than 0.5 µm.
Similar results were also observed for MAP2-123-MBP2 mutant. Only about 10 %
of the filaments were in the paired helical form. Most of the filaments had lengths
ranging between 0.5 and 1.5 µm, and no filament longer than 2.5 µm was observed
(Figure 2-4). MAP2-123 modB P3 and MAP2-123 modB P4 were the two most
51
Figure 2-2A. Montage of electron micrographs illustrating the SF morphology of
MAP2-123 [Module-A] polymers. Note the absence of periodic paired-helical structure.
These SFs tend to undulate, but are less curved than wild-type MAP2 SFs.
Figure 2-2B. Montage of electron micrographs illustrating the PHF-like morphology of
MAP2-123 [Module-B] polymers. Note the regular periodicity indicative of PHF
structure. (Bar = 0.1 mm)
52
120
100
80
%PHF
60
%SF
40
20
w
ild
ty
M
A
P2 pe
m
M
od
A
A
P2
M
m
A
P2
od
B
m
M
o
dB
A
P2
P1
m
M
od
A
B
P2
P2
m
M
o
dB
A
P2
P3
m
od
B
P4
0
A
PHF mass distribution
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
MAP2 MAP2 MAP2 MAP2 MAP2 MAP2
modA modB modB modB modB modB
P1
P2
P3
P4
B
Figure 2-3. A, Frequency of PHF formation, plotted as the number of PHFs divided by
the total number of PHFs and SFs (the latter values are listed in parentheses) for
filaments assembled from the indicated mutants; and B, Relative abundance of PHFs
(plotted as the total length of PHFs divided by the total length of PHFs and SFs) for
filaments assembled from the indicated mutants.
53
n=152
n=65
70
60
50
40
30
20
10
0
16
14
12
10
8
6
4
2
0
0- 0.5- 1.0- 1.5- 2.0- 2.5- 3.0- 3.50.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
0- 0.5- 1.0- 1.5- 2.0- 2.5- 3.0- 3.50.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
ModA
ModB
n=31
n=61
30
10
25
8
20
15
6
10
4
5
2
0
0- 0.5- 1.0- 1.5- 2.0- 2.5- 3.0- 3.50.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
0
0- 0.5- 1.0- 1.5- 2.0- 2.5- 3.0- 3.50.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
ModB-P1
ModB-P2
n=48
14
12
10
8
6
4
2
0
n=161
70
60
50
40
30
20
10
0
0- 0.5- 1.0- 1.5- 2.0- 2.5- 3.0- 3.50.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
ModB-P3
0- 0.5- 1.0- 1.5- 2.0- 2.5- 3.0- 3.50.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
ModB-P4
Figure 2-4. Length distribution of the MAP2 mutants containing the entire Module-A or
Module-B from tau or the single-residue mutations from Module-B.
54
Fig. 2-5. Comparison of amino acid sequence between MAP2-123 [Module-B]
(indicated as MmB) and tau-123 [Module-A] (indicated as TmA). In gray are the non
significant differences between the sequences outside of the modules and in red the
significant ones. In the black box are indicated the glycine residues of tau-123 [ModuleA]
substituted with the corresponding lysine of MAP2-123 [Module-B], to generate the
G326K mutant.
55
Figure 2-6. Montage of electron micrographs illustrating the SF morphology of tau-123
[Module-A] polymers. Note the absence of periodic paired-helical structure.
56
35000
30000
25000
G326K
TmA
20000
MmB
P4
15000
10000
5000
0
460
480
500
520
540
560
Figure 2-7. Fluorescent emission spectrum of thioflavin-S reacted in the presence of
polymers from different tau or MAP2 mutants (G326K = tau-123 [Module-A] point
mutant G326K TmA = tau-123 [Module-A], MmB = MAP2-123 [Module-B] and P4 =
MAP2-123 MBP4). Numerations refer to the longest human tau isoform.
57
MmB
LKNVKSKIGSTDNIKYQPKGGQVQIVTKKIDLSHVTSKCGSLKNIRHRPGGG
QVEVKSEKLDFKEKAQAKVGSLDNAHHVPGGGNVKIDSQKLNFREHA
- + -
QVKVKSE
+ + -
QVEVESE
- - -
E338K
K340E
Figure 2-8. Sequence of the microtubule-binding region of MAP2-123[Module-B]
mutant. In red are indicated the charge distributions in the module area and their change
in the two mutants E338K and K340E.
58
interesting point mutations. About 60 % of the filaments from MAP2-123 modB P3
mutant were PHF-like filaments and they were very long reaching an average length of
about 1.8 µm. Even more interestingly, 78 % of the filaments from MAP2-123 modB P4
were classified as PHFs. Although the average length 1 µm, no filament over 2 µm was
observed (Figure 2-4).
The polymer mass distribution for the mutants reflects the frequency of paired
helical filament occurrence (Figure 2-3B). Only about 5 % of total MAP2-123
[Module-A] was present in the paired helical form. On the contrary, 88 % of the polymer
mass in MAP2-123 [Module-B] protein was estimated to be PHF. Different values were
calculated for the different single-point mutants. Interestingly, MAP2-123 modB P3
relative abundance of PHF (75.3 % of polymer mass accounted for PHF) was
considerably high and comparable to the values shown for MAP2-123 [Module-B]
(88.0 %) and MAP2-123 modB P4 (80.4 %). The other two single-point mutants,
MAP2-123 MBP1 and MBP2, showed very little protein (7.0 % and 12.8 % of total mass
respectively) assembled with paired helical morphology.
Of all the mutants previously generated by our lab and analyzed in the previous
paragraph, the most interesting one was the MAP2-123 [Module-B] mutant, where all
four critical residues in the module-B motif were substituted in MAP2 MTBR, with the
corresponding one of tau (DeTure 1998). This particular mutant was able to produce
87 % of the filaments in the paired helical form after overnight incubation in the presence
of polyanionic facilitators.
To understand the significance of the Modules more fully, I generated tau-123
mutants in which residues inside the Modules were substituted by the corresponding
59
MAP2 residues. Of the three mutants generated (tau-123[Module-A];
tau-123[Module-B] and tau-123[Module-A/B]), tau-123[Module-A] was the one
anticipated to behave like MAP2-123[Module-B] mutant as these two mutants share a
high residues identity (Figure 2-5). Surprisingly, tau-123[Module-A] assembled poorly
in the presence of polyanions. Very few filaments were observed when the mutant was
incubated in the presence of either sulphated glycosaminoglycans or RNA. When
filaments were observed, the morphology was quite difficult to define, being the contour
of these filaments mostly homogeneous with no defined periodicity (Figure 2-6).
Analysis tau-123 [Module-A] polymerization was conducted through fluorescence
spectroscopy using thioflavin-S. As shown in Figure 2-7, the polymers formed in lower
abundance than MAP2-123 [Module-B]. Since the result was unexpected, we reasoned
that some other differences in charged residues outside of the two modules might play a
role in determining the morphology of the filaments produced. A possible candidate was
identified in residue 326 (where numeration refers to the 441-residue human tau isoform).
This residue is a glycine in the tau sequence and a lysine in the corresponding position in
MAP2 MTBR. Another mutant was generated from tau-123 [Module-A] carrying this
G326K extra substitution, and polymers formed after incubation in the presence of
polyanions were analyzed. Analysis of electron microscopy images from the mutant
G326K did not show evidence of PHF formation and when paired helical filaments were
identified, they were never in an amount comparable to the MAP2-123 [Module-B],
although the extra mutation contributed for a recovery in polymer mass produced in
respect to tau-123 [Module-A], as deducted from the fluorescence measurements with
thioflavin-S (Figure 2-7).
60
Investigation of the Role of Charges in ModuleB
The differences in charges that characterize the four residues in module-B seem to
be responsible for a “gain-of-function” in the sense that MAP2-123 [Module-B] acquired
the ability to form paired helical filaments after substitutions of those residues. The four
residues that are involved go from R, K, E and V in MAP2-123 to Q, E, K and E in
tau-123, changing the net charge from a +1 in MAP2 to a –1 in tau. We reasoned that
those differences in charge could be the key to interpret this change of behavior and the
acquired ability to form PHFs of MAP2-123 [Module-B].
To further investigate the significance of those charges two mutants were generated
from MAP2-123 [Module-B]. One mutant (E338K) in which the second charged residue
E was switched back to K (i.e., the corresponding one of MAP2-123) generating a
MAP2-123 Module-B mutant in which the net charge was +1 (as in wild type MAP2-123
Module-B) but the sequence of charges inside the Module-B motif was changed. A
second mutant was also generated (K340E) in which a positively charged K residue was
substituted with a negatively charged E generating a Module-B variant with a net charge
of –3 (Figure 2-8). If the sequence of charges plays indeed a role, a “loss-of-function”
mutation (i.e., the mutant would lose the ability to form PHF acquired with Module-B
substitution) or some change in polymer mass would be expected.
Samples from the two mutants were incubated in polymerizing conditions and
analyzed by electron microscopy and fluorescence spectroscopy. None of the two
mutants behaved differently from the MAP2-123 [Module-B]. The filaments formed in
the presence of polyanions were mostly PHFs as in MAP2-123 [Module-B] and the
polymer mass was absolutely comparable to the one produced by MAP2-123 [Module-B]
or MAP2-123 MBP4 which are usually in the same range (Figure 2-9).
61
Discussion
One of the main goals of in vitro studies on tau polymerization is to understand the
mechanisms that drive the protein toward the building of polymer blocks. To characterize
this reaction, more information is needed on which factors might be involved in the
filament assembly. The MAP2-123 mutants generated by this lab in previous studies
(DeTure 1998) proved to be an interesting insight into the mechanisms of tau
polymerization. As I proceeded to quantify and characterize the filaments generated by
the [Module-A] and [Module-A] mutants, it seemed more and more evident that the
morphology of filaments generated by tau polymerization depends on the sequence of the
protein, or more precisely on the distribution of charges inside the sequence, rather than
on other factors like post-translational modifications or intermolecular bond formations.
Von Bergen et al. (2000) showed that a small cluster of 43 residues (comprising the
third repeat of tau --second repeat in the three repeat forms-- plus few extra residues
flanking the repeat region) acquires pronounced β-structure in conditions of
self-assembly. Inside the 43 amino acid region, a small hexapeptide (VQIVYK)
coincides with the highest predicted β-structure potential in tau and may be a key factor
in driving the aggregation of tau into filamentous structures. The hexapeptide VQIVYK
is contained in [Module-A], confirming our prediction that this region of tau might
indeed be significant in the kinetics of filament formation (DeTure 1998). The von
Bergen study also showed that the filaments formed by these short peptides, although
able to seed PHFs, could only form straight filaments. This observation is consistent with
the results shown in this chapter for the images analysis of MAP2-123 [Module-A],
62
50000
40000
E338K
30000
MmB
20000
10000
0
450
500
550
20000
15000
K340E
10000
MmB
5000
0
450
500
550
Figure 2-9. Fluorescent emission spectra of thioflavin-S reacted in the presence of
polymers from different tau or MAP2 mutants (E338K = MAP2-123 [Module-B] point
mutant E338K, K340E = MAP2-123 [Module-B] point mutant K340E,
MmB = MAP2-123 [Module-B]). Numerations refer to the longest human tau isoform.
63
where 97 % of the filaments were in a straight form. This mutant although seemingly
resembling the wild type MAP2 MTBR in its polymerizing behavior, it is different from
the latter as the filaments formed by Module-A mutant were thicker and less wavy than
the corresponding one of the wild type MAP2. Furthermore the polymerization of
Module-A protein is accelerated by polyanions that, on the contrary, are known to inhibit
wild type MAP2 MTBR polymerization (DeTure et al. 1996).
The most interesting result of the image analysis study presented in this chapter is
the finding that MAP2-123 MBP3 (the single-point mutation involving the third charged
residue in Module B) ability to generate PHF-like filaments is comparable to that of
MAP2-123 MBP4. In the original study (DeTure 1998), MAP2-123 MBP4 was
considered the only interesting single point mutant with respect to PHF formation.
However more extensive morphology analysis in this chapter revealed that MAP2-123
MBP3 generated about 60 % paired helical filaments. This value is comparable to that
from MAP2-123 MBP4 mutant, in which about 78 % of the filaments generated are
PHFs. Furthermore, the relative abundance of protein aggregated into paired helical
filaments for the MAP2-123 MBP3 mutant is almost equal to that of the MBP3
single-point mutant and, even more interestingly, comparable to the amount calculated
for the MAP2-123 [Module-B], which is the most efficient PHF forming mutant (Figure
2-2B). As for the other two single-point mutants (namely MAP2-123 MBP1 and MBP2),
the image analysis confirmed that they have little or no ability to form PHF-like
filaments. The length distribution analysis shows that most of the filaments formed are
equally distributed between 0.5 and 1.5 µm. The only notable exceptions are MAP2-123
[Module-A] and MAP2-123 MBP3 that show a good number of filaments to be longer
64
than average reaching often 2.5 to 3 µm in length. The average length of the filaments
produced by these mutants is consistently higher than the one observed for wild type tau
MTBR, which seldom exceeds 0.5 µm.
To better evaluate the significance of the Modules, especially the Module-B a study
was conducted on the behavior of tau MTBR mutants in which critical residues in the
modules were substituted with the corresponding ones of MAP2. After comparing
sequences from tau MTBR and MAP2-123 [Module-B] I speculated that a tau-123
[Module-A] mutant would have to behave very similarly to the MAP2-123 Module-B, as
the significant differences in charged residues between these two mutants would be very
little (as shown in Figure 2-4). Surprisingly enough, tau-123 [Module-A] proved to be a
poor candidate for filament formation. And, when filaments were formed in the presence
of polyanions, very little or no PHFs were observed. This result was confirmed by
fluorescence measurement of total polymeric mass using thioflavin-S. As shown in
Figure 2-6, the fluorescent emission spectrum for tau-123 [Module-A] is considerably
lower than the one for MAP2-123 [Module-B] and MAP2-123 MBP4. When the
sequences of tau-123 [Module-A] and MAP2-123 [Module-B] are compared, only 5 other
residues outside of the modules carry significant differences (Figure 2-4, boxed residues).
It is possible that one of these five differences in charged residues still plays a role
in the assembly behavior of the protein. Another mutant (tau-123 MA-G326K) was
generated where a glycine in tau-123 [Module-A] was substituted with the corresponding
lysine residue of MAP2 (G326K where the numeration is referring to the longest isoform
of tau). This residue was chosen as a candidate because of the five differences in charge
outside the modules, it is the only one located inside the MTBR second repeat (third in
65
the four-repeat isoforms of tau). The second repeat is particularly important in filament
assembly as it contains the only cysteine of tau in the three repeat forms and it has been
shown to be the minimal sequence required for tau-tau interaction (Perez et al. 1996).
Most of the filaments observed under the electron microscope had straight morphology.
Although no improvement was observed in terms of filament quality the thioflavin-S
fluorescence measurements showed an increment in total filaments assembled from
tau-123 MA-G326K] (see Figure 2-6) in respect to tau-123 [Module-A]. The extra
mutation although recovering tau-123 [Module-A] in terms of total filaments assembled
did not produce results comparable to MAP2-123 [Module-B] and MAP2-123 MBP4.
The results obtained from these two mutants suggest that other residues outside of
the modules may be involved in filament assembly, but also underline the fact that factors
other than simple differences in charge distribution may have a significant role in this
pathological feature. A possibility could be that it is not just the difference in charges that
influences the assembly behavior of tau, but rather the distribution of charged residues
inside the sequence and more specifically the position in respect to each other.
The sequence of charges inside the module for tau goes: negative, positive, and
negative. If this sequence of charges, rather than the nature of the residues inside the
module, determines the filament assembly properties of MAP2-123 [Module-B], it should
be possible to observe significant changes by inverting the charges or changing them
completely. To investigate this possibility two more mutants were generated from
MAP-123 [Module-B]. One mutant (namely MAP2-123 MB-E338K) had the glutamate
from tau in position 338 (numbering referred to full length human tau) switched back to
MAP2 lysine so that the final sequence of charges inside the Module-B would now be:
66
positive, positive and negative. A second mutant (MAP2-123 MB-K340E) was also
generated where the lysine inside the Module-B was switched to a glutamate, so that the
sequence of charges inside Module-B would be changed to: negative, negative and
negative (see Figure 2-7 for a schematic of the mutations). We were expecting to
observe, at least in one of the two new mutants, a noticeable change in the filament
assembly pattern. Unfortunately both two mutants produced paired helical filaments in
morphology and amount comparable to the MAP2-123 [Module-B] (see Figure 2-9).
The previous work form this lab proved that some charged residues inside tau
sequence influence filament morphology driving it toward paired helical rather than
straight form. We speculated that this difference in assembly behavior might depend on
the location of the charged residues in the overall sequence and with respect to each
other. In this chapter a detailed characterization of the mutants previously generated by
the lab is presented. We also generated new mutants to further investigate the
significance of the modules. The assembly studies of these mutants prove that although
the residues in Module-B play an important role in filament morphology, other factors
may be involved in tau protein polymerization and the location of charges inside the
modules, although important, is insufficient to explain the peculiar behavior of the
PHF-producing mutant MAP2-123 [Module-B]. The microtubule-binding region of tau
is likely to interact with other parts of the protein during filament formation. The
difference between MAP2 and tau in the module regions could result in different
interactions between molecules ultimately determining a different packing scheme for the
subunits that build a filamentous aggregate. This in the end would generate paired helical
rather than straight filaments.
CHAPTER 3
RETENTION OF MICROTUBULE ASSEMBLY-PROMOTING CAPACITY OF TAU
AND MAP2 UPON CONVERSION TO DISULFIDE-CROSSLINKED
HOMODIMERS
Introduction
The biochemical basis of neuronal degeneration and death in Alzheimer’s disease
(AD) remains uncertain (Sandbrink et al. 1996). Brain tissue from deceased AD patients
exhibits extensive fibrillar pathology in the form of neurofibrillary tangles (or NFTs),
dystrophic neurites and neuropil threads. NFTs occur in greatest abundance in affected
cerebral cortical neurons, most frequently within the hippocampus and amygdala.
Lesions in these regions account for the loss of memory and language, as well as other
intellectual deficits. Tangles consist of paired helical filaments and straight filaments
(Kidd 1963, Terry 1963). A number of cytosolic proteins are found associated with
NFTs (Anderton et al. 1982, Trojanowski and Lee 1994, Wischik et al. 1988), but the
cytoskeletal protein tau is the core filament component (Trojanowski and Lee 1994,
Wischik et al. 1988). Several factors appear to underlie these pathological depositions,
but the cause of neuronal death in each disease appears to be multifactorial. In this
regard, evidence in each case for a role of oxidative stress is provided by the finding that
the pathological deposits are immunoreactive to antibodies recognizing protein
side-chains modified either directly by reactive oxygen or nitrogen species, or by
products of lipid peroxidation or glycoxidation (Castellani et al. 1996, Sayre et al. 1997).
Although the source(s) of increased oxidative damage are not entirely clear, the findings
67
68
of increased localization of redox-active transition metals in the brain regions most
affected is consistent with their contribution to oxidative stress.
Wille et al. (1992) found tau self-associates into anti-parallel dimers, and Schweers
et al. (1995) showed that oxidation of Cys-322 leads into dimers facilitated assembly of
PHF- and SF-like structures. Our laboratory previously discovered that similar filaments
could form from disulfide-cross-linked dimers of MAP2, another brain-specific
microtubule-associated protein (DeTure et al. 1996, Zhang et al. 1996). Polymerized tau
(Schweers et al. 1995) and polymerized MAP2 (DeTure et al. 1996, Zhang et al. 1996)
also exhibit the dye-binding properties considered to be a hallmark of Alzheimer NFTs.
These considerations suggest that unknown pathophysiologic factors divert tau protein,
but not MAP2, from its role in stabilizing neuronal MTs to a pathologic state favoring
self-polymerization into abnormal filament structures that may injure neurons and impair
their function. Three-repeat forms of tau and MAP2 both possess a single thiol located in
the second nonidentical repeat. MAP2’s second repeat is the main binding site for MT
interactions (Coffey et al. 1994, Joly et al. 1989, Joly and Purich 1990), whereas tau’s
first repeat as well as the first inter-repeat collectively contribute to the strength of
tau-MT binding interactions (Ennulat et al. 1989, Goode and Feinstein 1994). The fact
that the amyloidogenic Aβ peptide1–42 stimulates oxidative stress and protein disulfide
formation (Huang et al. 1999) adds even greater weight to the question of whether
disulfide-linked tau and MAP2 homodimers retain their capacity to interact with
microtubule components.
To address this question, the MT assembly-promoting properties of oxidized
dimers of 3-repeat-tau and MAP2 MTBRs were examined.
69
Material and Methods
Synthetic single-stranded oligonucleotides for cloning were made by Genemed
Synthesis Incorporated, and cDNA products were ligated into the pETh-3b vector for
cloning and expression. This vector was a derivative of pBR-322 and was a gift from Dr.
Donald McCarty at the University of Florida. Invitrogen One Shot competent cells or
DM1 competent cells from Gibco-BRL were used for cloning, and E. coli BL21 (DE3)
pLYS S competent cells from Novagen were used for protein expression. Agar, tryptone
and yeast extract were obtained from DIFCO Laboratories. Tris base, sodium acetate,
phenylmethylsulfonyl fluoride, dithiothreitol, sodium dodecylsulfate, ampicillin,
magnesium chloride, 2-(N-morpholino)ethanesulfonic acid, DNase, sodium chloride,
Folin reagent, thioflavin S and isopropyl-β-thiogalacto-pyranoside were from Sigma
Chemical Company. DNA high melt agarose, Wizard Minipreps, dNTPs, Lambda DNA
markers and certain restriction enzymes were obtained from the Bioproducts Division of
Fisher Scientific. Most restriction enzymes were purchased from New England Biolabs
while T4 DNA ligase, Taq DNA polymerase and chloramphenicol were purchased from
Boehringer-Mannheim. Microcon, Centricon and Centriprep by Amicon were used to
concentrate protein samples. Whatman Corporation provided the P11 phosphocellulose,
and Coomassie Brilliant Blue R 250 was from Crescent Chemicals. The cDNA
constructs for expressing MAP2-N123C and tau-N123C were cloned by previous
members of the Purich lab (Coffey 1994). Additionally, the shorter analogues
MAP2-123 and tau-123 were made by cassette mutagenesis.
Briefly, the MAP2-123 containing plasmid had been engineered to contain a unique
Afl II site at the 5’ end of the second repeat and an unique Stu I site at the 3’ end of the
70
second repeat (Ainzstein and Purich 1994). The MAP2-123 plasmid was digested with
Afl II and Stu I releasing a small inserted that could be separated from the plasmid by gel
electrophoresis. Synthetic oligonucleotides were designed which annealed to one
another, coded for the mutations of interest and changed the Stu I site by silent mutation.
These oligonucleotides when annealed had a single-stranded overhang on the 5’ end that
corresponded to the sticky end created by Afl II digestion and a blunt end on the 3’ end
corresponding to Stu I digestion. This insured the cassette was inserted in the correct
orientation when incubated with the digested and purified MAP2-123 plasmid. Ligations
from the mix were transformed into a methylation negative strain of DM1 cells, and
colonies were screened by the loss of the Stu I digestion site. Positive colonies were
picked and sequenced by the ICBR DNA sequencing core at the University of Florida.
Same procedure was used for the other constructs
Expression and Purification of Wild Type Tau123 and Module-B Mutants
MAP2-123[Module-B] and tau-123[Module-A] mutants, were expressed and
purified using a modified version of the protocol based on the heat stability and cationic
nature of the MAPs (Coffey et al. 1994). For expressing proteins, cDNAs in the pETh-3b
vector were transformed into E. coli BL21 (DE3) cells. Overnight cultures of 10 mL of
these cells were used to inoculate one-liter cultures of LB media containing 50 mg/mL of
ampicillin and 34 mg/mL of chloramphenicol. The cells were grown at 37° C until the
cultures reached an OD-600 of 0.4 to 0.5 at which time protein expression was induced
by bringing the media to 0.5 mM IPTG. This agent allows the bacterial gene expression
of T7 RNA polymerase, which then binds the T7 promoter found upstream of the cloned
cDNA. This resulted in massive transcription and translation of the cDNA of interest.
After 2 hours of protein expression the cells were harvested by centrifugation at 5,000 g
71
for 5 minutes. The pelleted cells were then resuspended in ice-cold 1 x MEM buffer (100
mM MES, 1 mM EGTA and 1 mM MgCl2 at pH 6.8), washed and pelleted again at 5,000
g for 5 minutes. Bacteria from one liter of culture were resuspended in 20 mL of cell
lysis buffer (100 mM Tris, 500 mM NaCl, 1 mM MgCl2, 1 mM PMSF, 20 units/mL
DNase and 1 mM DTT) and frozen at –20° C overnight. The pellets were then thawed
and sonicated with a Branson Model 450 Sonifier at 20 watts output at 60 % for four
two-minute sessions on ice. This step lysed the cells and fragmented the DNA. The
solution with the lysated bacteria was then heated at 80° C for 10 minutes before being
placed on ice for 20 minutes. The solution was then freed of denatured, aggregated
proteins by centrifugation at 40,000 g for 20 minutes. The supernatant from this spin
contained mostly heat-stable MAP2 or tau fragments in high salt. The sample was then
diluted with water to bring the salt concentration below 150 mM NaCl before loading it
onto a 1 mL phosphocellulose column equilibrated in 1x MEM. The solution was loaded
by gravity, and the column was washed extensively with 10 mL of 50 mM MEM. The
sample was eluted with 50 mM MEM containing 1 M NaCl and 1 mL fractions were
collected. The fractions with and OD-280 above 0.1 were pooled and then dialyzed into
2 x 200 volumes of 50 mM MEM at pH 6.8 for 16 hours. These samples were then
processed directly in filament assembly reactions or aliquoted and frozen at -20° C.
Sample purity was confirmed by SDS-PAGE, and the molecular weights of the
polypeptides was verified by matrix-assisted laser desorption mass spectroscopy. Some
samples contained lower-molecular-weight bands, presumably formed by incomplete
translation or proteolysis (Zhang 1997). The samples were finally isolated to purity on
HPLC using a Jupiter C4 reverse-phase column (Phenomenex).
72
Bacterial expression and purification of unlabeled and [3H]leucine-labeled human
tau and MAP2 MTBR’s were carried out as described previously (Coffey et al. 1994,
Schweers et al. 1995), except for the following modifications. After harvesting bacteria
by centrifugation, cells were resuspended and pelleted again at 1500 g for 10 min. They
were resuspended once more in 15 mL lysis buffer [100 mM Tris–HCl (pH 8.0), 0.5 M
NaCl, 1.0 mM MgCl2, 1.0 mM dithiothreitol, 21 units/mL DNase, and 0.5 mM PMSF].
Cells were lysed by overnight freezing/thawing, followed by sonication. This method
typically yielded 5 to 8 mg microtubule-associated protein (specific activity of 1000-2000
cpm/mg) that was more than 99 % pure, based on SDS-PAGE and Coomassie staining or
fluorography. For dimerization, purified tau or MAP2 was dialyzed into Tris-HCl (pH
6.8) buffer with three buffer changes over 9-16 h. The dialysis favors protein
dimerization as about 70 to 90 % of tau and MAP2 are in dimer form after this treatment.
Protein concentrations were determined using the Lowry method adjusted for the MAPs
(Coffey et al. 1994).
Tau and MAP2 MTBR Dimer Purification
After dialysis, a solution containing recombinant monomer and dimer was acidified
in 10 % TFA and injected at a flow rate of 1 mL/min into Hewlett-Packard HP1090A
HPLC using a Jupiter C4 reverse-phase column (Phenomenex) equilibrated with an
elution solvent made up of 4:1 v/v solution A (0.1 % TFA in water) and solution B
(0.085 % TFA in acetonitrile). Following injection, the column was developed from 5 to
50 min using a linear gradient from 7:3 v/v to 1:4 v/v solutions A and B. Dimer fractions
were collected, concentrated to dryness using a Savant Speedvac concentrator, and
redissolved in 0.1 M MEM buffer. Samples from the fractions eluted corresponding to
the elution time of the proteins were prepared for SDS-PAGE by adding sample buffer
73
without reducing agents. The bands were stained with Coomassie and compared to
dye-labeled medium molecular weight marker from Promega and the gel was 12 %
cross-linked acrylamide with 0.1% SDS.
To prepare monomer solutions, 200 mM tau or MAP2 MTBR were treated with 4
mM DTT for 1 h at room temperature. DTT was then removed by Sephadex G-25
filtration (Pharmacia, Piscataway, NJ), and standardized samples were used for
microtubule assembly experiments. More frequently, 4 mM DTT (final concentration)
was added directly to the assembly mixture 5 min prior to spectrophotometric
measurements of microtubule assembly. Both methods produced monomer in high yield
and purity, as evidenced by a single band at the monomer molecular weight in
SDS-PAGE in the absence of thiol reducing agents.
Tubulin Preparation and Polymerization
Bovine brain microtubule protein (containing both tubulin and MAPs) was purified
by cycles of temperature-dependent assembly/disassembly according to Karr et al. (Karr
et al. 1979). To obtain MAP-free tubulin, was used the phosphocellulose method of
Weingarten et al. (1975). Purified tubulin was aliquoted frozen in liquid nitrogen and
stored at -80° C until use. Microtubule assembly was carried out in 0.4 mL MEM buffer
[100 mM Mes, 1 mM MgSO4, 1 mM EGTA (pH 6.8)] with 1 mM GTP, and 60 mM KCl.
We used 24 µM tubulin for assembly in the presence of 5 µM tau MTBR and 15 µM
tubulin for assembly in the presence of 2 µM MAP2 MTBR. All reaction components
were mixed at 4° C, and assembly was initiated by transfer to a cuvette that was
prewarmed to 37° C in a thermoregulated Cary Model 210 spectrophotometer.
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Polymerization of tubulin to form microtubules was monitored turbidimetrically as
the increase in absorbance at 350 nm over a 20 to 25 min assembly period. For binding
measurements, MTs were assembled from tubulin (5 mg/mL), and Taxol (50 mM) was
then added for 15 min at 37° C. Each binding assay contained Taxol-stabilized MTs
(tubulin content 0.25mg/mL), 100 mM Mes, 1 mM EGTA, 1 mM MgCl, 1 mM GTP, 2.5
mM Taxol, and 60 mM KCl at pH 6.8 in a final volume of 50 or 100 mL. To remove
aggregated protein, [3H]MAP-2 or [3H]tau MTBR was always centrifuged at 100,000 g
for 15 min immediately prior to the experiment. In the binding experiments,
uncomplexed tau or MAP2 concentrations ranged from 0.5–2 mM. Assay samples were
warmed to 37° C, and Taxol-stabilized MTs were admixed with gentle pipetting to
minimize shearing. After 20 min, samples were transferred to centrifuge tubes (precoated
with 10 mg/mL BSA) and centrifuged at 100,000 g for 15 min at 37° C in a Beckman
AirFuge. The supernatant was removed, and the pellet was rinsed with MEM buffer and
resuspended in 30 mL 0.5 % SDS. The amount of microtubule-bound MTBR was
determined by scintillation counting of pellet fractions.
For salt desorption experiments, each 100 mL binding assay contained
Taxol-stabilized MTs (tubulin content = 1.5 mg/mL), 100 mM Mes (pH 6.8), 1 mM
EGTA, 1 mM MgCl2, 1 mM GTP, 2.5 mM Taxol, and 30 mM [3H]MAP2 or [3H]tau
MTBR at the KCl concentration indicated on the graph. Solutions were incubated at
37° C for 20 min, transferred to precoated centrifuge tubes and centrifuged at 100,000 g
for 15 min at 37° C in a Beckman Airfuge. Supernatant and pellet were determined
separately by scintillation counting. For electron microscopy, MT-containing samples
were added to carbon-coated 400 mesh copper grids for 30 s, followed by negative
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staining with 1-2 % uranyl acetate for 30 s. Grids were examined on a Hitachi H-7000
transmission microscope at 75 kV.
Results
As shown in Figure 3-1, tau and MAP2 share remarkable homology in their
microtubule-binding regions (MTBRs), especially in the three repeat sequences. The
ability of either protein to polymerize into PHF or SF structures depends on the formation
of an interchain disulfide bond in the dimer (DeTure et al. 1996, Schweers et al. 1995),
and this disulfide link forms readily when tau or MAP2 is dialyzed with constant stirring
in the presence of atmospheric oxygen. Because MAP2 and tau affinity for assembled
microtubules depends on the presence of the second repeat (Ennulat et al. 1989, Joly et al.
1989), and because this repeat contains the critical thiol for PHF and SF formation, the
scope of this research was to determine whether disulfide formation altered the ability of
these dimers to promote tubulin polymerization or modified their binding affinity toward
assembled microtubules. These experiments required highly purified samples of
monomeric and dimeric tau and MAP2. The HPLC elution profile shown for tau MTBR
in Figure 3-2A indicates that monomer and dimer display sufficiently different retention
times to permit their efficient separation and recovery. The purity of each tau component
was confirmed by SDS gel electrophoresis (Figure 3-2B), which shows that fractions 16–
18 contain substantial quantities of highly pure tau monomer and that fractions 19–21 are
rich in highly purified dimer. Except for shift in the peak elution times, virtually
identical HPLC elution profile was obtained for MAP2 MTBR, and SDS gel
electrophoresis likewise confirmed the purity of MAP2 monomer and dimer (Figure
3-2C). Isolated tau or MAP2 dimer could be used after removing the volatile elution
buffer and redissolving the protein in 100 mM MEM assembly buffer. Because similar
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Figure 3-1. General features of three-repeat tau and MAP2, including sequences of the
microtubule-binding regions used in this study.
77
Figure 3-2. A, HPLC purification of tau monomer and dimer; B, SDS gel electrophoresis
of HPLC-purified tau MTBR; C, SDS gel electrophoresis of HPLC-purified MAP2
MTBR.
78
treatment of the monomer favors some oxidation and dimerization, we routinely
included 4 mM dithiothreitol (DTT) in all experiments conducted with
monomer-containing samples. As shown in earlier studies (DeTure et al. 1996, Schweers
et al. 1995), DTT completely converts the dimer into monomer within minutes.
Furthermore, the presence of DTT is without effect on either the rate or extent of tubulin
polymerization into microtubules (MacNeal and Purich 1978).
Microtubule-associated proteins like tau and MAP2 stimulate tubulin
polymerization (MacNeal and Purich 1978, Sloboda et al. 1976), but little is known about
microtubule self-assembly in the presence of the corresponding disulfide-linked dimers.
The data presented in Figure 3-3A indicate that tau monomer is only slightly more
effective than cross-linked dimer in promoting tubulin polymerization. These data also
show that for tau dimer there is a modest degree of assembly overshoot, a phenomenon in
which initially there is a slightly higher extent of polymerization than later attained at
steady-state (Detrich et al. 1987). In the case of MAP2 (Figure 3-3B), we have observed
that the homodimer is consistently less effective than the monomer in stimulating
microtubule self-assembly. Nonetheless, the significant finding is that both tau dimer and
MAP2 dimer do promote microtubule assembly, suggesting that the dimeric species can
bind to microtubules.
To test whether tubulin had nonspecifically aggregated, rather than polymerized, in
the presence of tau dimer or MAP2 dimer, negative staining techniques and transmission
electron microscopy were used to examine the morphology (Figure 3-4) of structures
assembled in the presence of tau or MAP2 homodimers. The micrographs confirm that
virtually all of the assembled structures are microtubules. There was no
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Figure 3-3. A, Promotion of tubulin polymerization by tau monomer and disulfide-liked
dimer. B, Promotion of tubulin polymerization by MAP2 monomer and disulfide-liked
dimer.
80
evidence of MT-MT cross-linking or bundling. Moreover, the observed turbidity
amplitudes in Figure 3-4 are most consistent with microtubule assembly; had any
significant bundling occurred, the amplitudes would have been two to three times greater.
To determine the relative affinity of monomers and dimers for microtubules, the
amount bound versus [tau]free or [MAP2]free was measured. As shown in Figure 3-5C,
dimer and monomer forms display similar binding properties, suggesting that disulfide
cross-linking has no major effect on microtubule binding affinity. Because MAPs also
rely on electrostatic attraction to stabilize binding to microtubules (Purich and
Kristofferson 1984), the strength of tau and MAP2 dimer binding to MTs could be
assessed by determining the amount bound as a function of salt concentration.
Taxol-stabilized microtubules are used in this type of experiment to avoid any
confounding effects of salt concentration on the critical tubulin concentration. The
binding curve for tau monomer and dimer are almost superimposable (Figure 3-5A),
whereas the MAP2 dimer appears to bind with slightly higher affinity than its monomer
(Figure 3-5B). Taken together, the data in Figure 3-5 clearly demonstrate that dimers
have substantial affinity for assembled microtubules.
Discussion
The formation of tau-S-S-tau and MAP2-S-S-MAP2 homodimers and their
involvement as building blocks of Paired Helical and Straight Filaments led to this
investigation of dimer interactions with unpolymerized tubulin and assembled
microtubules. In considering how dimerization might promote paired helical or straight
filament formation, one might expect that any reduced capacity to interact with
assembled microtubules would create a thermodynamic driving force favoring PHF
and/or SF assembly. Surprisingly, although the sulfhydryl groups responsible for
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Figure. 3-4. Electron micrographs of microtubules assembled in the presence of MAP2
(top) and tau (bottom).
82
Figure 3-5. Affinity of tau and MAP2 monomers and dimers for microtubules. A, Salt
desorption of tau MTBR monomer and dimer from Taxol-stabilized microtubules; B, salt
desorption of MAP2 MTBR monomer and dimer from Taxol-stabilized microtubules;
and C, microtubule binding properties of tau and MAP2 monomers and dimers.
83
cross-linking lie in the middle of those repeats required for MT binding, disulfide bond
formation has only a modest effect on the ability of cross-linked dimers to stimulate
tubulin polymerization and to bind to microtubules. Our observations suggest that factors
other than disulfide cross-linking must be involved in the accumulation of neuropathic
filaments.
Although SH groups are maintained in a highly reduced state in healthy neurons,
there is mounting evidence that oxidative stress plays a role in the Alzheimer and related
neuropathies (Owen et al. 1996, Smith et al. 1996). Furthermore, recently published
experiments suggest that the Aβ-peptide exerts its cellular toxicity by directly trapping
molecular oxygen and producing hydrogen peroxide through reduction of metal ions such
Fe3+ and Cu2+ to Fe2+ and Cu 1+ (Huang et al. 1999). Other indications that oxidative
stress occurs in AD include an increased amount of protein carbonyls (Smith et al. 1991,
Smith et al. 1996), more extensive lipid peroxidation (Palmer and Burns 1994, Sayre et
al. 1997), formation of nitrated protein (Smith et al. 1997) and the accumulation of
mitochondrial and nuclear oxidation adducts (Mecocci et al. 1994). Thus, although the
accumulation of disulfide-linked tau and MAP2 homodimers have not been directly
demonstrated in AD neurons, highly abundant thiol-containing proteins, such as tau and
MAP2, are apt to be good targets during oxidative stress. Aberrant glycation of tau,
hyperphosphorylation, as well as defective intracellular targeting to axons have been
offered as potential factors, but there is no clear evidence that these processes actually
culminate in PHF or SF formation in vivo. One should not exclude the possibility that
oxidative dimerization may prime tau for one or more other posttranslational
modifications, such that filament formation is a multi-factorial process. In any case, our
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demonstration that tau and MAP2 homodimers readily promote tubulin polymerization
and bind to microtubules represents yet another clue in efforts to define the underlying
mechanism(s) leading to PHF, SF, and neurofibrillary tangle formation.
CHAPTER 4
REDOX PROPERTIES OF CYSTEINE RESIDUES IN THE MICROTUBULEBINDING REGION OF TAU PROTEIN
Introduction
Alzheimer's disease is a very complex clinical condition that does not appear to
have a single cause. One clear feature in the pathophysiology of this disease is oxidative
stress. Likewise, there is significant evidence that the pathogenesis of several other
neurodegenerative diseases, including Parkinson's disease, Friedreich's ataxia and
amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species and
mitochondrial dysfunction (Mark et al. 1997, Owen et al. 1996, Smith et al. 1996).
Oxidative stress, a process that has been implicated in both normal aging and in various
neurodegenerative disorders, may be a common mechanism underlying various forms of
cell death, including necrosis, apoptosis, and excitotoxicity.
Evidence that oxidative stress plays a role in aging and neurodegenerative diseases
comes from several studies. The abundance of protein oxidation products (carbonyls)
increases in aging, and can be detected in AD brains. The activity of glutamine
synthetase, an oxidation-vulnerable enzyme, is also significantly lowered in specific
regions of AD brains, again suggesting that AD may represent a specific brain
vulnerability to age-related oxidation (Smith et al. 1991). Also it has been shown that
redox-active transition metals increasingly localize in the brain regions most affected by
neurodegeneration. In particular, redox-active iron is associated with both the senile
plaques and neurofibrillary tangles. This lesion-associated iron is able to participate in in
85
86
situ oxidation and readily catalyzes an H2O2-dependent oxidation (Smith et al. 1997). A
commonly held scientific belief is that amyloid plaques contribute to or cause the
oxidative damage and inflammation that occur and, ultimately, destroy brain cells.
Recently Huang et al. (1999) provided evidence that the A-beta peptide of Alzheimer's
disease directly produces hydrogen peroxide through reduction of metal ions such as
Fe(III) or Cu(II). Within cells, glycoxidation adduction to and direct oxidation of amino
acid side-chains have also been demonstrated in neurofibrillary tangles. In particular,
advanced glycation end-products (AGEs) colocalize with tau paired helical filaments in
neurofibrillary tangles in sporadic Alzheimer disease. Since AGE-modified proteins
aggregate and generate reactive oxygen intermediates it could be possible that AGEs in
paired helical filament tau contribute to the oxidative stress observed in Alzheimer
disease (Yan et al. 1994). Ultimately, oxidative stress during certain neurodegenerative
disorders may lead to, or result from, a disturbed glutathione (GSH) homeostasis. This
tripeptide (γ-L-glutamyl-L-cysteinylglycine) is the most abundant thiol in neuronal cells,
where it reaches concentrations of 3-5 mM. GSH plays multiple roles in the nervous
system including free radical scavenger, redox modulator of ionotropic receptor activity,
and possible neurotransmitter. An important role for glutathione was proposed for the
pathogenesis of Parkinson's disease, because a decrease in total glutathione
concentrations in the substantia nigra has been observed in preclinical stages, at a time at
which other biochemical changes are not yet detectable (Owen et al. 1996). Evidence
correlating oxidative stress and diminished GSH status has been shown for Lou Gehrig's
disease or amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's
disease (Bains and Shaw 1997, Schulz et al. 2000). Certain forms of ALS arise from
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dysfunctional superoxide dismutase, again underscoring the connection of
neurodegenerational oxidative stress (Morrison and Morrison 1999).
Glutathione depletion would induce a generalized oxidation inside the neuronal
cells, which, among other effects, could lead to the formation of tau-tau homodimers.
Tau dimerization is required for the nucleation step leading to the self-assembly of
neuropathic filaments (Schweers et al. 1995). The cysteine thiol in the second repeat of
tau plays a particularly important role in the dimerization process, as it is the only
cysteine present in the three-repeat isoforms of the protein. Four-repeat tau isoforms,
which contain a second cysteine in the added repeat sequence, are less prone to filament
assembly. Instead, they tend to form intramolecular disulfide bridges that block
intermolecular disulfide formation (Schweers et al. 1995). Glutathione may exert a
protective effect on tau reacting with its cysteines and preventing the dimerization of this
MAP as well as of MAP2.
To investigate this hypothesis, I have analyzed the cysteine residue in the second
repeat inside the microtubule-binding region of tau with particular attention to this
residue reactivity with glutathione and the synthetic thiol, dithiothreitol (DTT).
Material and Methods
Synthetic singles-tranded oligonucleotides for cloning were made by Genemed
Synthesis Incorporated, and cDNA products were ligated into the pETh-3b vector for
cloning and expression. This vector was a derivative of pBR-322 and was a gift from Dr.
Donald McCarty at the University of Florida. Invitrogen One Shot competent cells or
DM1 competent cells from Gibco-BRL were used for cloning, and E. coli BL21 (DE3)
pLYS S competent cells from Novagen were used for protein expression. Agar, tryptone
and yeast extract were obtained from DIFCO Laboratories. Tris base, sodium acetate,
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phenylmethylsulfonyl fluoride, dithiothreitol, sodium dodecylsulfate, ampicillin,
magnesium chloride, 2-(N-morpholino)ethanesulfonic acid, DNase, sodium chloride,
Folin reagent, thioflavin S, isopropyl-β-thiogalacto-pyranoside, reduced and oxidized
glutathione were from Sigma Chemical Company. DNA high melt agarose, Wizard
Minipreps, dNTPs, Lambda DNA markers and certain restriction enzymes were obtained
from the Bioproducts Division of Fisher Scientific. Most restriction enzymes were
purchased from New England Biolabs while T4 DNA ligase, Taq DNA polymerase and
chloramphenicol were purchased from Boehringer-Mannheim. Microcon, Centricon and
Centriprep by Amicon were used to concentrate protein samples. Whatman Corporation
provided the P11 phosphocellulose, and Coomassie Brilliant Blue R 250 was from
Crescent Chemicals. The cDNA constructs for expressing tau-123 were made by cassette
mutagenesis from clones prepared by previous members of the Purich lab (Coffey 1994).
Briefly, the tau-123 containing plasmid had been engineered to contain a unique Afl II
site at the 5’ end of the second repeat and an unique Stu I site at the 3’ end of the second
repeat (Ainzstein and Purich 1994). The tau-123 plasmid was digested with Afl II and
Stu I releasing a small inserted that could be separated from the plasmid by gel
electrophoresis. Synthetic oligonucleotides were designed which annealed to one
another, coded for the mutations of interest and changed the Stu I site by silent mutation.
These oligonucleotides when annealed had a single-stranded overhang on the 5’ end that
corresponded to the sticky end created by Afl II digestion and a blunt end on the 3’ end
corresponding to Stu I digestion. This insured the cassette was inserted in the correct
orientation when incubated with the digested and purified tau-123 plasmid. Ligations
from the mix were transformed into a methylation negative strain of DM1 cells, and
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colonies were screened by the loss of the Stu I digestion site. Positive colonies were
picked and sequenced by the ICBR DNA sequencing core at the University of Florida.
Expression and Purification of Wild Type Tau123
Tau-123 MTBR constructs were expressed and purified using a modified version of
the protocol based on the heat stability and cationic nature of the MAPs (Coffey et al.
1994). For expressing proteins, cDNAs in the pETh-3b vector were transformed into E.
coli BL21 (DE3) cells. Overnight cultures of 10 mL of these cells were used to inoculate
one-liter cultures of LB media containing 50 mg/mL of ampicillin and 34 mg/mL of
chloramphenicol. The cells were grown at 37° C until the cultures reached an OD--600
of 0.4 to 0.5 at which time protein expression was induced by bringing the media to 0.5
mM IPTG. This agent allows the bacterial gene expression of T7 RNA polymerase,
which then binds the T7 promoter found upstream of the cloned cDNA. This resulted in
massive transcription and translation of the cDNA of interest. After 2 hours of protein
expression the cells were harvested by centrifugation at 5,000 g for 5 minutes. The
pelleted cells were then resuspended in ice-cold 1 x MEM buffer (100 mM MES, 1 mM
EGTA and 1 mM MgCl2 at pH 6.8), washed and pelleted again at 5,000 g for 5 minutes.
Bacteria from one liter of culture were resuspended in 20 mL of cell lysis buffer (100
mM Tris, 500 mM NaCl, 1 mM MgCl2, 1 mM PMSF, 20 units/mL DNase and 1 mM
DTT) and frozen at -20° C overnight. The pellets were then thawed and sonicated with a
Branson Model 450 Sonifier at 20 watts output at 60 % for four two-minute sessions on
ice. This step lysed the cells and fragmented the DNA. The solution with the lysated
bacteria was then heated at 80° C for 10 minutes before being placed on ice for 20
minutes. The solution was then freed of denatured, aggregated proteins by centrifugation
at 40,000 g for 20 minutes. The supernatant from this spin contained mostly heat-stable
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tau fragments in high salt. The sample was diluted with water to bring the salt
concentration below 150 mM NaCl before loading it onto a 1 mL phosphocellulose
column equilibrated in 1x MEM. The sample was loaded by gravity, and the column was
washed extensively with 10 mL of 50 mM MEM. The sample was eluted with 50mM
MEM containing 1 M NaCl and 1 mL fractions were collected. The fractions with and
OD-280 above 0.1 were pooled and then dialyzed into 2 x 200 volumes of 50 mM MEM
at pH 6.8 for 16 hours. The dialysis favors protein dimerization as about 80 to 90 % of
tau is in dimeric form after this treatment. These samples were then processed directly or
aliquoted and frozen at -20° C.
Sample purity was confirmed by SDS-PAGE, and the molecular weights of the
polypeptides was verified by matrix-assisted laser desorption mass spectroscopy. Some
samples contained lower-molecular-weight bands, presumably formed by incomplete
translation or proteolysis (Zhang 1997). Protein concentrations were determined using
the Lowry method adjusted for the MAPs (Coffey et al. 1994).
Tau-123 MTBR Dimer Purification
Similarly to the method presented in the previous chapter, a mixed protein solution
containing recombinant monomer and dimer as well as lower-molecular-weight fractions,
was collected after dialysis, acidified in 10 % TFA and injected at a flow rate of 1
mL/min into Hewlett-Packard HP1090A HPLC using a Jupiter C4 reverse-phase column
(Phenomenex). The column was equilibrated with an elution solvent made up of 4:1 v/v
solution A (0.1 % TFA in water) and solution B (0.085 % TFA in acetonitrile). Following
injection, the column was developed from 5 to 50 min using a linear gradient from 7:3
v/v to 1:4 v/v solutions A and B. The elution was collected in 1 mL fractions in a 2212
Helirac fraction collector (LKB-Pharmacia). Dimer fractions were collected,
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concentrated to dryness using a Savant Speedvac concentrator, and redissolved in 0.1 M
MEM buffer. Samples from the fractions eluted corresponding to the retention time of
the proteins were prepared for SDS-PAGE by adding sample buffer without reducing
agents.
The bands were stained with Coomassie and compared to dye-labeled medium
molecular weight marker from Promega and the gel was 12 % cross-linked acrylamide
with 0.1 % SDS. The fractions corresponding to the pure dimer where collected, dried in
a Savant Speedvac concentrator and redissolved in 0.05 M MEM buffer.
HPLC Analysis of Tau Dimer and Monomer After Reaction with Thiols
Tau purified dimer was incubated with different increasing amounts of reducing
thiols in 50 mM MEM buffer at pH 6.8 for 1 hour at room temperature after which 20 µL
samples from the reaction mixture were analyzed with HPLC. Reverse-phase HPLC was
performed on a Hewlett-Packard Series 1090A liquid chromatograph equipped with a
Jupiter C18 column (Phenomenex, CA). The tau-thiol mixture was eluted with a gradient
beginning with 90 % solvent A (0.1 % trifluoroacetic acid) and an increasing percentage
of solvent B (0.085 % trifluoroacetic acid/acetonitrile): 10-20 % in 5 min, 20-67 % in 20
min, 67-80 % in 5 min and return to 20 % in 5 min. The flow was maintained at
1 ml/min. The effluent was fed to the flow cell of a Hewlett-Packard Series 1090A diode
array detector. Absorbance was recorded at 220 and 280 nm wavelength simultaneously.
Peak integrations and area calculations were determined using HPLC ChemStation
software (Hewlett-Packard).
Tau-123-GSH Conjugation
In order to obtain a stable and sufficient amount of tau-glutathione conjugate,
HPLC purified tau in homodimeric form was reacted as follows: tau at final
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concentration of 5 mg/ml was mixed with 2-fold molar excess of reduced glutathione for
1 hour at room temperature and loaded by gravity onto a phosphocellulose column
equilibrated in 0.05 M MEM. The column was washed extensively with 10 volumes of
10 mM oxidized glutathione (glutathione dimer) in 50 mM MEM, deoxygenated by
boiling and fast cooling of sealed beaker or alternatively by flowing nitrogen gas in the
beaker. After the extensive wash with oxidized glutathione, tau protein was eluted from
the column with 50 mM deoxygenated MEM containing 1 M NaCl. The eluted protein
was dialyzed by spinning in microcon concentrating devices (Amicon) at 14000 g for
four times replacing the filtered volume at every spin with equal amount of 50 mM MEM
buffer containing 1 mM oxidized glutathione. The protein-thiol heterodimer solution was
then concentrated on a Microcon filtering device. After concentration, samples from the
tau-glutathione solution were run on SDS-PAGE in the absence of thiol-reducing agents.
The protein was mostly in monomeric form as shown from the SDS-PAGE. To verify
the presence of the glutathione associated to the observed tau monomer, samples of the
heterodimer were flown on a MALDI-TOF mass spectrometer. Alternatively the
tau-glutathione mix eluted from the phosphocellulose column was injected at a flow rate
of 1 mL/min into Hewlett-Packard HP1090A HPLC using a Jupiter C4 reverse-phase
column (Phenomenex). Conditions of column equilibration and elution were the same
used in the dimer purification protocol. The elution was collected in 1 mL fractions in a
2212 Helirac fraction collector (LKB-Pharmacia). The fractions corresponding to the
known tau retention time were than pooled together, concentrated to dryness using a
Savant Speedvac concentrator, and redissolved in 0.05 M MEM buffer. A sample from
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this final solution was run on SDS-PAGE. The tau-glutathione heterodimers prepared as
indicated were then used for filament assembly experiments.
Electron Microscopy
Carbon-coated grids were prepared by coating 400-mesh copper grids with grid
glue (i.e., 1 inch Scotch tape washed into 10 mL chloroform) to ensure proper adhesion
of carbon films onto the grids. Carbon films created by vapor adsorption of elemental
carbon onto mica strips were floated on water and then picked up with copper grids.
These carbon-coated grids were prepared freshly for each microscope session. Sample
was adsorbed on to the grids by floating a grid on a drop of sample for 30 seconds. The
nonadsorbed sample was then wicked off and the grid was negatively stained by floating
it on a drop of 1 % uranyl acetate or 1 % phosphotungstic acid for 30 seconds. Grids
were examined after 15 minutes on a Hitachi H-7000 transmission electron microscope at
75 kV.
Time-course of Monomer Formation and Tau-thiols Reactivity Experiments
Tau purified dimer at 500 mM final concentration were mixed with 2-fold molar
excess of reduced glutathione or 6- to 8-fold molar excess of dithiothreitol in 50 mM
MEM at pH 6.8 in 1.8 mL plastic tubes. The different samples were incubated at room
temperature for 1 hour after which time, were stored at 4° C (to slow down spontaneous
degradation) for the required time in the time-course experiments. To test the reactivity
of tau dimer with glutathione, tau and reduced glutathione were mixed in different ratios
from 1:1 to 1:10, tau-to-glutathione.
To investigate how rapidly the dimer was converted to monomer, tau and reduced
glutathione were mixed together at 1:1 molar ratio and samples were collected at
different incubation times from 10 to 120 minutes. Samples from the different
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experiments were then analyzed using Tris-tricine SDS-PAGE in absence of reducing
agents.
Results
Tau polymerization is preceded by the formation of disulfide-crosslinked
homodimers, which act to nucleate filament assembly (Schweers et al. 1995). Tau
possesses only one cysteine residue in its three-repeat isoforms (as shown in Figure 4-1).
The cysteine is located in the third repeat (second in the three-repeat forms), which is
very important for microtubule binding (Goode and Feinstein 1994). Coincidentally, this
repeat is also the minimal sequence required for tau-tau interaction in filament assembly
in vitro (Perez et al. 1996). The only other cysteine present in the sequence of full-length
tau is in the extra repeat in four-repeat isoforms. These isoforms are less prone to
filament formation, because they tend to form intramolecular disulfide links rather than
intermolecular bonds between two distinct tau molecules. Since the focus of this chapter
is on the redox properties of the cysteine residue inside the MTBR of tau, the experiments
were carried out on tau-123, a fragment of tau containing only the microtubule-binding
region. Time-course experiments in which tau was reacted either with glutathione or
dithiothreitol clearly suggest that GSH is more effective. This is a surprising result, in
view of the fact that DTT possesses the ability to form a dithiolane ring that stabilizes
product formation. As shown in Figure 4-2a, at time 0, tau-GSH and tau-DTT samples
are both mostly in monomeric form, as expected. Upon prolonged incubation (30 days),
however, the tau-DTT samples show increased amounts of dimer, whereas the tau-GSH
remains at or near its original amount. After 90 days, the tau-DTT sample is almost
completely reconverted to the tau-tau homodimer (Figure 4-2a bottom SDS-PAGE),
while tau-GSH persists mainly in its monomeric form. This result also hold true at
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Figure 4.1. Complete sequence of human adult tau. The microtubule-binding repeats are
underlined and colored in green. In the red box is the extra repeat absent in the
three-repeat isoforms of tau. The arrows indicate the position of the cysteines. Note that
in three–repeat tau, there is only one cysteine present.
96
concentrations of DTT that are four times higher than corresponding GSH ones. In fact,
GSH at a molar ratio of 1:1 with tau reduces the same amount of tau dimer as a 10-fold
molar excess of DTT (Figure 4-2b). The process is relatively slow; the reduction of tau
to monomer is still not complete after 10 minutes. After 30-40 minutes the generation of
monomer reaches a plateau, and there is no further apparent change in the reduction
process after 40 minutes (Figure 4-2c). This result prompted use of one-hour incubation
times for all the following experiments, to allow sufficient time to assure the maximum
reduction of tau by glutathione.
HPLC analysis of the product of tau-thiol interactions revealed another aspect of the
reduction properties of GSH in respect to DTT. Figure 4-3 shows different resolution
times for tau exposed to increasing amounts of thiols. Exposure of tau dimer to GSH
generates two species of monomers, compared to only species observed when the protein
is reacted with DTT, despite the amount of reducing thiol reacted with tau. This is to be
expected because of the following reaction:
We have previously characterized the chromatographic profile for tau monomer and
dimer (see previous chapter). The tau monomer generated in the reduction process has a
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lower retention time, preceding the dimer in the elution from the reverse-phase HPLC
column (Figure 4-3). Since the extra peak observed in tau-GSH reaction is eluted before
the regular tau monomer of DTT, a different form of monomer has to be generated when
tau is reacted with GSH. Two possibilities: (a) the extra peak could be a smaller
fragment of tau protein due to partial proteolysis. This hypothesis is ruled out by the
observation that samples from the HPLC purified fractions on SDS-PAGE show only
tau-123 in monomeric and/or dimeric form. (b) the extra peak represents indeed a second
monomer, made of tau stably bound to GSH. To verify this possibility the extra peak was
purified from HPLC and run in a MALDI-TOF mass spectrometer. As expected, the
molecular weight measured corresponds precisely to the sum of tau and GSH masses
(Figure 4-4). The amount of tau monomer generated by the two different reducing agents
was calculated as molar amount of tau monomer per molar amount of reducing thiol. The
amount of tau monomer generated by GSH is constantly higher than the amount
generated by DTT, being almost always double (Figure 4-5 and monomer-to-dimer ratios
in Figure 4-3). And this finding is somehow unexpected when we consider that DTT has
two reacting thiols against the only one present in GSH.
Tau-GSH conjugates were tested for filament assembly and compared to results
from tau homodimer polymerizations. We speculated that glutathione could exert a
protective effect over tau maintaining it in a monomeric form and thus inhibiting or
slowing down the assembly process. Surprisingly the tau bound to glutathione was still
able to form regular filaments as shown in Figure 4-6.
98
Figure 4-2. A, Time course experiments where tau is mixed to GSH or DTT and let to
incubate for up to 30 days. B, and C, show the reactivity tau with GSH in terms of
efficiency and reaction time.
99
Figure 4-3. HPLC analysis of tau reacted with glutathione or dithiothreitol. For each
chromatogram the concentration of the reducing agents is represented as percentage of
tau concentration. On top of each chromatogram is indicated the ratio between monomer
(M) and dimer (D) generated by the reaction. P-SH = tau monomer; P-S-S-P = tau
homodimer, P-S-S-G indicates tau conjugated to glutathione. The percent values indicate
the amounts of reducing agents per fixed amount of tau protein.
100
Figure 4-4. MALDI-TOF mass spectrometry of peak isolated from HPLC run of tau
reacted with glutathione. Note that the molecular weight calculated corresponds exactly
to the sum of tau and glutathione.
101
100
Tau Monomer ( µ M)
90
80
70
60
GSH
50
DTT
40
30
20
10
0
0
100
200
300
400
500
[Thiol], µ M
Figure 4-5. Amount of tau monomer generated by reaction of tau homodimer with
increasing amounts of glutathione or dithiothreitol and calculated as molar amount of tau
monomer per molar amount of reducing thiol.
102
Discussion
This investigation focuses on the ability of thiol-reducing agents to alter tau’s
ability to self-assemble. There is only one cysteine residue in the three-repeat forms of
tau. This cysteine is inside the second repeat of the microtubule-binding region
(conventionally named R3, as this is actually the third repeat when all four repeats are
present) (Figure 4-1). A second cysteine is contained inside the extra repeat
(conventionally named R2) of the MTBR, which is present only in the four-repeat
isoforms of tau. Four-repeat tau isoforms are less prone to filament assembly because
they tend to form intramolecular disulfide bonds, rather than intermolecular ones
(Schweers et al. 1995). This has also been shown in studies on the separated properties
of R2 and R3 repeats, in which it was shown that all R2 and R3 peptides bound
specifically to the tubulin peptide regardless of the state of phosphorylation or
dimerization. The isolated R2 and R3 units formed homodimers approximately in the
same rate. When the two peptides were mixed, however, the R2-R3 heterodimer was
formed preferentially over the homodimers (Hoffmann et al. 1997). Although similar in
sequence the R2 and R3 repeat seem to have different properties and the cysteines inside
the two repeat seem to have different reactivity. The reactivity of the cysteine present in
the three-repeat forms of tau was investigated as this may play a key role in tau
dimerization and consequent aggregation into filamentous structures.
While investigating the properties of tau cysteine residue, particular attention was
directed to its ability to react with other thiols. Glutathione presented itself as an obvious
candidate, being the most represented thiol in the neurons and the reducing agent of
choice in the cell. To compare the reactivity of tau cysteine with glutathione,
dithiothreitol was chosen, being one of the best synthetic reducing agents available.
103
Figure 4-6. Montage of electron micrographs illustrating different filaments assembled
from tau-GSH conjugate. The typical paired helical morphology is visible.
104
The results from these experiments show that tau reacts more stably and readily
with glutathione than with dithiothreitol. In time-course experiments, tau homodimers
were reduced to monomers in about 20-30 minutes and remained in such a reduced form
even after 90 days. On the contrary, tau reacted with dithiothreitol was oxidized back to
homodimeric form after the same period of time. The reaction is very efficient as a 1:1
molar ratio of glutathione-to-tau generates the same amount of monomer as a 10-fold
molar excess of GSH over tau (see Figure 4-2). This efficiency of reduction is better
shown by the analytical HPLC experiments. Tau-123 reacted with GSH is reduced to
two main monomeric forms: one corresponding to the protein with the -SH group of
cysteine available and a second monomer, not observed in the DTT reduced samples, in
which the cysteine of tau is stably bound to glutathione (Figures 4-3, 4-4). This is
expected since DTT tend to form a dithiolane ring stabilizing the reaction (see reaction
scheme in Results). Upon integration of the peaks from the analytical HPLC it was
possible to calculate the relative amounts of every species observed in the
chromatographs. The amount of tau monomer generated in the presence of glutathione
was constantly higher than the dithiothreitol one. The results clearly support the idea that
the only -SH group available in tau reacts more favorably with glutathione than with
other thiols like dithiothreitol. This finding is surprising, considering that dithiothreitol
has two thiol groups available for reaction, against the only thiol group present in GSH
(Figure 4-1b), doubling the chances of formation of a heterodimer with the cysteine of
tau.
It is possible that the cysteine inside the repeats of tau have been designed to
interact specifically with glutathione allowing this thiol to exert a protective action from
105
oxidative stress thus avoiding the formation of tau-tau homodimers, a process that
ultimately nucleates the assembly of straight or paired helical filaments. This idea could
be better explained by looking at the nature of tau sequence around its cysteine and the
nature of glutathione itself. Glutathione is a slightly acidic tripeptide with a net negative
charge. The cysteine in tau is flanked by a lysine on one side and a glycine on the other.
It may be that the positively charged lysine on one side of cysteine acts as a docking site
for glutathione, by interacting with its negative charge, thus favoring the creation of the
disulfide bridge. Unfortunately in vitro experiments where glutathione-bound tau was
incubated under polymerizing conditions, failed to prove this protective role of
glutathione as the heterodimeric tau could still generate filaments (Figure 4-6).
Nonetheless this reactivity of tau for glutathione could be more easily considered as a
protection mechanism in a cellular environment where glutathione is present in high
molar excess (3-4 mM cellular glutathione against an average of 4-10 µM tau). And this
idea would shed a clearer light on the relationship between oxidative stress, glutathione
depletion and neurofibrillary tangle formation in affected neuronal cells.
CHAPTER 5
A FLUORIMETRIC ASSAY OF TAU POLYMERIZATION
Introduction.
One impediment to the systematic investigation of tau polymerization in vitro is the
lack of a reliable method for measuring this process. Among the techniques used so far
are sedimentation (Arrasate et al. 1999, Schneider et al. 1999) and electron microscopy
(Goedert et al. 1996, Hasegawa et al. 1997, King et al. 1999, King et al. 2000, Nacharaju
et al. 1999, Wilson and Binder 1997). Although spectrophotometric measurement of the
turbidity of protein solutions is useful in tubulin polymerization studies, (Gaskin et al.
1974) turbidimetric analysis requires large filaments to obtain adequate light scattering.
For this reason, this technique is not sensitive enough to measure tau polymerization at
physiological protein concentrations (1-4 µM). Furthermore, other nonfilamentous
aggregates can reduce value of the method in assays of filament assembly.
By far, the most widely used method for assessing tau filament assembly is the use
of dyes that fluoresce upon binding to aggregates. Thioflavin-S and thioflavin-T had
already been successfully employed to measure amyloid plaques in samples from brains
of patients affected by Alzheimer’s disease (Naiki et al. 1989). Binding of thioflavin S to
paired helical filaments in Alzheimer neurofibrillary tangles has been used as a diagnostic
marker to confirm the disease on post mortem brain samples. The dye is known to
exhibit a characteristic fluorescence at 480 to 490 nm when bound to Alzheimer paired
helical filaments and filaments formed in vitro from recombinant tau fragments
(Schweers et al. 1995). Friedhorff et al. characterized the method for the measurement of
106
107
tau polymers in vitro (Friedhoff et al. 1998, King et al. 1999). Although the method is at
present widely utilized to determine the amount of tau polymer formed, it has some
intrinsic limitations. The mechanism by which the fluorophore thioflavin binds to the
polymer is not known; nor is known the stoichiometry of the reaction. Furthermore the
increase of fluorescence upon binding of the dye to the polymer is linear only up to
4-5 µM final concentration of the protein (Friedhoff et al. 1998).
Because there is at present no definitive method to study polymerization in vitro, I
have taken the initial steps in the development of an alternative polymerization assay.
This method relies on the covalent attachment of a fluorescent molecule to the N-terminal
residue of tau-123 fragment. The strategy used was originally exploited to label peptides
with an N-terminal serine residue. The serine is oxidized to an aldehyde and is then
conjugated with a hydrazine form of the fluorescent molecule yielding to the formation of
a hydrazone (Geoghegan et al. 1993, Geoghegan and Stroh 1992). In this chapter, I
document experiments that establish the feasibility of this approach.
Matherials and Methods
Genemed Synthesis Incorporated made synthetic single-stranded oligonucleotides
for cloning and mutations, and cDNA products were ligated into the pETh-3b vector for
cloning and expression. This vector was a derivative of pBR-322 and was a gift from Dr.
Donald McCarty at the University of Florida. Invitrogen One Shot competent cells or
DM1 competent cells from Gibco-BRL were used for cloning, and E. coli BL21 (DE3)
pLYS S competent cells from Novagen were used for protein expression. Agar, tryptone
and yeast extract were obtained from DIFCO Laboratories. Tris base, sodium acetate,
phenylmethylsulfonyl fluoride, dithiothreitol, sodium dodecylsulfate, ampicillin,
108
magnesium chloride, 2-(N-morpholino) ethanesulfonic acid, DNase, sodium chloride,
Folin reagent, thioflavin S, isopropyl-β-thiogalacto-pyranoside, sodium-m-periodate and
sodium borohydride were from Sigma Chemical Company. DNA high melt agarose,
Wizard Minipreps, dNTPs, Lambda DNA markers and certain restriction enzymes were
obtained from the bioproducts division of Fisher Scientific. Most restriction enzymes
were purchased from New England Biolabs while T4 DNA ligase, Taq DNA polymerase
and chloramphenicol were purchased from Boehringer-Mannheim. Microcon, Centricon
and Centriprep devices for protein sample concentration were made by Amicon.
Whatman Corporation provided the P11 phosphocellulose, and Coomassie Brilliant Blue
R 250 was from Crescent Chemicals. Sitting bridge crystallization cells and ancillary
material for polymerization experiments were obtained from Hampton Research.
Molecular Probes Incorporated manufactured the fluorescent molecules used in these
experiments. Acetonitrile and other reagents for HPLC purification of the proteins used
in these experiments were from Fisher Chemicals. The HPLC separation was run on a
Jupiter C18 reverse-phase column from Phenomenex Corporation.
The tau-123 expressed for use as controls in the experiments presented in this
dissertation were all subcloned from a cDNA isolated by reverse transcription and
polymerase chain reaction from total RNA isolated from retinoic acid-differentiated
human NT2 cells from Strategene.
Tau-123 was mutated to introduce a serine residue at the N-terminus of the protein
sequence. To obtain this mutant a PCR reaction was carried using 5’ oligonucleotide
coding for the mutations of interest and containing a unique Nde I restriction site outside
the coding sequence at the 5’ end. The 3’ oligonucleotide used in the PCR reaction is a
109
primer commonly used by this lab for general cloning of tau-123 and MAP2-123
fragments and contains a unique Eco RI restriction site outside the coding sequence at the
3’ end. The product from this reaction was a cDNA coding for three-repeat tau with an
extra serine at the N-terminus. The PCR product could be inserted into the Nde I and Eco
RI sites of the pETh-3b plasmid. Cloning simply involved digesting the PCR product
with Eco RI and Nde I, purifying the released fragment from the plasmid by gel
electrophoresis and ligating the PCR product into the plasmid. Transformed Invitrogen
One Shot competent cells were plated and colonies were collected for screening of their
plasmids. The strategy for screening was based on the presence of a unique Afl II site in
the middle of the tau-123 sequence otherwise absent in the original plasmid. Positive
colonies were grown in quantity and sequenced by the ICBR DNA sequencing core at the
University of Florida.
Expression and Purification T-123 with N-terminal Serine
S-MAP2-123[Module-B], and S-tau-123 mutants carrying the N-terminal serine as
well as tau-123 were expressed and purified using a modified version of the protocol
based on the heat stability and cationic nature of the MAPs (Coffey et al. 1994). For
expressing proteins, cDNAs in the pETh-3b vector were transformed into E. coli BL21
(DE3) cells. Overnight cultures of 10 mL of these cells were used to inoculate one-liter
cultures of LB media containing 50 mg/mL of ampicillin and 34 mg/mL of
chloramphenicol. The cells were grown at 37° C until the cultures reached an OD-600 of
0.6 to 0.7 at which time they were induced by bringing the media to 0.5 mM IPTG. This
agent allows the bacterial gene expression of T7 RNA polymerase, which then binds the
T7 promoter found upstream of the cloned cDNA. This resulted in massive transcription
and translation of the cDNA of interest.
110
After 2 hours of protein expression the cells were harvested by centrifugation at
5,000 g for 5 minutes. The cells were then washed by resuspending them with ice-cold
1x MEM buffer (100 mM MES, 1 mM EGTA and 1 mM MgCl2 at pH 6.8) and pelleting
again at 5,000 g for 5 minutes. Bacteria from one liter of culture were resuspended in 20
mL of cell lysis buffer (100 mM Tris, 500 mM NaCl, 1 mM MgCl2, 1 mM PMSF, 20
units/mL DNase and 1 mM DTT) and sonicated with a Branson Model 450 Sonifier at 20
watts output at 80 % for two two-minute sessions on ice. This step lysed the cells and
fragmented the DNA. This sample was then heated at 80° C for 10 minutes before being
placed on ice for 20 minutes. The solution was then freed of denatured, aggregated
proteins by centrifugation at 40,000 g for 20 minutes. The supernatant from this spin
contained mostly heat-stable MAP2 or tau fragments in high salt. The sample was
diluted with water to bring the salt concentration below 150 mM NaCl before loading it
onto a 1 mL phosphocellulose column equilibrated in 50 mM MEM. The sample was
loaded by gravity, and the column was washed extensively with 10 mL of 50 mM MEM.
The sample was eluted with 50 mM MEM containing 1 M NaCl and 1 mL fractions were
collected. The fractions with and OD-280 above 0.1 were pooled and then dialyzed into
2 x 200 volumes of 50 mM MEM at pH 6.8 for 16 hours. These samples were then
processed directly or aliquoted, fast-frozen in liquid nitrogen and stored at -20° C.
Sample purity was confirmed by SDS-PAGE, and the molecular weights of the
polypeptides was verified by matrix-assisted laser desorption mass spectroscopy. Some
samples contained lower-molecular-weight bands, presumably formed by incomplete
translation or proteolysis (Zhang 1997). These samples were further purified on HPLC
using a Jupiter C4 reverse-phase column (Phenomenex).
111
Oxidation of N-terminal Serine
To label the protein with the hydrazine form of the fluorescent probes obtaining a
hydrazone the N-terminal serine must be oxidized to an aldehyde. Oxidation was carried
by incubating 5 to 8 mg/mL tau123 with two-fold molar excess of sodium-m-periodate in
phosphate buffer at pH 7.0 for 20 minutes at room temperature. The chemical reaction is
illustrated in Figure 5-1a. The pH of the solution is paramount to the oxidation of the
targeted group (Geoghegan and Stroh 1992). In fact a lower pH of solution can facilitate
the oxidation of other residues not involved in the hydrazone formation. Although the
reaction of sodium-m-periodate with amino-alcohols is 1000 times faster than with any
other chemical group (Geoghegan and Stroh 1992, Nicolet and Shinn 1939, Offord
1990), oxidation of other residues may still occur if the pH of the solution is not
controlled. The exposure to sodium-m-periodate was limited to 20 minutes, to take
advantage of the faster interaction of the oxidizing agent with amino alcohols. After
incubation the sodium-m-periodate oxidizing effect was quenched by adding two-fold
molar excess of ethylene glycol to the solution. The mixture containing the oxidized
tau123 protein was acidified in 10 % TFA and injected at a flow rate of 1 mL/min into
Hewlett–Packard HP1090A HPLC using a Jupiter C4 reverse-phase column
(Phenomenex, CA) equilibrated with an elution solvent made up of 4:1 v/v solution A
(0.1 % TFA in water) and solution B (0.085 % TFA in acetonitrile). Following injection,
the column was developed from 5 to 50 min using a linear gradient from 7:3 v/v to 1:4
v/v solutions A and B. Dimer fractions were collected, concentrated to dryness using a
Savant Speedvac concentrator, and redissolved in 0.5 M acetate buffer at pH 4.5.
112
Figure5-1a. Periodate oxidation reaction. The amino alcohol group of the N-terminal
serine reacts with periodate generating an aldehyde with release of ammonia and carbon
dioxide. The reaction is 1000 times faster with amino alcohol than with any other
chemical group.
Figure 5-1b. Conjugation of fluorescent molecule to the protein. The reaction is a
nucleophilic attack of the nitrogen of the hydrazine to the carbon of the aldehyde with
formation of a semicarbazone. Two candidate molecules for the conjugation reaction are
shown. The asterisks show the position of the hydrazine in the fluorescent molecules.
113
Fluorescent Labeling of Tau123
S-tau-123 mutant with N-terminal serine was labeled by reacting the hydrazine
group of the florescent molecule of choice with the N-terminal aldehyde, generated as a
result of periodate oxidation of the protein. A solution containing 5 to 8 mg/mL purified
S-tau-123 and a five-fold molar excess of the fluorescent probe was incubated at room
temperature overnight in sodium acetate buffer at pH 4.5 in a sealed shielded container to
protect the fluorescent molecule from exposure to light and consequent photo bleaching.
Similarly to the oxidation of Ser-tau-123 with sodium-m-periodate, the hydrazone
formation reaction must also be carried in a pH-controlled environment. The optimal pH
of the reaction must be slightly acidic (pH 4.5-5) to facilitate the nucleophilic attack of
the amino group of the hydrazine to the carboxyl group of the aldehyde. A schematic of
the reaction is shown in Figure 5-1b. Three different fluorescent molecules have been
used in their hydrazine forms, Alexa488, Alexa350 and Lucifer Yellow. Of these three
Alexa350 was then chosen as the molecule of election for the fluorescence spectroscopy
measurements. After over night incubation reverse-phase HPLC of the sample was
performed on a Hewlett-Packard Series 1090A machine (Hewlett Packard) equipped with
a Jupiter C18 column (Phenomenex, CA). The fluorescently labeled tau was eluted with
a gradient beginning with 90 % solvent A (0.1 % trifluoroacetic acid) and an increasing
percentage of solvent B (0.085 % trifluoroacetic acid/acetonitrile): 10-20 % in 5 min,
20-67 % in 20 min, 67-80 % in 5 min and return to 20% in 5 min. The flow was
maintained at 1 ml/min. The effluent was fed to the flow cell of a Hewlett-Packard
Series 1090A diode array detector. Absorbance was recorded at 220 and at a second
wavelength varying according to the nature of the fluorophore attached to tau (345 nm for
Alexa350, 488 nm for Alexa488 and 427 nm for Lucifer Yellow). Peak integrations were
114
determined using HPLC ChemStation software (Hewlett-Packard). Chromatograms for
purifications of two of the dye employed in the reaction (Lucifer Yellow and Alexa350)
are shown in Figure 5-2.
In Vitro Assembly of Filaments with tRNA or Heparin
Samples (40 to 100 µM) were mixed with concentrations of tRNA or heparin
varying between 0.01 and 0.50 mg/mL in 1.5 mL Eppendorf tubes. The concentrations of
polyanion that worked best were between 0.01 and 0.05 mg/mL, which were lower than
those reportedly used for polymerization of full-length tau (Goedert et al. 1996, Kampers
et al. 1996, Perez et al. 1996). The samples were then incubated overnight at 37° C and
examined by electron microscope for filament assembly as described below.
Electron Microscopy
Carbon-coated grids were prepared by coating 400-mesh copper grids with grid
glue (i.e., 1 inch Scotch tape washed into 10 mL chloroform) to ensure proper adhesion
of carbon films onto the grids. Carbon films created by vapor adsorption of elemental
carbon onto mica strips were floated on water and then picked up with copper grids.
These carbon-coated grids were prepared freshly for each microscope session. Sample
was adsorbed onto the grids by floating a grid on a drop of sample for 30 seconds. The
nonadsorbed sample was then wicked off and the grid was negatively stained by floating
it on a drop of 1 % uranyl acetate or 1 % phosphotungstic acid for 30 seconds. Grids
were examined after 15 minutes on a Hitachi H-7000 transmission electron microscope at
75 kV. For polymer length measurements, digitally scanned images were analyzed using
Image-1 software (Universal Imaging Inc.).
115
Figure5-2. HPLC profiles of the products of conjugation reaction: Elution of Lucifer
Yellow (right) and Alexa 350 (left) coincide with the elution of tau. No other tau peaks
are observed in the chromatographic profiles.
116
Results
Many in vitro studies of tau polymerization focus on the kinetics of filament
formation. Several methods have been exploited to follow these reactions, but a
definitive model is still unavailable mostly do to the absence of a completely reliable
method. The most diffuse method to measure filament formation exploit the ability of
thioflavine to emit a characteristic spectrum once bound to protein aggregates such as tau
or MAP2 filaments (DeTure et al. 1996, Friedhoff et al. 1998). This method, although
widely used to measure polymerization kinetics, is not completely reliable due to the
absence of any information on the selectivity of the dye binding and the stoichiometry of
the reaction. Furthermore the measurement is linear only up to a limited concentration of
tau protein (1-4 µM) (Friedhoff et al. 1998). In an effort to provide a valid alternative to
the methods currently available, a mutant tau-123 fragment carrying a fluorescent label
on its N-terminus was generated. To obtain such a result a serine residue was introduced
at the N-terminus of the protein. The presence of an N-terminal serine in the mutant
tau-123 is expected according to the N-rule of E.coli (Hirel et al. 1989), which states that
the catalytic efficiency of the enzyme methionyl-aminopeptidase (and therefore the extent
of cleavage of the N-terminal E.coli formyl-methionine) decreases in parallel with
increasing of maximal side-chain length of amino acid in penultimate position.
According to this rule, when E. coli expresses a protein with serine in second position
90 % of f-methionine is cleaved by methionyl-aminopeptidase, being serine an amino
acid with relatively modest side-chain length (Hirel et al. 1989). To confirm the presence
of a serine as N-terminal residue in Tau123, a partial N-terminal amino acid Edman
analysis of purified mutant S-tau-123 was performed at the ICBR Protein core at
117
University of Florida. The analysis results show the sequence S-K-N-K-V-, proving the
presence of a serine as first residue of the mutant Ser-tau-123.
The product of the conjugation reaction was purified using HPLC. The
chromatographic profile of the purification shows that tau elutes completely in
conjunction with the absorbance peak of the fluorescent probe (Figure 5-2) and no other
tau peak is observed outside this elution time. If the conjugation were incomplete, two
species would be generated: an unlabeled tau-123 and a fluorescently labeled one. The
two species would have a distinct elution profile because of the hydrophobic nature of the
fluorescent probe conferring a different retention time to the labeled tau-123 in respect to
the unlabeled one. Since no other tau elution peak is observed outside of the one
coinciding with the fluorescent probe, it is reasonable to speculate that most if not all of
the protein is fluorescently labeled.
The fluorescently labeled tau-123 was assembled into filaments to determine if the
presence of the fluorescent dye would disrupt the protein ability to polymerize into
filaments in vitro. As shown in Figure 5-3 both S-tau-123 and S-MAP2-123 [ModuleB]
retain their ability to promote filament assembly under standard polymerization
conditions. For spectrofluorimetric measurements Alexa350 was chosen as the
fluorophore of election because its emission spectrum is more intense than the Lucifer
Yellow one and the molecule is considerably more stable, thus reducing the effect of
photo bleaching.
N-tau-123 conjugated with Alexa350 was incubated in the presence of polyanionic
promoters of filament assembly. In Figure 5-4 are shown the emission spectra of the
fluorophore-conjugated protein, at different incubation times. After 72
118
Figure 5-3. The fluorescent molecule does not interfere with the polymerization of tau or
MAP2 [Module-B]. The dark areas in the EM picture on the left are aggregates of
bundled filaments from which occasionally emerge some of the paired helical filaments
typical of the MAP2 [Module-B].
119
hours of incubation the emission spectra reaches a plateau and little or no emission
increase is observed after that time. Interestingly the maximum emission coincides with
the spectrum observed for equimolar amounts of free fluorophore. This observation led
me to believe that the polymerization reaction produces a progressive release of the dye
from tau-123 as the protein aggregates into filamentous structures. To confirm this
hypothesis tau-123 labeled with Alexa350 fluorophore was incubated overnight in the
presence of glycosaminoglycans as polymerization facilitators. Spectra at the Alexa350
emission wavelength were taken before and after incubation. After overnight
polymerization, the tau-123 solution was centrifugated at 100,000 g for 1 hour at 25º C,
and fluorescent measurements were taken of the supernatant. The centrifugation
precipitates the assembled filaments from the solution. Electron micrographs of the
solution before and after spinning clearly show that centrifugation spins down the
filaments and aggregates (Figure 5-5a and 5-5b). The fluorescent measurements show
that the fluorophore is completely in the supernatant after centrifugation (Figure 5-6)
confirming its release from tau after the polymerization process.
Discussion
Full-length tau polymerizes in vitro (Crowther et al. 1994, Montejo de Garcini and Avila
1987, Montejo de Garcini et al. 1986, Troncoso et al. 1993, Wilson and Binder
1995)(Goedert et al. 1996, Kampers et al. 1996, Perez et al. 1996)(Wilson and Binder
1997), but does not assemble as well as small fragments containing only the
microtubule-binding region containing the repeat region (Crowther et al. 1992, Schweers
et al. 1995, Wille et al. 1992). Assembly of recombinant material has the advantage that
all the proteins are in the same nonphosphorylated state, and they tend to assemble best
120
7.00E+05
6.00E+05
5.00E+05
4.00E+05
3.00E+05
2.00E+05
1.00E+05
0.00E+00
400
450
500
t0
t4
t6
t8
t10
t12
t16
t24
t30
t36
t48
t72
t96
Figure 5-4. Emission spectra of the Alexa350-conjugated tau-123 protein, at different
incubation times.
121
under oxidizing conditions at high salt and a variety of pH. Typically, these results show
that paired helical filaments form most easily as the tau gets trimmed away leaving only
the repeats (Schweers et al. 1995, Wille et al. 1992).
Most of the in vitro studies employing recombinant tau aim to understand the
kinetics of the polymerization reaction since much of this process remains still unclear.
One of the limitations encountered in these studies is represented by the scarce
availability of reliable methods to measure polymer formation. Many methods have been
used in the past years but all failed to provide the ultimate tool to determine the kinetics
of filament formation. Even the most widely used method, based on the ability of
molecules like thioflavins to increase their fluorescent emission upon binding to tau
polymers (Friedhoff et al. 1998, King et al. 1999, Schweers et al. 1995), present intrinsic
limitations. The mechanism by which the fluorophore thioflavin binds to the polymer is
not known; nor is known the stoichiometry of the reaction. Furthermore the increase of
fluorescence upon binding of the dye to the polymer is linear only up to 4-5 µM final
concentration of the protein (Friedhoff et al. 1998).
The study described in this chapter attempts to develop a new method to follow tau
filament formation in an effort to provide a valid alternative to thioflavin measurements.
The method is based on the creation of a fluorescently labeled tau where the
fluorophore is covalently attached to the N-terminus of the protein (Gaertner et al. 1992,
Geoghegan et al. 1993, Offord 1990). To obtain the covalent labeling the target protein
must have a serine residue as the first residue at the N-terminal end. This serine acts as a
α-amino alcohol and can be oxidized to an aldehyde by reaction with
sodium-m-periodate. Although periodate is considered a powerful oxidizing agent,
122
undesired oxidation of other residues can be easily avoided by running the reaction in a
controlled pH environment (Geoghegan and Stroh 1992). Furthermore the reaction of
periodate with amino-alcohol is 1000 times faster than with any other functional group in
the protein (Nicolet and Shinn 1939, Offord 1990). Oxidation of other residues can be
limited or avoided by limiting the exposure to periodate to a short time.
Once the labeled tau-123 was obtained, assembly reactions were conducted in the
presence of polyanions. The fluorescently labeled tau can still form filaments (Figure
5-3), but it tends to form large aggregates in the polymerization solution. A possible
explanation for this behavior resides in the hydrophobic nature of the fluorophore, which
in aqueous solution probably tends to aggregate dragging the protein in the process.
Although aggregation could represent an advantage by increasing the speed of polymer
formation, it makes more difficult to detect the filaments by electron microscopy. This
problem can be avoided by incubating labeled tau with equal amounts of unlabeled one,
thus obtaining better electron micrographs.
The tendency of the labeled protein to aggregate is most likely an initial
phenomenon during filament assembly since fluorescence data suggest that the probe is
then released from the protein as a result of the polymerization. Fluorescent emission
spectra of Alexa350 taken at different incubation times to follow the polymerization
process show increased fluorescence from time 0 to time 72. After 72 hours the
fluorescent emission reaches a plateau and this is expected since the maximum
polymerization time for tau and tau fragments in the presence of polyanions is considered
2 to 3 days. The maximum emission observed corresponds to the values measured for
123
Figure 5-5. Electron micrographs of the solution containing polymerized tau-123
conjugated with Alexa350. (a) EM field from polymerization solution, before
ultracentrifugation and (b) EM field from the solution supernatant after spinning.
Centrifugation clearly spins down the filaments and leaves undefined aggregates in the
supernatant.
124
5.00E+05
t0
t24 bef. spin
4.00E+05
t24 supernatant
3.00E+05
2.00E+05
1.00E+05
0.00E+00
400
450
500
Figure 5-6. Fluorescent measurements of solution containing polymerized tau-123
conjugated to Alexa350. The measurements were taken at time 0, after 24 hr incubation
in the presence of polyanions. A third measurement was taken of the supernatant after
centrifugation at 100,000 g for 1 hour.
125
1.2
(Ft – F0)/∆Fmax
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
Time (hours)
Figure 5-7. Release of fluorescent molecules from tau-123 upon its polymerization.
Tau-123 is conjugated with Alexa350 and the emission is measured at different times.
The emission are plotted as: (total fluorescence – fluorescence at time 0)/ maximum
fluorescence – fluorescence at time x).
126
equimolar amounts of the fluorophore alone. This result indicates that the increased
emission observed during polymerization could be caused by release of the dye from tau
as the protein aggregates into the filamentous structures. Fluorescent measurements of
the assembly solution before and after centrifugation verified this hypothesis (Figure 5-5
and 5-6). The increased fluorescence can be easily explained by the idea that the
fluorescent molecule when attached to tau is masked by the protein structure itself thus
decreasing its total emission. The reaction shows a first order behavior when the
fluorescent emission is plotted as (Ft – F0)/∆Fmax (Figure 5-7).
The reaction that generates the hydrazone is a reversible reaction due to the instability of
the carbazone double bond. Attempts to stabilize the hydrazone by reducing the double
bond with borohydrate produced great amounts of precipitation in the solution frustrating
any attempt to obtain a clear, stable conjugate. Furthermore, boric acid (a product of
borohydrate decomposition) tends to bind tightly to proteins. The originalgoal of
creating a stable fluorescently labeled tau to follow polymerization was only
incompletely achieved. Even so, this labeling technique promises to become a useful tool
to measure the kinetics of filament assembly, simply by determining the kinetics of the
fluorescent molecule release from tau upon its aggregation into filamentous structures.
CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTIONS
Alzheimer’s disease (AD) is the most frequent form of dementia, and the disorder
is linked to the accumulation of extracellular neuritic plaques and intracellular
neurofibrillary tangles (NFTs). This dissertation focused on the molecular mechanisms
underlying NFT assembly form the microtubule-associated protein tau. Tau pathology is
also a characteristic observation in several other neurodegenerative disorders, and
mutations in the tau gene are responsible for frontotemporal dementia and parkinsonism
linked to chromosome 17 (FTDP-17).
Tau promotes tubulin polymerization, stabilizes microtubules, and maintains
neuronal integrity, including axonal transport and axonal polarity. Tau protein is
abundant in both the central and peripheral nervous systems. In brain it is predominantly
found in neurons concentrated in axons. In adult human brain six tau isoforms are
produced from a single gene through alternative mRNA splicing (Andreadis et al. 1992).
In the C-terminal part of tau, three or four tandem imperfect repeats are present,
containing domains important for binding to microtubules. The alternatively spliced
exon 10 encodes the additional fourth repeat. Alternative splicing of tau is
developmentally regulated as in immature brain only the transcript encoding the shortest
isoform with three repeats is expressed but in adult cerebral cortex all six isoforms are
present (Goedert et al. 1989, Kosik et al. 1989). Tau is posttranslationally modified by
phosphorylation in a dynamic process, and it has been suggested that this is an additional
mechanism to regulate tau function (Goedert et al. 1991).
127
128
The mechanism by which tau protein stops acting as a microtubule-stabilizing
factor and starts building neurofibrillary structures is still largely unknown.
Understanding the biochemistry of tau polymerization may foster the development of
new therapeutic approaches to inhibit the formation and accumulation of tau polymers
inside the affected cells. Of all the studies involving tau and its role in neurodegenerative
diseases, many were conducted in vitro using tau constructs and fragments. In particular,
many studies involved solely the C-terminal part of the protein containing the
microtubule-binding region, as this has been demonstrated to be the core component of
PHFs (Goedert et al. 1988, Wischik et al. 1988). Previous work from this laboratory
demonstrated that a small stretch of residues in the microtubule-binding region is
paramount to the morphology of the filaments formed in tau. MAP2, which forms only
straight filaments in vitro and has never been found in the neurofibrillary tangles, could
form paired helical filaments when a small sequence from tau was substituted to the
corresponding one in MAP2. Many MAP2 mutants containing different tau residues
alternatively substituted were generated. The studies carried on some of those mutants
led us to believe that the information concerning the ability of tau to form aberrant
filaments and the morphology of the filaments formed is indeed stored in the sequence of
the protein itself or more precisely in the distribution of charges in specific areas of the
microtubule binding region (DeTure 1998).
The first part of this study dealt with a characterization of MAP2 mutant
polypeptides previously generated by our laboratory. To better understand the
importance of the residues contained in the stretches of residues named Module-A and
Module-B, polymerization data from the MAP2 mutants resembling tau behavior in vitro
129
were analyzed to quantify and to characterize the morphology and frequency of the
different polymers formed. Two of the single-point MAP2-123 mutants showed the most
interesting results (MAP2-123-MBP3 and MAP2-123-MBP4) as only two of the charged
residues inside MAP2-123 [Module-B] play a role in determining the morphology of
filaments assembled and they produce paired helical filaments in amounts and
morphology comparable to the main mutant MAP2-123[Module-B], a finding partially
overlooked in the original work. To further assess the importance of the residues
contained in Module-B and the role they play in determining the morphology of tau and
MAP2 polymeric filaments, other point mutations changing the distribution of charges in
the Module-B were generated. Furthermore, the first part of this work aimed to address
the significance of the Modules by generating tau mutants that have the Module-A and B
motifs switched to the correspondent ones of MAP2. Although the tau mutants did not
produce novel findings on the mechanisms of polymerization, they yielded interesting
new hints about the possible role of some of the residues lying outside Module-A and B.
It is evident from my results that the distribution of charges in the amino acid sequence
may be necessary, but insufficient, for the polymerization of tau. The mutant
tau-123[Module-A] failed to produce comparable results with the MAP2-123[Module-B]
despite the fact that they share high sequence identity (see Figure 2-4). The partial
recovery in terms of amount of aggregates produced, obtain with
tau-123[Module-A]G326K in which another tau residue outside of Modules was
substituted with the correspondent MAP2 one, indicates that other residues outside of
modules may also be involved in the self assembly of tau.
130
Another important aspect of tau involvement in neurodegenerative diseases is
connected to the dimerization of the protein as a nucleating step in the formation of
abnormal fibrillary polymers. A pathological feature accompanying AD is oxidative
stress. Several studies demonstrated that amyloid plaques formation is associated with
oxidative stress in areas of the brain affected by AD. Products of oxidation like
glycoxidation adduction to and direct oxidation of amino acid side chains have been
demonstrated in the lesions and neurons of AD (Mecocci et al. 1994, Palmer and Burns
1994, Sayre et al. 1997, Smith et al. 1996). Furthermore A-beta peptides may generate
oxidative stress in the extracellular environment surrounding the affected neuronal cells,
as they have been shown to directly produce hydrogen peroxide through metal ion
reduction (Huang et al. 1999). Oxidation stress within affected cells could explain the
dimerization of tau through disulfide bridge formation, a key nucleating step in the
filaments formation (Schweers et al. 1995). It has been hypothesized that dimerization of
tau and MAP2 following oxidative stress of the cell could impair the ability of these
proteins to interact with microtubule, thereby blocking their stabilizing effect. To better
understand the significance of the dimer formation I analyzed the ability of tau and
MAP2 dimers to interact with microtubules and to promote tubulin assembly and
compared it with the monomers interaction. The results clearly show that tau
homodimers readily promote microtubule assembly and maintain an affinity for
microtubules that is comparable to the affinity of monomeric tau.
Tau homodimers are generated through formation of a covalent disulfide bridge
between cysteine residues of two separated proteins. There are only two cysteines in
each of the four-repeat isoforms and one cysteine in the three-repeat isoforms. Because
131
of this difference, three-repeat forms are more easily oxidized into a dimer, since
four-repeat isoforms tend to form more intermolecular disulfide bonds. The difference in
number of cysteine residues between three- and four-repeat tau isoforms suggests a
different role for these residues rather than a structural one (i.e., involvement in tertiary
structures formation). To better understand the significance of the cysteine located in the
second repeat of the microtubule-binding region and its role, the redox properties of the
three-repeat forms of tau was investigated in this work. The reactivity of three-repeat tau
MTBRs versus different reducing agents, in particular glutathione and dithiothreitol was
analyzed. Glutathione is the most abundant thiol in the cell. It is particularly abundant in
neuronal cells where it can reach concentrations around 3-4 mM. It is the most important
cytosolic reducing agent and its role in maintaining the cell redox state is very well
known. In many neurodegenerative diseases, decreased levels of intracellular reduced
glutathione accompany oxidative stress and glutathione depletion has been linked to
Parkinson and other neurodegenerative diseases (Bains and Shaw 1997, Owen et al.
1996, Schulz et al. 2000). If glutathione, among other functions, was to be involved in
the maintenance of the reduced state of tau, its depletion during oxidative stress could
drive some of tau molecules to form homodimers thus starting the nucleation step
involved in the polymerization process. The ability of tau to interact with glutathione or
dithiothreitol was analyzed in this work to define the redox properties of the cysteine in
the second repeat of tau. The results show high reactivity of tau for glutathione, even
when compared with dithiothreitol, one of the most efficient synthetic reducing agents
available. It is possible that the cysteine inside the repeats of tau have been designed to
132
interact specifically with glutathione allowing this thiol to exert a protective action from
oxidative stress and formation of tau-tau homodimers.
One of the main problems encountered by researchers in studying tau
polymerization in vitro has been the lack of a reliable quantitative method for measuring
filament assembly. Techniques such as sedimentation assays (Arrasate et al. 1999,
Schneider et al. 1999), qualitative electron microscopy (Goedert et al. 1996, Hasegawa et
al. 1997) and quantitative electron microscopy (King et al. 1999, Wilson and Binder
1997) have been utilized for this purpose. Alternative methods involve the
spectrophotometric measurement of turbidity of the protein solution after polymerization
is induced (Gaskin et al. 1974) as well as 90° light scattering (Mukherjee and Lutkenhaus
1999). The method most frequently used and widely accepted as a standard tool for
polymerization measurement involves the use of dyes that fluoresce upon binding to the
polymeric structure. The most commonly used dyes are thioflavin-S and thioflavin-T
which exhibit a characteristic fluorescence at 480 to 490 nm when bound to Alzheimer
paired helical filaments and filaments formed in vitro from recombinant tau fragments
(Friedhoff et al. 1998, Schweers et al. 1995). Although commonly used to measure
polymerization in vitro, the method presents some limitations. The mechanism by which
the fluorophore thioflavin binds to the polymer is not known; nor is known the
stoichiometry of the reaction. Furthermore the increase of fluorescence upon binding of
the dye to the polymer is linear only up to 4-5 µM final concentration of the protein
(Friedhoff et al. 1998).
The final part of this research dealt with the development of an alternative method
to measure polymerization of tau in vitro using a fluorescently labeled tau generated
133
through the covalent binding of a fluorescent molecule to the N-terminal residue of
tau-123 fragment. Although this method requires further development, the initial results
were encouraging as they clearly suggest that this method could represent a valid
alternative to follow polymerization kinetics of tau in vitro.
All of this research resulted in a better understanding of the biochemical
mechanisms behind tau complex polymerization reaction, which ultimately generates the
pathological neurofibrillary tangles while at the same time provided some new insights
on the redox properties of this protein. Future research developments could follow
several different directions. An optimization of the fluorescent method to measure the
polymerization could ultimately provide an invaluable tool for in vitro studies of this
process. The same principle could be than applied to every polymerization study. The
fluorescently labeled protein of interest could be studied employing more accurate
measurement methods such as Near-Field Optical Microscopy (NFOM), in conjunction
with the labeling method I used for this research. By adjusting concentration of the
fluorescently labeled protein to sub-micromolar amounts and by controlling the
conjugation of the sample to the NFOM probe it should be possible, in principle to follow
the polymerization reaction with extreme accuracy.
The relation between tau and glutathione should also be further investigated. It
should be fairly easy to confirm if indeed tau sequence around cysteine has been designed
to facilitate the protein interaction with glutathione. Mutants with alanine substituted to
the lysine residue flanking the cysteine in tau can be made and reacted with glutathione.
According to the idea of a lysine with docking function for glutathione, the
lysine-to-alanine mutant should show less reactivity than the wild-type tau. Furthermore,
134
analogs of glutathione without the extra carboxyl group and thus less acidic should react
less readily with cysteine of tau than the regular glutathione molecule. One such a
molecule is an ester analog of glutathione where an ester is conjugated to the carboxyl
group of the γ-glutamyl residue.
Finally, more studies should be done on the residues in Module-B with particular
reference to their possible relation with naturally occurring mutations linked to
frontotemporal dementias, especially the two mutations (V337M and E342V) that occur
inside the Module-B sequence (Lippa et al. 2000, Spillantini et al. 1998).
APPENDIX
ELECTRON MICROSCOPY
The electron microscope is an extremely valuable tool for the study of protein
structures. Instrumentation has markedly improved in the past 20 years, and sample
preparation methods have been developed and optimized to use electron microscopy for
studies of protein assemblies and other macromolecules. Scanning Electron Microscopy
(SEM) and Transmission Electron Microscopy (TEM) are the two different microscopy
modes developed as the mainstream investigative tools in biology and biochemistry. For
the purpose of this dissertation a brief description of the Transmission Electron
Microscopy will be presented.
Transmission Electron Microscopy
In the normal transmission mode, an electron beam is emitted from a source and
accelerated toward an anode at ground potential. The source of emission is a tungsten
filament made of various types of materials. During passage through the specimen,
electrons are deflected from their path by electrostatic interactions primarily with the
nuclei of the atoms where positive charge is concentrated. A condenser lens with an
aperture system to control the angular width of the beam is used to guide the beam onto
the specimen. Two types of lenses are used in electron microscopy, magnetic (most
common) or electrostatic. In these types of lenses, incoming electrons are focused by
interaction with a magnetic field or electrostatic potential distribution along the axis. The
specimen is followed by an objective lens that serves to magnify the image about
100-fold. To prevent large numbers of scattered electrons from blurring the image
135
136
produced by the transmitted electrons, an aperture is placed after the objective lens, thus
improving contrast of the image. Finally, one or two projector lenses are used to produce
an image magnified about 1000 to 200,000 x on a photographic plate or fluorescent
screen. The common limitations of this instrument are the same as in other microscopes.
Image quality is limited by spherical and chromatic aberrations and by diffraction effects
as well as variations in the field strength of the lenses, effect of the electron beam on the
sample, and scattering from contamination.
Resolution and Contrast.
Electron microscopes capable of 10 Å resolution have been available for nearly 60
years now. Many microscopes that are commercially available can achieve point-to-point
resolution of 3 Å or less on ideal specimens. Even when the instrument is in top
operating condition, resolution will be limited to the degree to which the operator can
correct astigmatism and achieve focus. In light microscopy, lack of proper focus results
in a fuzzy image that, although it may be unsatisfactory in resolving details, is unlikely to
be misinterpreted. Defocused electron micrographs, on the other hand, are often
interpreted erroneously. If the objective-lens field is asymmetrical the image will be
focused at different levels with greatest difference in two mutually perpendicular
directions. Such an image is said to be “astigmatic”. Contour phenomena are produced
by Fresnel diffraction that occurs when the electron beam encounters abrupt
discontinuities in refractive index. These discontinuities also exist at the edge of certain
particles to produce contrast effects that are superimposed on the subject image.
Another complicating phenomenon is phase contrast, which gives rise to an
apparent background structure even in regions of constant scattering power containing no
137
projecting edges. The apparent granularity increases with defocusing and at focus
becomes comparable in size to the resolution limit of the instrument.
Combinations of phase contrast and contour phenomena at images of negatively
stained molecules result in spurious contrast, which may be incorrectly interpreted as a
subunit structure in a protein molecule.
With proper care it is possible to achieve 5 to 10 Å resolution routinely in electron
microscopy; however the problem of contrast remains. Although macromolecules can be
visualized directly in the electron microscope when placed on an appropriately thin
support, it has not generally been possible to visualize a difference in the scattering of
electrons passing through different regions of an enzyme, as would be required to “see”
subunit detail. To overcome this problem contrast can be enhanced artificially to produce
sufficient intensity difference between portions of the molecule thus allowing
visualization of the subunit structure.
Shadow Casting.
The specimen to be shadowed is prepared by spraying or placing a drop of the
sample solution on the electron microscope grid (a fine mesh copper screen) that has been
previously coated with a support film of collodion, Formvar, or carbon. After
evaporation of the solvent, the grid is placed in a vacuum evaporator, and metal atoms
evaporated from a hot filament are caused to impinge on the molecules of the sample at a
suitable angle. Metal deposit thus build up on surfaces directly exposed to the filaments
and are completely absent on the protected side of the molecules. When examined in the
electron microscope, the shadowed specimen scatters electrons in regions where metal
deposits have formed. The protected areas cause little scattering and the resulting
138
transmitted beam produces a darkening of the photographic plate in these regions. This
appears as a lighter area on the screen and as a shadow on the plate.
Positive Staining.
Biological macromolecules contain many charged groups that will bind ions of
opposite charge. Certain ions containing heavy metals with high electron scattering
power as useful as electron stains; the binding of such stains to particular sites of the
molecules of the sample is termed positive staining. In order to achieve sufficient
contrast, many heavy metals must be located within a given volume element. The
method is particularly successful in the case of molecular aggregates in which the
molecules are ordered in such a way that strong stain-binding regions adjoin and bands of
stain are observed. The results obtained on various collagen aggregates are examples of
the application of this method for the solution of molecular problems. Positive staining
of specimens such as these is analogous to the classic staining procedures of thin
sections.
Negative Staining.
The visualization of protein subunit structure by electron microscopy has most
frequently and successfully been accomplished by employing some modification of
procedures collectively termed negative staining. The preparative procedures are
relatively easy and they can yield to excellent results in protein studies. An appropriate
concentration of sample is mixed with phosphotungstic acid or uranyl acetate. The
mixture is applied to the thin carbon film supported on a copper grid, and most of the
solution is withdrawn by touching the edge with filter paper to leave a thin liquid film
behind. As this film dries, the staining substance solidifies as a glassy, almost
structureless, electron dense layer. Surface tension forces at the surface of a protein
139
molecule usually cause preferential buildup of the stain around the protein, and stain
enters the crevices between protein subunits. Alternatively in thicker areas of stain, the
solid protein effectively prohibits the formation of an electron dense layer in the areas of
its molecular domain. Scattering of the electron beam in the vicinity of the protein
domain is determined by the relative amount of stain and protein. The solidification of
the stain appears to precede the total dehydration of the protein, thereby affording the
additional advantage of apparent structural preservation for molecules that are markedly
affected by direct drying. The most common molecules used for negative staining of
proteins are phosphotungstate, uranyl acetate and uranyl formate. The uranyl molecules
are smaller than phosphotungstic acid in size and may penetrate into narrow crevices to
outline subunit detail not seen with phosphotungstic acid. Negative staining may be used
to advantage for the observation of general molecular shape in molecules and molecular
aggregates. More recently, it has become a valuable tool for the determination of subunit
stoichiometry and the probable symmetry of oligomeric proteins. Determination of
structural models from electron micrographs of macromolecules visualized by negative
staining must be done with caution. Negative staining is the elective choice for the
detection and structural investigation of tau filaments.
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BIOGRAPHICAL SKETCH
Luca Di Noto was born in Ancona, Italy on November 30, 1963. He grew up with
his parents and brother, in Falconara Marittima, Italy and graduated from Liceo
Scientifico Livio Cambi in 1982. He majored in Biological Sciences at the University of
Urbino, Italy.
Upon receiving his bachelor’s degree from University of Urbino in 1993, Luca
worked at the University of Ancona, Italy, in the Institute of Biology and Genetics where
he worked on Glutathione and the Glyoxalases system under the supervision of Dr.
Giovanni Principato, Ph.D. In 1995 Luca was awarded a research fellowship at the
Institute of Experimental Animal Sciences of Umbria and Marche in Ancona, Italy. In
1996, he began his doctoral work under the supervision of Dr.Daniel L. Purich, Ph.D. in
the department of Biochemistry and Molecular Biology at the University of Florida.
Upon receiving his Ph.D., Luca will begin his postdoctoral work at the National
Heart, Lung, and Blood Institute in Bethesda under the supervision of Dr. Rodney L.
Levine, M.D., Ph.D.
162