PHOSPHORYLATION AND SEQUENCE DEPENDENCY
OF NEUROFILAMENT PROTEIN OXIDATIVE
MODIFICATION IN ALZHEIMER DISEASE
By
QUAN LIU
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Adviser: Dr. George Perry
Department of Pathology
CASE WESTERN RESERVE UNIVERSITY
January, 2005 CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
______
candidate for the Ph.D. degree *.
(signed)______(chair of the committee)
______
______
______
______
______
(date) ______
*We also certify that written approval has been obtained for any proprietary material contained therein.
I grant to Case Western Reserve University the right to use this work, irrespective of any copyright, for the University’s own purpose without cost to the University or to its students, agents and employees. I further agree that the
University may reproduce and provide single copies of the work, in any format other than in or from microforms, to the public for cost of reproduction.
Quan Liu
(sign)
iii
DEDICATION
To my parents, my lovely fiancée, and family members
Be proud of me for having double “D”s and partially it is yours.
iv TABLE OF CONTENTS
Title Page i
Signature Sheet ii
Copyright wavier. iii
Dedication. iv
Table of Contents. 1
List of Tables 5
List of Figures 6
Acknowledgements 9
List of Abbreviations 11
Abstract 15
Chapter 1 General Introduction 16
1.1 Introduction of Alzheimer Disease 17
1.1.1 Clinical 17
1.1.2 Pathology 17
1.1.3 Etiology. 18
1.1.4 Pathogenesis and Hypothesis models. 19
1.1.5 Research Relevance: Oxidative Stress and AD Pathogenesis. 24
1.2 Introduction of Neurofilament Proteins 27
1.2.1 General Information 27
1.2.2 History 28
1.2.3 Structure 28
1.2.4 Expression. 30
1 1.2.5 Assembly 32
1.2.6 Transport 34
1.2.7 Posttranslational Modifications 35
1.2.8 Degradation. 37
1.2.9 Functions. 38
1.2.10 Animal Models.. 38
1.2.11 Neurofilaments and Neurodegenerative Diseases. 45
1.2.12 Summary. 52
1.3 Introduction of Tau Protein 54
1.3.1 General Information 54
1.3.2 History. 54
1.3.3 Tau gene and Tau Expression. 55
1.3.4 Tau Protein Structure 56
1.3.5 Posttranslational Modifications of Tau. 56
1.3.6 Tau Degradation 58
1.3.7 Tau Functions. 59
1.3.8 Tau Polymerization. 60
1.3.9 Animal Models 61
1.3.10 Tau and Neurodegenerative Diseases (Tauopathies) 64
1.3.11 Summary. 69
1.4 Introduction of Oxidative Modification. 70
1.4.1 Free radical Theory of Aging 70
1.4.2 Free Radicals. 70
2 1.4.3 The Primary Sources of Free Radicals 71
1.4.4 Physiological Damages Caused by Free Radicals 73
1.4.5 Antioxidant Systems. 73
1.4.6 Oxidative Stress in The Nervous System 76
1.4.7 HNE and HNE Modification in AD 80
1.4.8 Summary. 80
1.5 Research Relevance 83
Chapter 2 Oxidative Modification of Neurofilament Proteins 84
2.1 Introduction 85
2.2 Materials and Methods. 94
2.3 Results 99
2.4 Discussion 111
Chapter 3 Cellular Protective Function of Neurofilament Heavy Subunit 121
3.1 Introduction 122
3.2 Materials and Methods. 128
3.3 Results 132
3.4 Discussion 140
Chapter 4 Oxidative Modification of Tau Protein Contribute to NFT
Formation. 144
4.1 Introduction 145
4.2 Materials and Methods. 155
4.3 Results 159
4.4 Discussion 165
3 Chapter 5 Conclusion and Future Work. 169
References. 176
Appendix Publications, Abstracts and Conferences 235
4 List of Tables
Table 1.1 Comparison of the structures of neurofilaments
and other type IV filaments 29
Table 1.2 The summary and comparison of NF subunits
knock-out and overexpression mouse models 42
Table 2.1 Comparison of all the proteins and peptides in
these studies. 116
Table 2.2 Protein database search for KSP repeats. 117
Table 2.3 All the control peptides used in these studies
did not show high level of HNE adduct. 118
5 List of Figures
Figure 1.1 Oxidative stress is a prominent central event
in AD pathogenesis. 25
Figure 1.2 Comparison of the structures of neurofilam-
ents and other type IV filaments. 31
Figure 1.3 Schematic model of neurofilament assembly 33
Figure 1.4 Regulatory functions of NF phosphorylation 36
Figure 1.5 Hypothetical relationships among neurofil-
ament proteins, oxidative stress, cell injury
and tissue damage 39
Figure 1.6 The structure of HNE and its adducts with amino acids. 81
Figure 2.1 Phosphorylation state of NFH regulates
level of HNE-NFH adduct 100
Figure 2.2 Tau protein did not show the similar effect. 101
Figure 2.3 C-terminus of NFH is very reactive with HNE. 102
Figure 2.4 Nonphospho-20 AA KSP peptides are not
able to be intensively modified by HNE. 104
Figure 2.5 Phospho-AKSPV peptide is modified by HNE. 105
Figure 2.6 Synthetic K-S-P peptides with and without
phosphation show different reactivity with
HNE 106
Figure 2.7 Synthetic K-S peptides with and without
phosphorylation did not show significant
6 difference in the formation of HNE adduct. 108
Figure 2.8 HPLC-ESI-MS/MS confirms the removal
of HNE from the HNE adduct after depho-
sphorylation of the sample 109
Figure 2.9 Hypothetical model for the phosphorylation-
regulated HNE modification in KSP motif. 121
Figure 3.1 HNE treatment induced higher level of
NFH in M17 cells 133
Figure 3.2 HNE treatment induced higher phosphoryl-
ation level of NFH. 134
Figure 3.3 MAPK were activated in M17 cells after
HNE treatment 135
Figure 3.4 M17 differentiated cells with higher level of
NFH showed significant protection form HNE
cytotoxicity with LDH cytotoxicity assay 137
Figure 3.5 M17 differentiated cells with higher level of
NFH showed significant protection form HNE
cytotoxicity with LDH cytotoxicity assay 138
Figure 3.6 N2A cells showed significant protection with
overexpressing NFH using trypan blue staining. 139
Figure 4.1 Summary of the specificity of seven NFT antibodies. 161
Figure 4.2 Recognition of various τ forms with and without
dephosphorylation by antibodies to NFT. 162
7 Figure 4.3 Enhancement (fold increase) of antibody
recognition by HNE modification 163
Figure 4.4 Immunocytochemistry on adjacent serial
sections with the antibodies to NFT. 164
8 ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my dissertation advisor, Professor
George Perry, for his never-ending guidance and support during my Ph.D. research. He has continuously provided me with enthusiasm, vision, and wisdom, and inspired me from the beginning to the end. He also created an environment that gave me the flexibility to explore new ideas, while helping me to make critical decisions whenever the project was at a crossroad. He is an outstanding researcher to have as a role model for a
Ph.D. student.
I also want to thank my committee chair Professor Mark A. Smith, who provided knowledge and skills for me anytime during my Ph.D. studies, and motivated me to improve my basic knowledge in all aspects. It is he that really made me to write scientific papers and accomplish all the things in my Ph.D. training process.
I have also had the good fortune to work with Professor Lawrence M. Sayre, who has helped me in every aspect of the thesis, which is deeply appreciated. I admire him for his broad knowledge, creative thinking, and deep insight in experimental design and ability for solving problems. Discussions with Professor Sayre directly led to the results of the neurofilament studies in chapter 2.
I also thank Professor Shu G. Chen, who really helped me with the equipment and in producing good data. He also directly helped me with the methods and experimental design, which put me on the right track so many times. He has always been supportive for my research work and contributed extensively to my Ph.D. research.
9 It has been a privilege to work together with these intelligent and friendly people in my laboratory and co-laboratories. They have given me all the support from time to time. Here I send my gratitude to Sandra L. Siedlak, Peggy L.R. Harris, Beth Kumar,
Xiongwei Zhu, Kazuhiro Honda, Paula I. Moreira, Dandan Wang, De Lin, David R. Sell, and Dr. Ricardo B. Maccioni, Dr. Jesus Avila, Dr. Mervyn J. Monteiro, Dr. Don W.
Cleveland, Dr. Robert Salomon, Dr. Vincent Monnier, Dr. Michael Kinter, Dr. Touradj
Solouki, Dr. Michael Strong, and so many other people that have already contributed to my Ph. D. research work. I really had a good time with them and had been honored to have all these colleagues to work with.
Last, I would like to thank my family. All the family members are so supportive for my studies, my personal life and my future plans, in spite of they are thousand miles away or nearby. They are the best family members I know in my life. Surely they are worthy of my most sincerely appreciation.
This work has been supported by the NIH and Alzheimer Association. Their assistance is gratefully acknowledged.
10 List of Abbreviations
Aβ amyloid-β protein
AβPP amyloid-β precursor protein
AD Alzheimer’s disease
ALE advanced lipoxidation end-product
ALS Amyotrophic Lateral Sclerosis
AP alkaline phosphatase
AR alkoxyl radical
BME. β-mercaptoethanol
CaM. calmodulin MARK kinases
Cdk5. Cyclin-dependent kinase 5
CNBr cyanogen bromide
CNS central nervous system
CMT. Charcot-Marie-Tooth Disease
CSF. cerebrospinal fluid
CWRU Case Western Reserve University
DNPH. 2,4-dinitrophenylhydrazine
ECL enhanced chemiluminescence
ERK1/2DAB 3,3’-diaminobenzidine
FRTA free radical theory of aging
FTDP-17 Frontotemporal Dementia with Parkinsonism
Linked to Chromosome 17
GSH glutathione
11 GSK3. glycogen synthase kinase 3
HF hydrofluoric acid
HNE. 4-hydroxy-2-nonenal
H2O2 hydrogen peroxide
HOCl hypochlorous acid
HPLC-ESI-MS/MS high performance liquid chromatography-electrospray
ionization-mass spectrometry/mass spectrometry
IF intermediate filament
JNK Jun N-terminal kinase
KSP Lysine-Serine-Proline
LDH. lactate dehydrogenase
MALDI Matrix-assisted Laser Desorption/Ionization mass
spectrometry
MAPs. microtubule-associated proteins
MAPK Mitogen-Activated Protein Kinase
MF microfilament
MT microtubule
NF-L. neurofilament proteins light subunit
NF-M neurofilament proteins medium subunit
NF-H. neurofilament proteins heavy subunit
NEFL neurofilament proteins light subunit gene
NEFM. neurofilament proteins medium subunit gene
NEFH. neurofilament proteins heavy subunit gene
12 NFPs. neurofilament proteins
NFTs. neurofibrillary tangles
NOR. nitric oxide radical
O-GlcNAc O-linked N-acetylglucosamine
PBS phosphate-buffered saline
PD Parkinson's Disease
PHF paired helical filaments
PKA protein kinase A
PKC protein kinase C
PLC phospholipase C
PNS peripheral nervous system
PP2A protein phosphatase 2A
PR. peroxyl radical
PSP. Progressive supranuclear palsy
RA retinoic acid
ROS Reactive oxygen species sGAGs sulfoglycosaminoglycans
SDS-PAGE. Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis
SOD superoxide dismutases
SCZ Schizophrenia
TBS Tris-buffered saline
TCA trichloroacetic acid
13 TEM. transmission electron microscopy
TMAO trimethylamine N-oxide
UMLS Unified Medical Language System
14 Phosphorylation and Sequence Dependency of Neurofilament Protein Oxidative
Modification in Alzheimer disease
Abstract
By
QUAN LIU
Protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) are prominent features of oxidative damage in neuronal cell bodies in Alzheimer disease
(AD). Such adducts are also seen in axons of normal as well as diseased individuals.
These HNE adducts are constant throughout the axons and the aging process of humans, mice, and rats, indicating that HNE adduction is physiological and regulated from birth to senility. In axons, HNE is primarily adducted to the neurofilament heavy subunit (NFH) and the neurofilament medium subunit (NFM), and limited to lysine residues.
Interestingly, we found that phosphorylation is essential since formation and removal of
HNE adducts are controlled by the NFH/M phosphorylation state. Studies using immunochemistry, synthetic peptides, mass spectrometry and chemical stabilization of
HNE adducts, demonstrated that XKSPX, the most repeated sequences in NFH/M, are the major component in neurons highly susceptible to phosphorylation regulated aldehyde adduction. To our knowledge, this is the first study to directly show phosphorylation can regulate NFH-HNE levels in axons and the multiple KSP repeats are the critical motifs which preserve the phosphorylation-dependent regulation. This study provides the new evidence that indicates signal transduction could modulate oxidative modification in brain through activation of kinases and phosphatases.
15
Chapter 1
General Introduction
16 1.1 Introduction of Alzheimer Disease
1.1.1 General Information
Alzheimer’s disease (AD) was first reported by Dr. Alois Alzheimer, a Germany
doctor, during the 1907 neurology conference, in which he described a 51-year-old
woman developing rapid memory degeneration and died with severe dementia several
years later (Alzheimer, 1907; Alzheimer et al., 1995). Although the disease was once
considered rare, it is now established as the leading cause of dementia. An estimated 4
million individuals in the United States have Alzheimer’s disease with an annual expense
of about $70-$100 billion. Also as many as 350,000 more individuals develop the disease
each year (Mayeux et al., 2003). There is still no cure for AD, and the only therapy
known as cholinesterase inhibitors or anti-cholinesterase drugs including Reminyl
(galantamine), Aricept (donepezil hydrochloride) and Exelon (rivastigmine) act to slow
the disease progression (Alzheimer’s Association, 2004). In October 2003, Namenda
(Memantine), a new drug as a NMDA receptor antagonist, was approved by FDA to be
used on moderate to severe Alzheimer patients (Alzheimer’s Association, 2004).
Alzheimer’s disease involves the parts of the brain that control thought, memory,
and language. After last two decades of intensive study, more information has been
obtained, but the cause of AD is still unknown. There are several major theories such as beta amyloid toxicity (Selkoe, 2000), tauopathy (Lee et al., 2001), inflammation (McGeer et al., 2001; Weiner et al., 2002), oxidative stress (Markesbery, 1997; Christen, 2000;
Picklo et al., 2002, Perry et al., 2002), all of which have been discussed in the literature
intensively.
1.1.2 Clinical
17 The risk of Alzheimer’s disease varies from 12% to 19% for women over the age
of 65 years and 6% to 10% for men (Seshadri et al., 1997). On average, AD patients live
for about 8 years after they are diagnosed, although the disease can last for as many as 20
years. The areas of the brain that control memory and thinking skills are affected first, but as the disease progresses, cells die in other regions of the brain. Eventually, the person with AD will need complete care. If the individual has no other serious illness, the loss of brain function itself will cause death.
1.1.3 Pathology
The hallmarks of Alzheimer’s disease are two distinctive lesions in the patient’s brain, senile plaques and neurofibrillary tangles (NFTs) (Alzheimer et al., 1995). They were identified by silver staining of the AD patient’s brains. In addition, other pathological changes are detected in the AD patient’s brains, such as neuronal and dendritic loss, neurotrophic threads, dystrophic neuritis, granulovacuolar degeneration,
Hirano bodies, cerebrovascular amyloid, and atrophy of the brain (Smith, 1998).
Senile plaques are described originally as “millary foci” (Alzheimer et al., 1995).
They are spherical extracellular lesions with 10-200 µm in diameter with a central core made up by bundles of 6-10 nm amyloid-β protein (Aβ) (Smith, 1998). In the peripheral region of the senile plaques, Aβ protein, amyloid-β precursor protein (AβPP), τ, and neurofilament proteins are reported (Perry et al., 1988).
NFTs are a major intracellular protein aggregation found in AD brains. They are located primarily in the cerebral cortex, especially in the large pyramidal neurons in the hippocampal and frontotemporal regions (Smith, 1998). NFTs are composed of bundles
of paired helical filaments (PHF), the major component of which is the
18 microtubule-associated protein τ (Grundke-Iqbal and Iqbal, 1989; Lee et al., 1991).
Moreover, neurofilament proteins are also reported (Perry et al., 1988). In PHF, τ is abnormally hyperphosphorylated (Grundke-Iqbal et al., 1986; Trojanowski and Lee,
1995; Lee et al., 2001), ubiquitinated (Iqbal and Grundke-Iqbal, 1997; Mori et al., 1987;
Perry et al., 1987), oxidized (Mattson, 1995; Takeda et al., 2000), proteolytically processed and aggregated into filaments (Lee et al., 2001; Lovestone and McLoughlin,
2002; Perez et al., 2002). Hyperphosphorylation of τ renders it unable to bind to microtubules and therefore unable to promote or maintain microtubule assembly (Baudier and Cole, 1987). Moreover, hyperphosphorylation makes τ more resistant to proteolytic degradation, which may play a key role in neurofibrillary degeneration in AD patients
(Iqbal et al., 1998; Eidenmuller et al., 2000).
1.1.4 Etiology
A Age
Age is the single greatest risk factor in the etiology of AD. AD rarely occurs in people younger than 60 years old, and affects 10-15% of individuals over 65 years old and up to 47% of individuals over age of 80 (Evans et al., 1989). This predominance of age as a major cause in AD etiology indicates that some age-related events are closely involved in the development of the disease. However, the specific aging process related to AD pathogenesis is unknown.
B Oxidative Stress (OS)
As one of the primary theories of aging, free radicals (Oxidative stress) may play a major role in the AD pathogenesis. Oxidative stress is a potential source of damage to
DNA, lipids, sugars and proteins within cells, imbalance between the intracellular
19 production of free radical species and antioxidant defense mechanisms results in oxidative stress (Perry et al., 2002). In a post-mitotic environment such as neurons, oxidative stress can be treated as a marker of age-related deterioration in cellular homeostatic mechanisms. Since the neurons have a diminished capacity to deal with redox imbalance, even minor cellular stresses have the ability to lead to irreversible injury and such contribute to the pathogenesis of neurodegenerative disease. Reactive oxygen species (ROS), which are the primary mediators of oxidative injury, cause damage to lipids, sugars and amino acid side-chains (Picklo et al., 2002). For example, oxidative deamination of lysine and deguanidination of arginine results in protein-based aldehyde groups that can be detected by 2,4-dinitrophenylhydrazine (DNPH) (Levine et al., 1994). However, it appears that most DNPH-detectable carbonyls found on proteins are resulted from modification by lipid and sugar oxidation products, which act as secondary toxins. Such toxic carbonyl species have been shown to play a role in the pathophysiology of AD (Smith et al., 1994a; Yan et al., 1994; Sayre et al., 1997;
Castellani et al., 2001).
C Genetics
Although familial AD only accounts for a small percentage of AD cases and the majority of the AD cases are spontaneous. There is a strong evidence related with the genetic component in AD occurrence. Up to 35% of the AD patients have affected first degree relatives (Breitner and Folstein, 1984). Now the greatest correlated genetic factor is the polymorphism of ApoE gene, which may correlate with 50% of the AD patients especially for ApoE4 alleles, but the mechanisms are unknown (Ashford, 2004; Teter,
20 2004). Mutations in beta amyloid protein and presenilin 1 and 2 genes are considered as critical factors in the AD pathogenesis.
ApoE genes
ApoE genes are found to be related with AD occurrence. ApoE is an abundant
34-kDa glycoprotein that is synthesized and secreted mainly by astrocytes and microglia in the central nervous system (CNS). It is well established that ApoE, specifically the E4 allele of ApoE, is a major genetic risk factor for the more common, late-onset form of
AD (Saunders et al., 1993; Rebeck et al., 1993). ApoE genotype also seems to be a determinant of brain Aβ burden in individuals affected with AD (Strittmatter et al., 1993;
Schmechel et al., 1993). Now the polymorphism of ApoE gene is considered the greatest correlated genetic factor in AD pathogenesis (Ashford, 2004; Teter, 2004).
Amyloid β precursor protein
Aβ protein is the major component of senile plaque cores and it is derived from a precursor protein called amyloid β precursor protein (AβPP). Amyloid β precursor protein gene is located on chromosome 21 (21q11-22) (St George-Hyslop et al., 1987;
Tanzi et al., 1987). The normal function of AβPP is unknown, but it is shown to be involved in several or more broad physiological functions in neurons. The mutations in
AβPP results in a great increase of the Aβ protein production, which may cause the extracellular protein aggregation (Selkoe 1997, 2001). The AβPP mutant mice have an overproduction of Aβ protein and demonstrate senile plaque formation and synaptic deficits without NFTs pathology, which indicates a key pathological role of the mutation in AβPP protein (Games et al., 1995; Hsiao et al., 1996).
Presenilin 1 and 2
21 The majority (~70%) of early-onset familial AD cases are associated with
mutations in two genes, presenilin 1 and presenilin 2, which are located on chromosome
14 and 1 respectively (Rogaev et al., 1995). Over 50 different pathogenic mutations in
presenilin 1 gene and 3 mutations in presenilin 2 gene have been described (Checler,
1999). There is considerable homology between the gene products of presenilin 1 and
presenilin 2, which are transmembrane proteins of 463 and 448 amino acids respectively,
both have six to nine hydrophobic membrance-spaning domains (Rogaev et al., 1995).
The physiological functions of these two proteins are unknown, SEL-12, one of the
homolog proteins in Caenorhabditis elegans involved in the Notch receptor pathway
(Sherrington et al., 1995), indicates a potential role for preseniline involving in Notch pathway. Nonetheless, other possible roles include ion channel transport, protein processing, or cellular trafficking functions (Czech et al., 2000). In AD, it is thought mutations in these proteins contribute to the disease by affecting the processing of AβPP, which leads an increase of the Aβ burden in both AD patients and transgenic mice
(Citron et al., 1997; Scheuner et al., 1996).
D Other Factors
CARDIOVASCULAR DISEASE AND RELATED VASCULAR RISK
FACTORS Hyperlipidemia, hypertension, diabetes, and related factors usually leading to the occurrence of heart disease or strokes have been identified as putative antecedents to
Alzheimer’s disease (Breteler, 2000).
DOWN SYNDROME Adults with Down’s syndrome develop the neuropathological changes of Alzheimer’s disease by age 40, but not all patients become demented. The risk of Alzheimer’s disease associated with a family history of Down’s
22 syndrome is increased two to three fold.
ALCOHOL Individuals who drank wine in moderate amounts daily were less
likely to develop Alzheimer’s disease than both heavier drinkers and abstainers
(Orgogozo et al. 1997). The risk reduction associated with alcohol is possibly related to its antioxidant properties or its effects on lipid metabolism.
EDUCATION AND EARLY LIFE EXPERIENCE Several studies showed that
the risk of Alzheimer’s disease among poorly educated individuals or individuals in poor
living condition is significantly higher than those among well-educated persons (Stern et
al., 1994; Hall et al., 2000).
MENTAL AND LEISURE ACTIVITY Mental and physical exercises are
positive factors with a lower risk of AD (Lindsay et al., 2002).
SMOKING Smokers had a two- to four-fold increase in risk of Alzheimer’s
disease, particularly those individuals without an APOE-4 allele (Merchant et al., 1999;
Ott et al., 1998).
TRAUMATIC HEAD INJURY There is an increase of the risk of Alzheimer’s
disease related with traumatic head injury (Plassman et al., 2000).
ANTI-INFLAMMATORY AGENTS Alzheimer’s disease was found to be less
frequent among individuals who used anti-inflammatory agents (in t’ Veld et al., 2001;
Weggen et al., 2001).
ANTIOXIDANTS That antioxidants have been proposed as a protective factor
for poor memory and dementia are based on their oxidative stress-reducing effect in vitro.
Studies show that healthy individuals consuming dietary or supplemental antioxidants
demonstrate inconsistent and small difference (Engelhart et al., 2002; Morris et al., 2002).
23 Therefore, if at an earlier stage and long-term usage of antioxidant need to be further
explored.
HORMONE REPLACEMENT The use of estrogen by postmenopausal women
has been associated with a decreased risk of Alzheimer’s disease (Baldereschi et al., 1998;
Waring et al., 1999). Women using hormone replacement had about a 50% reduction in the disease risk.
1.1.5 Research Relevance: Oxidative Stress and AD Pathogenesis
There is profound evidence showing that the oxidative damage is the very
earliest neuronal and pathological changes of AD (Perry et al., 1998; Nunomura et al.,
2000, 2001; Pratico et al., 2001). In addition, many AD risk factors are related with
production of free radicals in their hypothetical models (Figure 1.1). Indeed, this early
role is borne out by clinical management of oxidative stress, which appears to reduce the
incidence and severity of AD (Sano et al., 1997; Stewart et al., 1997). Many markers of
oxidative damage are present in susceptible neurons even in the absence of
neurofibrillary pathology (Nunomura et al., 1999). A recent time course study in Aβ
transgenic mice confirmed these findings: lipid peroxidation is significantly increased in
7–8-month-old Tg2576 mice, proceeding any apparent Aβ deposition or increase of Aβ
levels (Pratico et al., 2001).
Oxidative stress may play a role in the development of neuritic abnormalities
since paired helical filaments (PHF) are more often found in neurites with membrane
abnormalities indicative of extensive lipid peroxidation (Praprotnik et al., 1996).
24
Figure 1.1 Oxidative stress is a prominent central event in AD pathogenesis.
Many AD risk factors are related to oxidative stress and most of the AD risk factors will directly cause production of free radicals.
25 Moreover, crosslinking of proteins by oxidative processes may lead to the
resistance of the lesion removal in AD even though they are extensively ubiquitinated
(Cras et al., 1995). Moreover, this resistance of neurofibrillary tangles to proteolysis
might play an important role in the progression of AD (Smith et al., 1998). Lipid
peroxidation is marked by significantly increased levels of thiobarbituric acid reactive
substances (TBARS), malondialdehyde (MDA), 4-hydroxy-2-transnonenal (HNE), and
altered phospholipid composition (Sayre et al., 1997). Oxidation of sugars is marked by
increased glycation and glycoxidation (Vitek et al., 1994; Smith et al., 1994, 1995;
Castellani et al., 2001).
Tau and neurofilament proteins have already been found in NFTs with
predominant oxidative modifications. It is of particular interest to study the role of oxidative modification of these proteins in neurons, or more specific, to examine whether it is directly related to pathogenesis of AD.
26 1.2 Introduction of Neurofilament Proteins
The function of neurofilaments, the major component in large myelinated neurons, has not been well understood even though they were discovered as structures
over 100 years ago. Recent studies have suggested that neurofilaments are closely related
to many neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson
disease, Alzheimer disease, and diabetes. Using in vitro assays, cultures, and transgenic
mice, these studies provided new insights into neurofilament function. The function of
each subunit, the relationship of neuroflaments with other cytoskeletal elements, and the
clinical significance, are topic of increasing attentions.
1.2.1 General Information
Neurofilaments (NF) are intermediate filaments of neurons that are considered to
add rigidity, tensile strength, and possibly intracellular transport guidance to axons and
dendrites (UMLS, 2004). Exclusively expressed in neurons, NFs are one of the
members of the cytoskeleton proteins that act together to form and maintain the cell
shape and facilitate the transport of particles and organelles within the cytoplasm.
Based on the differences in diameter and protein components, cytoskeletal
polymers are classified into three groups: microtubule (MT) (~24 nm), microfilament
(MF) (~6-8 nm), and intermediate filament (IF) (~10 nm). MTs, which are composed of
tubulin, are responsible for maintaining cell shape, organelle and vesicle movement, and
the formation of the spindle fibers during mitosis. MFs, which are composed
predominantly of actin, are responsible for cellular movement, muscle contraction,
mechanical strength, and cytokinesis. IFs are composed of different proteins and are
prominent in cells that withstand mechanical stress. Moreover, IFs are the most insoluble
27 components of the cells. While the polymer compositions of MTs and MFs in all tissues are similar, IF polymers differ in different tissues and cells. Based on the homology in molecular structure, five types of IFs have been identified and NFs belongs to the type IV
IFs (Table 1).
1.2.2 History
As early as the 1830s, neuronal networks had already been described (Valentin,
1836; Purkinje, 1838). The discovery of the silver staining method in the late nineteenth century resulted in a clear vision of NFs (Apathy, 1897; Cajal, 1903), which also led to the characterization of neurofibrillary tangles and senile plaques by Alois Alzheimer
(Alzheimer, 1906). With the development of electron microscopy in 1931 (Max Knoll and Ernst Ruska in Germany), the molecular structures of NFs were further defined as filaments ~10 nm in diameter and present exclusively in neuronal cells. For a long time, it has been known that NFs are involved in several neuronal diseases. In the past decade,
NFs have been linked to other human diseases, which will be discussed here. With the development of specific antibodies, transgenic animal models, and molecular genetic methodologies, the study of NFs has advanced to the molecular level.
1.2.3 Structure
Together with peripherin, α-internexin, and nestin, NFs belong to type IV IFs and share common sequence structures (Table 1). A central α-helical rod domain of about
310 amino acids is flanked by a globular N-terminal region and non α-helical carboxy-terminal sidearm domains. The central rod domains including region 1a, 1b, and
2 contain highly conserved motifs and every seventh residue is hydrophobic, which
28
Table1.1 Intermediate filaments include five defined types and other undefined types. Their molecular weights are varied and they are found in different cell types.
Neurofilament subunits belong to type IV intermediate filaments.
29 facilitates the formation of α-helical coiled-coil parallel homodimers or heterodimers. A linker region aligns the hydrophobic residues. These properties are characteristics of the intermediate filaments and are essential for their proper assembly (Fig 1.2).
NFs are composed of three subunits, and these subunits are defined by their molecular weights: NF-L (light), NF-M (medium), and NF-H (heavy), which are 62 kDa,
97 kDa, and 125 kDa respectively, as predicted from the DNA sequences (Shaw, 1991).
These subunits exhibit higher molecular weights on SDS-PAGE: 68 kDa, 160 kDa and
205 kDa respectively, due to the enriched negatively charged amino acids (glutamic acid) in their sequences and post-translational modifications such as phosphorylation and glycosylation (Shaw, 1991; Dong et al., 1993). NFs constitute the most abundant structures in large myelinated neurons, and can account for 13% of total proteins and
54% of the Triton-insoluble proteins in some neurons (Morris and Lasek, 1984). More detailed description of all intermediate filaments structures is recorded in several reviews
(Shaw, 1991; Fuchs E. and Weber, 1994; Al-Chalabi and Miller, 2003).
1.2.4 Expression
The three NF subunits are expressed during distinct stages of vertebrate development, triggered by neuron differentiation (Shaw and Weber, 1982; Nixon and
Shea, 1992). Initially, NF-L is expressed at the beginning of neuronal differentiation and overlapped with the expression of α-internexin and peripherin (Carden et al., 1987;
Willard et al., 1983). NF-M is expressed shortly after with the emergence of neurite formation whereas NF-H is expressed later when axonal radial growth is required for nervous system maturation (Carden et al., 1987; Willard et al., 1983). The genes coding for human NFs include NEFL gene (NF-L protein), NEFM gene (NF-M protein),
30
Figure 1.2 Comparison of the structures of neurofilaments and other type IV
filaments. The neurofilament subunits and other type IV IFs all include α-helical rod domain and are varied in N- and C- termini. A unique character of NF-M and NF-H is that the C-termini of them have multiple KSP repeats that are heavily phosphorylated.
Human NFM has 13 KSP and human NFH has 43/44 KSP, and most of them are phosphorylated. In addition, here shows the posttranslational modifications including phosphorylation and glycosylation on NF subunits (Summarized from Shaw 1991; Dong et al., 1993; Fuchs and Weber 1994).
31 and NEFH gene (NF-H protein). NEFL and NEFM genes are closely located on
chromosome 8, 8p21 (Myers et al., 1987; Strausberg 2002); NEFH gene is at
chromosome 22, 22q12.2 (Lees et al., 1988). Expression of mRNAs of NF subunits is
consistent with their protein levels in cultured neuroblastoma cells and NF-L and NF-M
mRNAs are expressed several days before the appearance of NF-H mRNA in these cells
(Breen and Anderton, 1991) It appears that the NF-L and NF-M genes are coordinately
regulated while the NF-H gene is expressed independently later.
1.2.5 Assembly
NF assembly is not clearly understood yet, and it is directly related to the
functions of the three subunits and the filament. In vivo studies have demonstrated that
NFs are obligate heteropolymers requiring NF-L to form a proper polymer with either
NF-M or NF-H (Lee et al., 1993). NF formation is believed to start with the dimerization of NF-L with either NF-M or NF-H subunits. The highly conserved rod domains of the
NF subunits are coiled together in a head-to-tail fashion to form a dimer. Two coiled dimers overlap with each other in an anti-parallel, half-staggered manner forming the tetramers. Finally, eight tetramers are packed laterally and longitudinally together in a helical array, forming a ropelike 10 nm filament (Fuchs and Weber, 1994, Heins et al,
1994, Fuchs and Cleveland, 1998; Herrmann and Aebi, 2000). In the cross section of NF, there are ~32 molecules though the number may change in different stages or conditions
(Herrmann et al., 1999). Theoretically, before the synthesis of NF-H, NFs are exclusively composed of NF-L and NF-M (Carden et al., 1987). The C-termini of NF-M and NF-H are not in the coils but they form the side-arms of the NFs (Perrone Capano et al., 2001)
(Figure 1.3). (See related study in Chapter 2)
32
Figure 1.3 Schematic model of neurofilament assembly. The neurofilament assembly process includes, two NF subunits (NF-L and either NF-H or NF-M) form head-to-tail coiled-coil dimmers, anti-parallel half-staggered tetramers, protofilament, and 10nm neurofilament. There are around 32 molecules in the cross section and hyperphosphorylated side-arms formed by C-termini of NF-H and NF-M stick out of the stem of the filament.
33 NF-L is essential for the precise assembly of NFs (Ching and Liem, 1993; Lee et
al., 1993, Zhu et al., 1997). NF-M participates in the formation of cross-bridges,
stabilization of the filament network, and the longitudinal extension (Elder et al., 1998a,
1999a, b; Jacomy et al., 1999). NF-H also contributes to the formation of cross-bridges and may interact with microtubules/microfilaments and other cytoskeletal elements
(Elder et al., 1998b 1999b; Jacomy et al., 1999). However, recent studies showed NF-H side arms are not critical for the radial growth of the axons (Rao et al., 2002, Al-Chalabi et al., 2003). NF-M and NF-H by themselves do not form filaments in vivo (Ching and
Liem, 1993; Lee et al., 1993, Nakagawa et al., 1995, Zhu et al., 1997), while NF-L can form homopolymer in vitro (Geisler et al., 1981; Liem et al., 1982) and in cells transfected with NF-L (Carter et al., 1998), but not in mice (Jacomy et al., 1999).
Although NFs are obligate heteropolymers in vivo in rodent, this may not be the case in human (Carter et al., 1998).
In contrast to NF subunits, α-internexin and peripherin self-assemble into homopolymers and co-assemble with NF subunits (Cui et al., 1995; Beaulieu et al., 1999a, b). Many CNS neurons also contain varying levels of α-internexin, a type IV intermediate filament subunit (Fliegner and Liem, 1991) expressed abundantly in embryonic neurons but at lower levels postnatally (Kaplan et al., 1990; Fliegner et al., 1994). Although
α-internexin colocalizes with NFs and forms filaments with each of the triplet proteins in
vivo (Ching and Liem, 1993), the composition of the filaments is still not known in vivo.
Peripherin, a type III intermediate filament, is expressed in lower motor, autonomic, and sensory neurons and co-assembles with the NF subunits (Beaulieu et al., 1999a, b).
1.2.6 Transport
34 After their synthesis in the perikarya, neurofilament proteins (NFPs) are quickly translocated into the axons and assemble into filamentous structures. In radioisotopic labeling studies, neurofilament proteins move at a rate of 0.2–1 mm/day in the axon with the slow components whereas other organelles such as membrane vesicles can move at much faster rate of 200–400 mm/day (Lariviere and Julien 2004). (See more details in
Chapter 2)
1.2.7 Posttranslational Modifications
Two major posttranslational modifications, phosphorylation and glycosylation, are involved in the formation and functions of NFs.
A. Phosphorylation (See more details in Chapter 2) (Figure 1.4)
B. Glycosylation.
In addition to phosphorylation, NFs are also modified post-translationally by
O-linked N-acetylglucosamine (O-GlcNAc) on serine and threonine residues (Dong et al.,
1993, 1996), which is the simplest protein modification with sugars (Hart 1989). Initially discovered from murine lymphocytes (Torres et al., 1984), this modification has been found in all the compartments of eukaryotic cells (Holt et al., 1986). Similar to phosphorylation, the O-GlcNAc modification (O-GlcNAcylation) is highly abundant and dynamic (Hart et al., 1995; Hart et al., 1993; Haltiwanger et al., 1992; Chou et al., 1992).
NF-H, NF-M, and NF-L are modified by O-GlcNAc in vivo to stoichiometries of at least 0.3, 0.15, and 0.1 mol GlcNAc/mol of protein at both termini (Dong et al., 1996)
(Fig 1). Although the stoichiometry of O-GlcNAcylation on isolated NFPs is very low compared with phosphorylation, it seems likely that only a subset of NF subunits is modified by O-GlcNAc, and the stoichiometry in those subunits is probably much higher.
35
Figure 1.4 Regulatory functions of NF phosphorylation. N-terminal phosphorylation has both inhibitory and promotional effect for the C-terminal phosphorylation in NFs, and N-terminal phosphorylation can also control the NFs translocation and assembly in axons.
36 It is worthy of mentioning that this O-GlcNAcylation is most likely in a consensus of PX0-4(S/T) motif (X is usually a hydrophobic residue) similar to the motifs used by proline-directed kinases and phosphatases (X[S/T]PX or XP[S/T]X) (Hart et al.,
1995).
The proximity or competition of the O-GlcNAcylation sites to the phosphorylation sites in the head domain and the importance of the head domain in NF assembly (Gill et al., 1990; Wong et al., 1990; Chin et al., 1991) indicates that
O-GlcNAcylation may also play a role in NF assembly. This regulation could be exerted through either direct or indirect (e.g. by affecting phosphorylation) influence on the structure of the head domain. It is possible that O-GlcNAc is added to NFPs by
O-GlcNAc transferase (Haltiwanger et al., 1992) right after their synthesis and prior to their assembly into filaments and transport into the distal part of axons, and is removed by an N-acetyl-β-D-glucosaminidase subsequently (Dong et al., 1993).
By replacing phosphates with O-GlcNAc, the interactions between NFs may switch from a repulsive one to an associative one, leading to close packing of NFs, which happens in nodes of Ranvier. Therefore, it seems likely that organization of NFs is regulated by kinase/phosphatase (Nixon 1993; Xu et al., 1994) and O-GlcNAc transferase/N-acetyl-β-D-glucosaminidase (Haltiwanger et al., 1992; Dong et al., 1994).
The dynamic O-GlcNAcylation (Hart et al., 1993; Hart et al., 1995) and phosphorylation
(Nixon 1993) could therefore regulate proper NF assembly and dynamics, and the abnormalities in either of them could contribute to some of the motor neuron diseases in which NFs accumulate aberrantly (Xu et al., 1994; Cleveland et al., 1995).
1.2.8 Degradation
37 Many neurons extend their axons over great distances, up to 1 meter in human
sciatic nerves, to form synapses with appropriate receptor cells. To maintain the
physiological functions of the nerves, certain proteins need to have long lifetimes to span
over the axon. NFPs are among one of them. In the transport process NFP degradation is
not detected (See more details in Chapter 2).
1.2.9 Functions (See more details in Chapter 3)
There are several aspects of NF function. These include the basic function of neurofilament to support axonal structure, detrimental effects of NF accumulations, NF protective effects (Figure 1.5), and the possible function of KSP repeats.
1.2.10 Animal Models
Many animal models have contributed to the studies of NFs in establishing NF subunits functions. The first model is the Japanese quail discovered in 1991 (Quiverer)
(Yamasaki et al., 1991) with a spontaneous mutation in NF-L at amino acid 114, a nonsense mutant, which generates a truncated protein of only 113 amino acids of NF-L out of total 556 amino acids and is incapable of forming NFs (Ohara et al., 1993).
Homozygous mutants contain no axonal NFs and exhibit a mild generalized quivering. In
these animals, radial growth of myelinated axons is severely attenuated (Yamasaki et al.,
1991) with a consequent reduction in axonal conduction velocity (Sakaguchi et al., 1993).
The second model is the transgenic mouse reported in 1994, expressing a
NF-H/β-galactosidase fusion protein in which the C-terminus of NF-H was replaced by
β-galactosidase (Eyer and Petersen, 1994). NF inclusions in the perikarya of neurons, depletion of axonal NFs, and reduced calibers were found. Later on, Zhu et al (1997) have shown that mice lacking NFs due to a targeted disruption of the NF-L gene have
38
Figure 1.5 Hypothetical relationships among neurofilament proteins, oxidative stress, cell injury and tissue damage. Neurofilament especially NFH as the most abundant protein in neurons may work as the scavenger for oxidative stress, which protects the other critical factors from oxidative attack.
39 reduced axonal calibers and delayed maturation of regenerating myelinated axons.
To demonstrate the NF subunits functions, we will only compare the NF subunit knock-out and overexpressing mice in this paper (Table 1.2). To see more transgenic mice comparisons, other reviews are recommended (Julien 1999; Lariviere and Julien,
2004)
NF-L Knockout Mice The targeted disruption of the NF-L gene in mice confirmed the importance of NF-L in NF assembly (Zhu et al., 1997). The NF-L knock-out mice had no overt phenotype. In the absence of NF-L, the levels of NF-M and
NF-H subunits are dramatically decreased to ~5% of normal level and unable to assemble into 10 nm filaments. As a result, these mice have a scarcity of IF structures, severe axonal hypotrophy, and no large-sized myelinated axons (Zhu et al., 1997). Levels of other cytoskeletons like tubulin are increased possibly as a compensating effect. While the NF-L gene knock-out mice provide definite proof that NFs are a major determinant of axonal caliber and NF-L is required for NFs formation, the specific roles of NF-M and
NF-H subunits remain unclear. The reduced levels of NF-H and NF-M in NF-L-/- mice
are probably the result of an enhanced proteolytic turnover of unassembled or
disorganized NF proteins in the absence of NF-L (Zhu et al., 1997; Williamson et al.,
1998; Beaulieu et al., 1999a; Levavasseur et al., 1999).
NF-H Knockout Mice Several laboratories have recently reported the characterization of NF-H null mice (Rao et al., 1998; Zhu et al., 1998; Kriz et al., 2000).
Surprisingly, the absence of NF-H has little effect; the mice phenotype, the NF number, and the calibers of motor axons during development were all normal. With the C57Bl6 strain, Rao et al (1998) and Zhu et al (1998) reported only a slight reduction in the caliber
40 of myelinated axons from the ventral roots of NF-H knockout mice, maybe the small changes are due to the increase of NF-M protein level and phosphorylation levels. MTs increased two fold with a small decrease in actin level. In the mean time, the null mutant
NF-H in 129J strain mice described by Elder et al (1998) exhibited a more pronounced reduction in the caliber of axons. In addition, they reported little difference in NF-L and
NF-M levels. These discrepancies are most likely attributable to differences in the mouse genetic backgrounds. In any case, the combined results suggest that NF-H has a minor effect on the radial growth of axons and may be a key factor interacting with MT/mf.
NF-M Knockout Mice A clearer conclusion comes from the analysis of NF-M knockout mice. Two independent studies have shown that disruption of NF-M has a more severe effect than disruption of NF-H on the radial growth of large myelinated axons
(Elder et al., 1999; Jacomy et al., 1999). The absence of NF-M did not cause any phenotypic changes in mice but caused an increase in NF-H level and its phosphorylation.
However, the severe decreases in NF-L level (>50%) caused axonal NF content decrease more than 50%, resulting in axonal atrophy. Comparing NF-H-/- mice and NF-M-/- mice, it showed that NF-M is more critical in axonal radial growth.
NF-M/H Double Knockout Mice Moreover, double knockout mice lacking both
NF-M and NF-H subunits, showed hind-limb paralysis (Jacomy et al., 1999; Elder et al.,
1999). The absence of NF-M/H causes the sequestering of unassembled NF-L proteins in the neuronal perikarya, no NF formation in axons, increase of MT by 2 fold, and loss of about 24% of ventral root axons (Jacomy et al., 1999; Elder et al., 1999). The analysis of
NF-M-/-, NF-H-/- double knockout mice demonstrates that the high molecular weight subunits are required for the in vivo assembly of NFs and normal axonal growth.
41
Table 1.2 The summary and comparison of NF subunits knock-out and overexpression mouse models. “-” means not found in the original papers.
42 NF-L Overexpressing Mice Studies of transgenic mice have found that
overexpressing mouse NF-L to ~4 fold leads to a striking ALS-like pathology, in which most of the mice died before 3 weeks postnatally with only 1/3 to 2/3 of the body weight compared with normal mice (Xu et al., 1993). Also strikingly, the only two mice surviving over 3 weeks finally recovered slowly to normality after 9 months. The transgenic mice showed intensive NF aggregation in all the compartments of neurons with depleted rough endoplasmic reticulum (RER), neuron degeneration in both dorsal and ventral roots, phosphorylated NF-H in cell bodies, and severe muscle atrophy, which resembles the human ALS pathology. This study confirmed that overexpression of NF-L and NF aggregation in mice can directly cause abnormality and degeneration of motor neurons.
NF-M Overexpressing Mice The NF-M overexpressing transgenic mice appeared normal (Wong et al., 1995). With about 2 fold increase of total (WT plus mutant) NF-M level in the mice, accumulation of NFs was detected in cell bodies and axons. The axonal caliber of neurons decreased about 50% with unchanged NF-L level and 50% decrease of WT NF-H and WT NF-M levels. This caliber reduction is a result of growth retardation rather than a total growth inhibition, which was restored to the normal level with time, and it is correlated with reduction of WT NF-H and WT NF-M. No neuron degeneration was detected. Because the nearest inter-neurofilament spacing did not change, this reduction may be strictly correlated with NF numbers.
NF-H Overexpressing Mice Overexpressing 1.2 to 4.5 fold of WT NF-H in transgenic mice showed no overt phenotypes or neuron loss (Marszalek et al., 1996). The level of NF-M in axons were correlatively reduced with increased NF-H level, and NF-L
43 level decreased by 20%-40% in axons. Axonal caliber reduction was correlated with
NF-H overexpression level, more than 3 fold of which totally inhibited large axon
growth. This caliber reduction can also be considered as strictly correlated with NF-M
level or NF numbers.
In all these studies, only mice overexpressing >3 fold NF-L and NF-M/H double
knockout mice showed severe phenotypes. Taken together, these data clearly demonstrate
all the NF subunits functions: expression of NF-L is responsible for NF assembly, and
NF-M/H subunits are required for NF assembly in vivo; NF-M is more critical for axonal
growth than NF-H, and the latter may be working as a key factor for NF interaction with
other cytoskeletal polymers. The nearest inter-neurofilament spacing does not change
when NFs are present in the axons, but to achieve the long-ranged “cross bridges”, the
side chains of NF-H and other key factors are definitely required. Expression of NF-M
and NF-H are co-regulated in an inverse manner in motor axons, and they are competing
for the transport and assembly with NF-L. In large caliber axons, microtubule content
does not correlate with axonal diameter as closely as does NF content (Friede et al., 1970,
Hoffman et al., 1984), but microtubule or other IFs are more significant in maintaining the diameter of medium- and small-sized axons where they are the major cytoskeletal
components.
Noteworthy, studies other than those above also provide solid evidence for the
NF function. Recently, studies using NF-H truncated mutant mice (Rao et al., 2002)
demonstrate that neither C-terminal tail of NF-H nor its phosphorylation is important for
axonal caliber. Transgenic mice expressing human NF subunits (Tu et al., 1995, Gama
Sosa et al., 2003) or mutations in mouse NFs (Lee et al., 1994, Rao et al., 2002) all show
44 severe motor neuron degeneration, which indicate that non-WT NF forms may be the
major factors for neuronal degeneration. In addition, studies with SOD mutant mice that
utilize human NF subunits provide more important information (Julien et a., 1987, Cote
et al., 1993, Collard et al., 1995, Meier et al. 1999).
1.2.11 Neurofilaments and Neurodegenerative Diseases
Abnormal accumulation of NFs is detected in many human neurodegenerative
disorders including ALS, AD, dementia with Lewy bodies, Parkinson disease, and
diabetes, etc. Many alterations can potentially lead to accumulation of NFPs, including
deregulation of NFPs synthesis, defective axonal transport, abnormal phosphorylation, and proteolysis. Studies in transgenic mouse also showed that NFs affect the dynamics and function of other cytoskeletal elements, such as microtubules and actin filaments
(Zhu et al., 1998, Ahlijanian et al., 2000). It is widely believed that NF abnormalities in neurodegenerative disorders are the hallmark of neuronal dysfunction.
A. Amyotrophic Lateral Sclerosis (ALS)
ALS, a motor neuron disease and also called Lou Gehrig's disease, was first identified in 1869 by the noted French neurologist Jean-Martin Charcot. ALS affects as many as 20,000 Americans with 5,000 new cases occurring in the United States each year, typically with an onset at an age between 40 and 60. Men’s risk is about 1.5 times to that of women. ALS is usually fatal within 5 years after diagnosis. It is a progressive neurodegenerative disease that attacks motor neurons in the brain and the spinal cord.
The progressive degeneration of the motor neurons in ALS eventually leads to neuron death and loss of related muscle movements. Patients of ALS become partially or totally paralyzed, but most of the patients’ cognitive functions remain unaffected. There is no
45 cure but the Food and Drug Administration (FDA) recently approved riluzole, a drug that
has been shown to prolong the survival of ALS patients.
A key neuropathological hallmark of ALS is intraneuronal aggregates of NFs in
degenerating motor neurons (Carpenter 1968; Averback 1981; Delisle and Carpenter
1984; Manetto et al., 1988; Munoz et al., 1988; Leigh et al., 1989; Murayama et al.,
1992). Although the reason for the NF aggregates is unclear, transgenic mouse models which overexpress any of the NF subunits (NFL, NFM, and NFH) will provoke motor
neuropathy characterized by the presence of abnormal NF accumulations resembling those found in ALS (Cote et al., 1993; Julien et al., 1995; Wong et al., 1995; Xu et al.,
1993). Remarkably, the motor neuropathy in transgenic mice overexpressing human
NF-H was rescued by restoring a correct stoichiometry of NF-L to NF-H subunits with the co-expression of human NF-L transgene (Meier et al., 1999). It has been proposed that such alterations in NF homeostasis are directly relevant to the pathogenesis of ALS
(Williamson et al., 1998).
Interestingly, abnormal NF inclusions are often associated with decreases in levels of NF-L mRNA. NF-L mRNA is selectively reduced by up to 70% in degenerating
neurons of ALS and AD (McLachlan et al., 1988; Bergeron et al., 1994; Wong et al.,
2000; Menzies et al., 2002). Decrease of NF-L mRNA level occurs with decreases of
α-internexin or peripherin mRNA levels, suggesting an absolute alteration of the
stoichiometry of NF expression in ALS (Bergeron et al., 1994; Wong et al., 2000).
Also codon deletions or insertions in the KSP regions of NF-H including a large
deletion of five KSP repeats have been detected in a small number of sporadic cases of
ALS, (Figlewicz et al., 1994; Tomkins et al., 1998; Al- Chalabi et al., 1999).
46 Taken together, these observations have suggested a significant alternation in
NF expression in ALS.
B. Alzheimer’s Disease (AD)
AD is a central nervous system neurodegenerative disease. It was first described
in 1906 by German physician Dr. Alois Alzheimer. Although the disease was once
considered rare, it is now established as the leading cause of dementia. An estimated 4
million individuals in the United States have Alzheimer’s disease with an annual expense
of about $70-$100 billion, and as many as 350,000 individuals develop the disease each
year (Mayeux R., 2003). The risk of Alzheimer’s disease varied from 12% to 19% for
women over the age of 65 years and 6% to 10% for men (Seshadri et al. 1997). On
average, AD patients live about 8 years after they are diagnosed, although the disease can
last for as many as 20 years. The areas of the brain that control memory and thinking
skills are affected first, but as the disease progresses, cells die in other regions of the
brain. Eventually, the person with Alzheimer’s will need complete care. If the individual
has no other serious illness, the loss of brain function itself will cause death. There is no
cure and the only therapy known as cholinesterase inhibitors or anti-cholinesterase drugs including Reminyl (galantamine), Aricept (donepezil hydrochloride) and Exelon
(rivastigmine) provide symptomatic relieves but do little to slow the disease progression.
After the last two decades of intensive studies, more information has been yielded, but the cause of AD is still unknown. Many hypotheses have been proposed, such as amyloid-β cascade (Selkoe, 2000), tauopathy (Lee et al., 2001), inflammation
(McGeer et al., 2001; Weiner et al., 2002), oxidative stress (Markesbery, 1997; Christen,
2000; Picklo et al., 2002, Perry et al., 1998, 2002).
47 Cytoskeleton disruption is a prominent feature and a secondary event followed by
oxidative damage in Alzheimer disease (Smith et al., 1995; Nunomura et al., 2001;
Ahlijanian et al., 2000). NFT as the hallmark of AD is composed of abnormally modified
τ, NFs, and other cytoskeleton proteins. The cause for the aberrant biochemical processes that transform normal τ and NF into abnormal filaments is not understood.
In AD, inappropriate hyperphosphorylation of proteins such as τ, NF is prominent, which is likely due to perturbation in the balance between kinases and phosphatases activity (Gong et al. 2000). MAP kinase (Trojanowski et al., 1993), GSK-3
(Mandelkow et al., 1992), and CDK 5 (Lew et al., 1994; Maccioni et al., 2001) pathways are known to be involved in τ and NF phosphorylation in neurons. One of the earliest changes noted in AD is accumulation of hyperphosphorylated τ and NF in normal perikarya (Sternberger et al., 1985; Manetto et al., 1988; Sobue et al., 1990; Cleveland et al., 2001). This abnormal hyperphosphorylation may cause the protein aggregation in
NFT in AD neurons. The role of NFT and senile plaques in AD are still actively disputed.
C. Parkinson's Disease (PD)
PD is a progressive disorder of the central nervous system. The disease was originally described in 1817 by an English physician, James Parkinson, who called it
"Shaking Palsy." It is affecting more than 1.5 million people in the United States. Men and women are similarly affected. The frequency of the disease is considerably higher in the over-60 age group, and the average duration of illness is about 9 years (Hely et al.
1999).
PD is caused by the degeneration of the pigmented neurons in the substantia nigra of the brain, resulting in decreased dopamine availability. Clinically, the disease is
48 characterized by a decrease in spontaneous movements, gait difficulty, postural instability, rigidity and tremor. Only in the 1960's, however, pathological and biochemical changes in the brain of patients were identified, opening the way to the first effective medication for the disease. Administration of the drug levodopa, a dopamine precursor, has been the standard treatment for Parkinson's disease.
The major pathological change of PD is an accumulated protein inclusion called the Lewy body , composed of numerous proteins include the essential constituent of
α-synuclein protein, the three NF subunits (Galloway et al., 1992), ubiquitin and proteasome subunits (Trimmer et al., 2004). Electron microscopy and biochemical evidence indicates that the abnormally phosphorylated NFs form a non-membrane bounded compacted skein in the neuronal soma in the affected neurons.
In familial PD, mutations in the Parkin gene is the major cause, in which over
20 different mutations have been identified (Abbas et al. 1999, Leroy et al. 1998, Lucking et al. 2000, Lim et al. 2002).
More recently, a point mutation has been reported in the region of the NEFM gene coding for the rod domain 2B of NFM in an individual with Parkinson’s disease
(Lavedan et al., 2002). The base pair change results in substitution of serine for glycine at residue 336, and probably disrupts assembly. The patient developed Parkinson’s disease at the very young age of 16. Although this is an isolated report and three other unaffected family members also carried the mutation, it is possible that this mutation is responsible for the early-age onset of an aggressive form of PD. It is already known that mutations in the same region of neurofilament proteins might cause a peripheral motor nerve axonal loss in NEFL and central dopaminergic loss in NEFM. It has also been found that NF-L
49 mRNA decreased in PD correlating with the severity of the disease (Hill et al., 1993).
D. Charcot-Marie-Tooth Disease (CMT)
CMT is the most common inherited neurological disorder. It was discovered in
1886 by three physicians, Jean-Marie-Charcot, Pierre Marie, and Howard Henry Tooth.
Approximately 150,000 Americans are affected by the condition. CMT affects both
motor neurons and sensory neurons to the muscles, and the patients slowly lose normal
use of their feet/legs and hands/arms as nerves to the extremities degenerate. The loss of
nerve function in the extremities also leads to sensory loss. The ability to distinguish hot
and cold is diminished as well as the sense of touch. There are several types of CMT but
the simplest classification is into types 1, 2, and 3. Types 1 and 3 are due to demyelization, and type 2 is an axonal disease. CMT is generally inherited in an autosomal dominant pattern. At present there is no cure for CMT, although physical therapy and moderate activity are often recommended to maintain muscle strength and endurance.
Several families have now been identified in which heterozygosity for mutations of the NEFL gene on chromosome 8 are associated with CMT2 (Mersiyanova et al., 2000; De Jonghe et al., 2001; Georgiou et al., 2002; Jordanova et al., 2003).
The first of these changes is a substitution of proline at residue 8 with glutamine. The second change is a switch of leucine to a proline residue at position 333 in the highly conserved rod domain 2B.
These NEFL CMT2 mutations disrupt neurofilament assembly and axonal transport, which probably underlies the disease mechanism. A mouse model with a mutation in the same coil, leucine to proline at residue 394, develops severe selective
50 motor neuron death due to disruption of neurofilament assembly (Lee et al., 1994).
Georgiou et al. (2002) identified a novel NF-L missense mutation (Cys64Thr) that caused
the disease in a large Slovenian CMT2 family. This mutation results in a proline to serine
substitution at codon 22 (Pro22Ser) and the mutation showed complete co-segregation
with the dominantly inherited CMT2 phenotype in this family. A similar Pro22Thr was also detected in unrelated Japanese patients with CMT disease (Yoshihara et al., 2002).
Jordanova et al. (2003) found six pathogenic mis-sense mutations and one 3-bp inframe deletion in the NF-L gene in 323 patients with different CMT phenotypes.
Some types of CMT are caused by defects in proteins expressed in the Schwann cell, including the connexin 32 (CX32/GJB1) genes. Such mutations result in aberrant myelination and altered neurofilament phosphorylation. In such cases, therefore, abnormalities of neurofilament phosphorylation occur downstream of the primary pathological process but are still upstream of the end point.
E Diabetes
Diabetes is a disease in which the body does not produce or properly use insulin. The cause of diabetes is still a mystery, although both genetics and environmental factors such as obesity and lack of exercise appear to play roles. There are 18.2 million people in the United States, or 6.3% of the population, develop diabetes. It is estimated that at least 20.1 million Americans have pre-diabetes, in addition to the 18.2 million with diabetes. About 65% of deaths among people with diabetes are due to heart disease and stroke.
Diabetes is associated with a symmetrical distal axonal neuropathy. The neuropathy is predominantly in sensory neurons or dorsal root ganglia. There is an
51 increase in phosphorylation of NFs in lumbar dorsal root ganglia of rats with streptozocin-induced diabetes, but not in other neural cell types (Fernyhough et al.,
1999). This is probably the result of activation of c-Jun N-terminal kinase (JNK), which is a NFs kinase. Pathologically, neurites from sympathetic ganglia are swollen with disorganized aggregates of NFs and other proteins, including peripherin (Schmidt et al.,
1997). Many of these studies show that NFs abnormalities can be the primary cause of the disease and that NFs changes may not simply be a passive marker of pathological processes.
The neuropathy associated with diabetes includes well-documented impairment of axonal transport, a reduction in axon caliber and a reduced capacity for nerve regeneration. All of those aspects of nerve function rely on the integrity of the axonal cytoskeleton. NFPs mRNAs are selectively reduced in the diabetic rat and posttranslational modification of at least one of the NFPs is altered. There is some evidence that altered expression of isoforms of protein kinases may contribute to these changes. Tubulin and actin aberrant modifications are also found (McLean WG., 1997)
The list of diseases involved with NF abnormalities will likely increase and the common mechanism for all the protein aggregation diseases may soon be recognized
(Caughey and Lansbury, 2003).
1.2.12 Summary
In this review, we summarized as many as eleven aspects of NF, which may provide some help for the overall knowledge of the readers. Future studies will be focusing on functions, interactions, and pathology of neurofilament in normal axon development and neurodegenerative diseases, which will provide more knowledge and
52 value for NF. Hopefully, in the recent future, some effective therapeutic method for these neurodegenerative diseases will be available.
53 1.3 Introduction of Tau Protein
1.3.1 General Information
Neurons are cells with a very complex morphology that develop two types of cytoplasmic extensions, axons and dendrites. Neural transmission occurs through these
processes, and therefore, any abnormal changes in neuronal morphology may affect their
normal function and induce pathological events.
Microtubules are very dynamic structures, and in proliferating cells such as neuroblasts (neuron precursors), their probability of assembly is the same as that of depolymerization in all directions. This equilibrium results in the cell maintaining a
sphere-like morphology. However, during the differentiation of a neuroblast into a neuron
(Mitchison et al., 1988), the microtubules become stabilized in specific directions,
thereby generating the cytoplasmic extensions that will become the axon and the dendrites (Mitchison et al., 1988).
Many specific proteins serve to stabilize microtubules, which include the microtubule-associated proteins (or MAPs) MAP1A, MAP1B, MAP2, and tau. An
asymmetric distribution of MAPs (Matus, 1988) is observed in mature neurons (Craig and
Banker, 1994), and tau is preferentially localized in axons (Binder et al., 1985). Present mainly in the axon of a neuron, tau has been related to axonal microtubule function, both
alone and in synergy with other MAPs (Gonzalez-Billaul et al., 2002).
1.3.2 History
Tau protein was discovered almost simultaneously in the United States and
Europe as a protein that lowers the concentration at which tubulin polymerizes into microtubules in the brain (Cleveland et al., 1977a, b; Fellous et al., 1977). At the same
54 time, other high-molecular-weight MAPs were also found to influence the cycles of
microtubule assembly and disassembly in vitro (Shelanski et al., 1973).
1.3.3 Tau Gene and Tau Expression
The tau cDNA was first isolated from a mouse brain expression library (Lee et al., 1988), and subsequently, it was cloned from other species including goat (Nelson et al., 1996), chicken (Yoshida et al., 2002), bovine (Himmler et al., 1989), and human
(Goedert al., 1992). More recently, tau sequences have been described in a number of distinct species (Nelson et al., 1996).
The human tau gene is located on chromosome 17 (Neve et al., 1986), where it occupies over 100 kb and contains at least 16 exons (Andreadis et al., 1992). Right beside
GC-rich 5'-region, a single untranslated exon exists (exon-1) (Andreadis et al., 1996).
Upstream of this exon there are several DNA sequences that contain consensus binding
sites for promiscuous transcription factors such as AP2 or SP1. Tau is mainly expressed in neurons, and an interaction with a neural specific factor has been proposed (Sadot et al., 1996). Nevertheless, the neural specific expression of the protein could also be due to
the presence of possible silencer elements in non-neural cells (Kosik et al., 1997).
In the central nervous system (CNS), alternative splicing of exons 2, 3, and 10
results in the appearance of six tau isoforms. Because exon 10 encodes for one of the regions involved in the binding of tau to microtubules, alternative splicing of exon 10 produces tau isoforms, with either three (tau 3R without exon 10) or four (tau 4R with
exon 10) tubulin/microtubule binding regions. In chicken, an extra tubulin binding region
appears (tau 5R) (Yoshida et al., 2002). In the peripheral nervous system (PNS), there is a
55 high-molecular-weight tau isoform expressing the exon 4A, which yields a protein known
as big tau with an approximate size of 100 kDa (Goedert et al., 1992).
Different tau isoforms are characteristic during brain development. Isoforms lacking exon 10 are found at early developmental stages, or in specific cell types like granular cells of dentate gyrus (Goedert et al., 1989). The determinants of exon 10 splicing have been studied in detail (D’Souza et al., 2002), and this splicing seems to be regulated by the phosphorylation of splicing factors (Hartmann et al., 2001). Other tau isoforms that lack exon 2, or exons 2 and 3 (Himmler et al., 1989),have also been described. Indeed, exons 2, 3, and 10 are alternatively spliced and are adult brain specific.
Exons 2 and 3 are alternatively spliced cassettes; exon 2 can appear alone, but exon 3
never appears independently of exon 2 (Andreadis et al., 1995). Furthermore, in humans there is little (if any) expression of exons 6 and 8.
1.3.4 Tau Protein Structure (See more details in Chapter 4)
1.3.5 Posttranslational Modifications of Tau
Several post-translational modifications have been described for tau protein
including phosphorylation, glycation, glycosylation, oxidation, cross-linking, ubiquitinylation, deamidation, and nitration. The mostly studied one is phosphorylation.
A. Tau Phosphorylation
In the 1980s, tau was found as a phosphoprotein (Ihara et al., 1986), and all these
studies focused on the serine/threonine phosphorylation of the tau protein. Recently, one
study has focused on phosphorylation on its tyrosine residues (Williamson et al., 2002).
(See more details in Chapter 4)
B. Tau Glycation
56 Proteins with a relatively long half-life can be modified at lysine residues by nonenzymatic reactions involving the condensation of a sugar aldehyde or ketone group
with the ε-NH2-groups of the lysines (Maillard et al., 1912). Advanced glycation end products can be formed through irreversible changes of these products that can result in the cross-linking of the modified proteins (Eble et al., 1983). It has already been shown
that Tau isolated from PHF is glycated (Ledesma et al., 1994), and this glycation might be involved in the aggregation of PHF into more complex aggregates (neurofibrillary tangles) (Ledesma et al., 1995). Moreover, glycated tau could generate oxygen free
radicals that are capable of disturbing neuronal function (Smith et al., 1995).
C. Tau Glycosylation
Both N- or O-glycosylation of tau has been reported, with N-glycosylation
occurring in hyperphosphorylated tau (Wang et al., 1996) whereas O-glycosylated
occurring in unmodified tau (Arnold et al., 1996). This relationship between
phosphorylation and O-GlcNAc glycosylation of tau proteins may play a role in the nuclear localization of tau. The transport of tau into the cell nucleus was reduced with a decrease in the incorporation of O-GlcNAc into tau (Lefebvre et al., 2003). The effects of
N-glycosylation on the phosphorylation of tau have also been studied (Liu et al.,
2002a,b,c).
D. Tau Ubiquitinylation
Ubiquitin is a small protein with 76-amino acid and associates with proteins to be degraded in an ATP-dependent manner (Hershko et al., 1998). Ubiquitinylated tau has already been found in aberrant aggregates such as inclusion bodies found in Pick's or
Parkinson's diseases (Mayer et al., 1989) and in some types of paired helical filaments
57 (PHF) found in AD (Morishima et al., 1993). This indicates that these abnormal tau
aggregates areproduces through unsuccessful or unfinished cellular degradation process.
E. Tau Oxidation
The presence of one or two cysteines in the tau isoforms lacking or containing exon 10 has raised the possibility of the formation of dimmers of tau through protein crosslinking via disulfide bond formation. The oxidation of tau could promote the formation of disulfide bond and result in its aberrant aggregation (Schweers et al., 1995).
Recently, in relation to tau oxidation, the formation of dityrosine crosslinkings (Giunta et al., 2002) and tyrosine nitration has been described in a tau molecule (Mailliot et al.,
2002).
F. Other Modifications of Tau
Tau truncation has been defined as the cleavage at the glutamic acid residue 391
(Wischik et al., 1997). This modification could facilitate aberrant tau aggregation
(Wischik et al., 1997). The deamidation of tau at asparagine (or glutamine) residues has
also been described (Watanabe et al., 2000) and it could play a role in tau aggregation
(Montejo de et al., 1986).
It has been proposed that tau may become cross-linked through the enzymatic reaction modulated by transglutaminase (Miller et al., 1995). Moreover, modifications involving a conformational change of tau protein, and that could involve proline cis-trans
isomerization, could also occur (Wedemeyer et al., 2002).
1.3.6 Tau Degradation
In vitro studies have already showed that tau could be degraded by different
proteases, such as lysosomal enzyme calpain (Johnson et al., 1989). However,
58 phosphorylated tau is more resistant to proteolysis by calpain degradation than unphosphorylated tau (Wang et al., 1995). Ubiquitination is not necessary for tau degradation through the ATP-dependent proteasome pathway (26S proteasome). There is evidence that tau can be degraded by the 20S proteasome without ubiquitination (David et al., 2002) and monoubiquitinylated tau (or with the addition of fewer than 5 moieties) can
be subjected to lysosomal degradation (Murphey et al., 2002). It is unclear whether polyubiquitinylated tau could be degraded by the ATP-dependent proteasome pathway.
While proteins like β-catenin need to be phosphorylated by GSK3 to be degraded, it looks
like tau protein is the opposite case. In addition, tau cleavage by caspases has been reported recently (Rohn et al., 2002).
1.3.7 Tau Functions
Weingarten et al. (1975) did the pioneer work clearly showing that tau facilitates
tubulin assembly. Later on it was found that tau both stabilizes polymerized microtubules
and nucleates microtubules (Drubin et al., 1986; Bre et al., 1990; Drechsel et al., 1992)
and that tau could also suppress microtubule dynamics (Bre et al., 1990; Panda et al.,
1995).
By depleting tau using antisense oligonucleotides, it was seen that tau is involved
in neurite outgrowth (Caceres et al., 1990, 1991). Additionally, in nonneuronal cells, the expression of tau induced the formation of long cytoplasmic extensions (Knops et al.,
1991) and resulted in microtubule stabilization and bundling (Lee et al., 1992; Knowles et al., 1994). Since the tau binding site on the tubulin molecule overlaps with that for other proteins like the molecular motor kinesin, tau could also influence axonal transport processes (Ebneth et al., 1998, Terwel et al., 2002).
59 The tau-deficient mouse produced by gene targeting has been seen to be quite normal (Harada et al., 1994), and the only identified differencescompared with wild-type are a decrease in numbers of microtubule in small caliber axons (Harada et al., 1994),
muscle weakness, and some behavioral deficits (Ikegami et al., 2000). This may be explained by the compensation for the lack of tau by other proteins, such as MAP1A, which has been found to increase in mice lacking tau (Harada et al., 1994). Consistently,
defects in axonal elongation were found in mice lacking both MAP1B and tau (Takei et
al., 2000).
Little is known about the function of tau as a membrane-associated protein
(Arrasate et al., 1997, Brandt et al., 1995a) and its nuclear localization (Brady et al.,
1995, Greenwood et al., 1995). As tau also binds to many different proteins with diverse
functions, it may therefore play different roles in the wide variety of processes in which
those proteins are involved.
1.3.8 Tau Polymerization
Oxidation could play a role in tau aggregation. Oxidation of cysteine to produce
disulfide cross-linking favors tau self-assembly in tau 3R molecules, where a single
cysteine is present, but not in tau 4R molecules, where the presence of two cysteines may
permit the formation of intramolecular disulfide bonds (Barghorn et al., 2002). In vitro
studies have shown that tau can be assembled through oxidation processes (Fenton's reaction) in the presence of iron (Troncoso 1993). Also, fatty acids like arachidonic acid can induce tau polymerization in vitro (Abraha et al., 2000, Wilson et al., 1997). This
type of polymerization could be related to the possible interaction of tau with plasma membrane components (Arrasate et al., 2000, Brandt et al., 1995). Another lipid
60 peroxidation molecule-HNE has been shown facilitating tau aggregation in vitro and in cells (Perez et al., 2000, 2002). (See more details in Chapter 4)
1.3.9 Animal Models
Transgenic animals expressing genomic human tau, as well as mice carrying cDNAs encoding either the largest or the smallest CNS isoform of human tau have been generated. In addition, transgenic mice carrying tau cDNA with the missense mutations found in FTDP-17 patients have also been obtained. Finally, an additional strategy to
generate animal models of tauopathies has been to overexpress the kinases responsible for tau hyperphosphorylation.
Mice have been generated that overexpress the genomic sequence of human tau, containing the coding sequence, intronic regions, and the regulatory regions of the gene
(Duff et al., 2000). The human tau is distributed in neurites and at synapses but is absent from cell bodies, and no neuropathological lesions were reported in mice of up to 8 months of age.
The polymers assembled from tau only contain the four-repeat isoforms among
majority of the FTDP-17 patients, (Heutink et al., 1989), and in several FTDP-17 families, the only tau mutations found have been those that affect the splicing of exon 10, increasing the ratio of four-repeat with respect to three-repeat isoforms (Heutink et al.,
1989). Taking these observations into account, it was suggested that the four-repeat forms of tau may favor fiber formation, and thus mice have been generated to test this hypothesis that carry cDNAs encoding either the largest or the smallest CNS isoform of human tau (Gotz et al., 1995). On the other hand, several transgenic animals
overexpressing the shortest isoform have been obtained but using different transgene
61 promoters. With the murine 3-hydroxy-methyl-glutaryl CoA reductase promoter (Brion et
al., 1999), hyperphosphorylated somatodendritic transgenic tau was detected although
NFTs did not appear to form in these animals. The level of expression of the same tau
isoform was increased by using the murine PrP promoter (Ishihara et al., 1999).
Transgenic lines with high levels of overexpression were not viable while lines with less
than 10-fold overexpression of tau protein developed inclusions in cortical and brain stem
neurons. These inclusions were most abundant in spinal cord neurons and were correlated with axon degeneration, diminished microtubules, reduced axonal transport in ventral roots, spinal cord gliosis, and motor weakness. NFT-like inclusions (detected by histochemistry using dyes such as Congo red and Thioflavin S) were detected in the same
transgenic mice at 18–20 mo of age (Ishihara et al., 2001). In addition, filaments were isolated from detergent-insoluble tau fractions (Ishihara et al., 2001).
The first transgenic mice carring cDNAs encoding the largest human brain tau
isoform and showing low levels (10%) of overexpression (Gotz et al., 1995) has been studied. Transgenic human tau protein was present in nerve cell bodies, axons, and dendrites. Tau was phosphorylated at sites that are hyperphosphorylated in PHFs, although filaments were not detected. The murine Thy 1.2 promoter has been used by two groups to drive the expression of the cDNA encoding the longest human brain tau isoform, although on different genetic backgrounds. In the first case, axonal degeneration
in the brain and spinal cord was detected in the mice (Spittaels et al., 1999). Axonal
dilation with accumulation of neurofilaments, mitochondria, and vesicles was also observed. The axonopathy and the accompanying dysfunction of the animals’ sensory
motor capacities were transgene-dosage dependent. The mice generated by the second
62 group (Probst et al., 2000) contained numerous abnormal, tau-immunoreactive nerve cell bodies and dendrites. In addition, large numbers of pathologically enlarged axons containing neurofilament and tau-immunoreactive spheroids were present, especially in
the spinal cord.
The third approach to generate animal models of tauopathies is based on that
mutations in tau are associated with FTDP-17 (Clark et al., 1998, Goedert et al., 1998,
Grover et al., 1999). Recently two groups have reported the generation of transgenic mice
expressing mutant human tau containing the P301L mutation (Gotz J et al., 2001, Lewis
et al., 2000), which is located in the tubulin-binding domain and reduces the affinity of tau for microtubules. These animals developed NFTs mainly in the spinal cord, and
similar to the previous transgenic models developing some neuropathological symptoms
encouragingly reminiscent of the human disease. In the second transgenic model published, short filaments of tau could be isolated from the brains of the transgenic mice
(Gotz J et al., 2001).
More recently, another transgenic mouse model containing the P301L mutation
has been characterized (Sahara et al., 2002), as well as one containing the P301S mutation
(Allen et al., 2002). In the latter model, abundant tau filaments within a PHF structures were observed. Interestingly, in the transgenic mouse expressing the tau P301L mutation, tau filaments were only observed in old female mice but not in their male counterparts.
This could be due to a decrease in the amount of tau in male mice (Bu et al., 2002).
Filaments were also found in transgenic mice expressing the mutation R406W of human
tau (Tatebayashi et al., 2002), a mutation that decreases the phosphorylation of tau at the
site recognized by Ab PHF-1 (Perez et al., 2000). Finally, it has been shown that
63 filaments are formed in transgenic mice expressing mutant (V337M) human tau
(Tanemura et al., 2002).
In summary, a significant body of data demonstrates that a large excess of normal
or mutated human tau can produce some of the cellular changes observed in tauopathies, but this may not be sufficient for the formation of the mature neurofibrillary aggregates
observed in the human diseases. Nevertheless, some transgenic lines do develop NFT-like
structures as well as short PHF-like filaments. What these approaches seem to indicate is
that, first, tau is linked to neurodegeneration, second, that neurons with long axons, such as those present in spinal cord, seem to be especially susceptible to alterations in tau. We have recently produced a transgenic mouse expressing three FTDP-17 missense mutations in tau: G272V, P301L, and R406W (Lim et al., 2001). Ultrastructural analysis of mutant tau-positive neurons revealed a pre-tangle appearance; filaments of tau were found as well as increased numbers of lysosomes displaying aberrant morphology similar to those found in AD (Lim et al., 2001).
1.3.10 Tau and Neurodegenerative Diseases (Tauopathies)
Tauopathies are considered as a group of disorders resulting from abnormal tau
phosphorylation, abnormal levels of tau, abnormal tau splicing, or mutations in the tau
gene. In some tauopathies, like AD or Down's syndrome, the tau pathology is associated with other cerebral changes.
A. AD
AD is the most common and the best-studied tauopathy. The disease results in widespread atrophy in the brain that begins in the temporal and parietal lobes. It leads to
problems in short-term memory, a function associated to the temporal lobe, visual and
64 spatial dysfunction, and poor performance of over-learned tasks related to the functions of
the parietal lobe (Wilhelmsen et al., 1999). Tau aggregates from AD are composed of the
six CNS tau isoforms in their phosphorylated form. The six tau isoforms are as follows:
1) those containing exons 2, 3, and 10, plus all the constitutive exons (Baker et al., 1999);
2) those having exons 2 and 3; 3) those containing exons 2 and 10; 4) those having only
exon 2; 5) those with only exon 10; and 6) only containing the constitutive exons. This combination of hyperphosphorylated tau isoforms results in the appearance of three major electrophoretic bands with a mobility corresponding to that of proteins with a relative molecular weight of 68,000, 64,000, and 60,000 (Delacourte et al., 1999; Greenberg et al., 1992; Lee et al., 2001). Furthermore, AD brains contain higher quantities of tau compared with unaffected controls (Khatoon et al., 1992, 1994).
The best-known tauopathy is AD, in which two main pathological structures are formed in the brains of patients: senile plaques (composed of the β-amyloid peptide), and neurofibrillary tangles (NFTs). These NFTs are made up of by paired PHF comprising of hyperphosphorylated tau (Grundke-Iqbal et al., 1986, Ihara et al., 1986). The number of
NFT has been correlated with the degree of dementia in this disease (Arriagada et al.,
1992), and thus there is great interest to study how these structures are formed. The formation of PHF from tau molecules may involve different steps and could include tau phosphorylation (although this may not be essential), a conformational change in the
protein, and finally polymerization. If indeed phosphorylation does facilitate tau assembly
into PHF, it will be of interest to know which kinases (and phosphatases) contribute to the phosphorylation of tau molecules to be reached.
B. Corticobasal Degeneration
65 Corticobasal degeneration is a disorder characterized by cognitive disturbances
like aphasia and apraxia, moderate dementia, and motor disturbances such as rigidity,
limb dystonia, and tremor. Pathological analyses have indicated frontoparietal atrophy
and glial and neuronal tau inclusions. Tau is also present in hyperphosphorylated form,
but only the 68,000- and 64,000-molecular weight forms can be detected by
electrophoresis.
C. Downs' Syndrome
Downs' syndrome is due to the trisomy of chromosome 21 that results in the
defective growth and maturation of the brain, producing a cognitive impairment and
dementia at 50 years of age. In this disorder, tau is also hyperphosphorylated yielding a
pattern similar to that of AD.
D. Frontotemporal Dementia With Parkinsonism Linked to Chromosome 17
In this disorder, the patient displays frontotemporal atrophy, with neural loss, gliosis, and cortical spongiform changes in the lobes. Those result in behavioral changes, language deficit, and hyperorality. Tau inclusions were observed in neuron and glia cells, and two types of hyperphosphorylated tau form were detected. In some cases, the pattern is similar to that of AD, with the 68,000-, 64,000-, and 60,000-molecular weight forms being detected, while in other cases the pattern is like CBD and only the 68,000- and
64,000-molecular weight forms were found. This is due to intronic mutations that result in the forced expression of exon 10 (Grover et al., 1999; Hutton et al., 1998; Spillantiniet al., 1998; Varani et al., 1999). On the other hand, mutations resulting in the deletion of lysine-280 lead to reduced splicing of exon 10 (Rizzu et al., 1999), while the G342V
mutation may affect the splicing of exons 2 and 3 (Lippa et al., 2000). Some factors
66 involved in the alternative splicing of tau have been already identified (D’Souza et al.,
20002), and more than 25 mutations in tau have been identified in exons 1, 9, 10, 11, 12,
and 13. These mutations may effect RNA splicing or the protein levels of tau. These tau
(missense) mutations preferentially occur in the microtubule binding region (exon 10), decreasing binding of the mutated protein to microtubules (Hasegawa et al., 1996, Hong et al., 1998) or affecting the binding of tau to other proteins that bind to that region of tau
(Goedert et al., 2000). Furthermore, tau self-assembly was increased in most of these
mutated tau proteins (Arrasate et al., 1999).
E. Pick's Disease
Pick's disease is a dementia that produces disturbances in language and behavior
and is associated with frontal lobe atrophy. It provokes changes in the character of the
patient and in their relationships with others, as well as depression. This disorder is characterized by the presence of cytoplasmic tau inclusions in neurons of the frontal lobe, known as Pick bodies. The granular cells of the dentate gyrus are also affected. The appearance of inclusions is in agreement with the appearance of 64,000- and
60,000-molecular weight hyperphosphorylated tau, indicating the absence of exon 10 expression. This exon is not expressed in tau present in the granular cells of the dentate gyrus (Goedert et al., 1989), suggesting that the degeneration of selective neuronal
populations in different tauopathies reflects the physiological pattern of tau isoforms expressed.
F. Postencephalic Parkinsonism
This disorder appears to be the consequence of an encephalic parkinsonism found
in patients that survived the Spanish influenza pandemia (Geddes et al., 1993). Different
67 brain regions are affected, but tau inclusions are mainly found in the hippocampus and the
putamen. The electrophoretic pattern for hyperphosphorylated tau is similar to that of AD.
G. Progressive Supranuclear Palsy
Progressive supranuclear palsy (PSP), also known as the Steele, Richardson, and
Olszewski disorder, is characterized by supranuclear gaze palsy, prominent postural instability, and dementia in the later stages. Tau inclusions have been found in neuronal and glial cells, with both astrocytes (tufted astrocytes) and oligodendrocytes (coiled bodies) being affected. Recently, as in FTDP-17, missense mutations have been found in
PSP patients with a monogenic (familial) origin (Delisle et al., 1999), and in fact to date,
mutations in Tau have been identified in PSP (J. G. De Yebenes, personal communication), CBD (Bugiani et al., 1999), and Pick's disease (Murrell et al., 1999).
Some specific tau polymorphisms may be a risk factor for PSP (De Yebenes et al., 1999), and these polymorphisms are based on the different numbers of repeats of a dinucleotide repeat (TG) present in the intron between exons 9 and 10 (see above). Individuals containing 11 dinucleotide repeats (tau allele A0) have been seen to have a greater risk of developing PSP.
H. Niemann-Pick Type C Disease
Niemann-Pick type C disease is an autosomal recessive lysosomal lipid storage disorder, mainly caused by mutations within the NPC-1 gene (Vanier et al., 1996). The clinical features are motor disturbances (due to cerebellar dysfunction) and dementia
(Vanier et al., 1991). The pattern of hyperphosphorylated tau is similar to that of AD.
I. Other Tauopathies
68 Other tauopathies involving hyperphosphorylated tau include parkinsonism with dementia, myotonic dystrophy, prion diseases with tangles, Blint disease (an ophthalmologic disorder), dementia pugilistica, dementia with tangles only or amyotrophic lateral sclerosis, and parkinsonism-dementia complex of Guam. These have been well-documented in the reviews of Buee et al. (2000) and in that of Ingram and
Spillantini (Ingram et al., 2002).
1.3.11 Summary
Tau is a microtubule-associated protein that is not essential for mammalian development, probably due to functional redundancy and the presence of other microtubule-associated proteins. However, in pathological situations tau may be
hyperphosphorylated and assembled in an aberrant way. Because of these modifications,
neural toxicity are augmented, resulting in the appearance of neurological disorders, mainly dementias, which are collectively known as tauopathies. Different types of neurons are damaged in different tauopathies, and the elucidation of the mechanisms
underlying the basis for this specificity will probably be the goal of several laboratories
studying in neurodegeneration.
69 1.4 Introduction of Oxidative Modification
1.4.1 Free Radical Theory of Aging
Aging is the seemingly inevitable decline in physiologic function that occurs over time. For all living organisms, the ultimate ending of aging is the same: death. At least four major theories of aging have been proposed to explain most or all of the cause of biological aging:
A. The free radical theory of aging (FRTA) (Harman, 1956);
B. The mitochondrial theory of aging (Harman,1972);
C. The membrane hypothesis of aging (Zs-Nagy, 1978);
D. The cross-link theory of aging (Kleinsek, 2002).
Dr. Denham Harman, the establisher of the free radical theory of aging, has defined aging as the increased probability of death with the increase of the age of an organism increases, and the accumulation of diverse adverse physiologic changes. The
FRTA was first proposed by Dr. Harman almost 50 years ago in November 1954
(Harman, 1992), and his groundbreaking original paper on the FRTA was published in
1956 (Harman, 1956). Free radicals, the highly reactive, small oxygen-containing molecules, play key roles in the mitochondrial, membrane and cross-link theories, as well as in the FRTA.
1.4.2 Free Radicals
Free radical is simply a molecule carrying an impaired electron. All free radicals are extremely reactive and ready to acquire an electron in any way possible. In the process of acquiring an electron, the free radical will attach itself to another molecule, thereby modifying it biochemically (Bradford and Allen, 1997). However, as free radicals
70 acquired an electron from the other molecules, they convert these molecules into free radicals, or break down or alter their chemical structure. Thus, free radicals are capable of damaging virtually any biomolecule, including proteins, sugars, fatty acids and nucleic acids (Leibovitz and Siegel, 1980). Harman points out that free radical damage occurs to long-lived biomolecules such as collagen, elastin and DNA; mucopolysaccharides; lipids that make up the membranes of cells and organelles such as mitochondria and lysosomes; components of blood vessel walls; and proteins and lipids that combine and accumulate as large pigment- lipofuscin (Harman, 1984).
The main radicals are superoxide radical, hydroxyl radical, hydroperoxyl radical, alkoxyl radical (AR), peroxyl radical (PR) and nitric oxide radical (NOR) (Jacob, 1995).
Other molecules that are technically not free radicals, but act much like them, are singlet oxygen, hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) (Jacob, 1995).
Collectively, the free radicals and non-free radical mimics are called oxidants or reactive oxygen species.
Free radicals are extremely short-lived because of their extreme reactivity. The longest half-life is 1-10 seconds for nitric oxide radical; the shortest (and most deleterious) is only one nanosecond (10 -9 sec) for hydroxyl radical (Jacob, 1995). A broad range of the chief diseases of aging, including cancer, heart/artery disease, essential hypertension, Alzheimer’s disease, aging immune deficiency, cataracts, diabetes,
Parkinson’s disease, arthritis and inflammatory disease, as well as aging itself, are now believed to be caused in whole or part through free radical damage (Harman, 1984;
Jacob, 1995; Ames et al., 1993; Pierrefiche and Laborit, 1995).
1.4.3 The Primary Sources of Free Radicals
71 There are four primary sources of oxidants formed within living organisms. The major source of free radicals and oxidants is the mitochondrial generation of ATP energy using oxygen. A small percentage (2-3% or less) of oxygen in mitochondria is inadvertently converted to superoxide radical, which can in turn generate hydrogen peroxide, hydroxyl radical, and all other free radicals (Beckman and Ames, 1998, Ames et al., 1993; Pierrefiche and Laborit, 1995). A second source of oxidants, especially hydrogen peroxide, is the peroxisomes, organelles that degrade fatty acids (Beckman and
Ames, 1998; Ames et al., 1993). A third source of oxidants is cytochrome P450 enzymes.
These enzymes help cells, especially in the lungs and liver, detoxify a broad range of potentially toxic food, drug and environmental pollutant molecules. Superoxide radical is a by-product of many of these detoxification reactions (Beckman and Ames, 1998; Ames et al., 1993).
Finally, white blood cells (phagocytes) attack germs with a mixture of oxidants including superoxide radical, hydrogen peroxide, nitric oxide radical, HOCl, and hydroxyl radical (Beckman and Ames, 1998; Leibovitz and Siegel, 1980; Ames et al.,1993). This may create serious free radical problems, especially in those suffering a chronic immune-activation condition, such as AIDS, chronic candidiasis, protozoal infections, chronic fatigue syndrome, etc. (Beckman and Ames, 1998 ; Ames et al.,
1993). In addition, various biomolecules including hydroquinones, flavins, catecholamines, thiols, pterins and hemoglobin, may spontaneously auto-oxidize and produce superoxide radical (Leibovitz and Siegel, 1980).
From outside the body, polluted urban air, cigarette smoke, iron and copper salts, some phenolic compounds found in many plant foods, and various drugs may all
72 contribute free radicals or provoke free radical reactions (Leibovitz and Siegel, 1980;
Ames et al.,1993).
1.4.4 Physiological Damages Caused by Free Radicals
Free radicals and ROS lead to several damaging effects as they can attack lipids, protein/
enzymes, carbohydrates, and DNA in cells and tissues. They induce undesirable
oxidation, causing membrane damage, protein modification, DNA damage, and cell death
induced by DNA fragmentation and lipid peroxidation. This oxidative damage/stress,
associated with ROS is believed to be involved not only in the toxicity of xenobiotics but
also in the pathophysiological role in aging of skin and several diseases like heart disease
(atherosclerosis), cataract, cognitive dysfunction, cancer (neoplastic diseases), diabetic
retinopathy, critical illness such as sepsis and adult/acute respiratory distress syndrome,
shock, chronic inflammatory diseases, organ dysfunction, deep injuries, production of
nitric oxide by the vascular endotheliums, vascular damage caused by ischaemia
reperfusion (Halliwell et al, 1984). Iron changes have been detected in multiple sclerosis,
spastic paraplegia, and amyotrophic lateral sclerosis, which reinforces the belief that iron
accumulation is a secondary change associated with neurodegeneration in these diseases,
although it could also be related to gliosis (glia might produce free radicals) in the
diseased area, or the changes in the integrity of the blood brain barrier caused by altered vascularisation of tissue or by inflammatory events (Gutteridge et al., 1986).
1.4.5 Antioxidant Systems
Endogenous Antioxidants
Biological systems have evolved with endogenous defense mechanisms to help
protect against cell damage induced by free radicals. Glutathione peroxidase, catalase,
73 and superoxide dismutases (SOD) are antioxidant enzymes, which metabolize toxic
oxidative intermediates. They require micronutrient as cofactors such as selenium, iron,
copper, zinc, and manganese for optimum catalytic activity and effective antioxidant
defense mechanisms (Halliwell et al., 1992; 2001). SOD, catalase, and glutathione
peroxidase are three primary enzymes, involved in direct elimination of active oxygen
species (hydroxyl radical, superoxide radical, and hydrogen peroxide). In addition,
glutathione reductase, glucose-6-phosphate dehydrogenase, and cytosolic GST are
secondary enzymes, which help in the detoxification of ROS by decreasing peroxide
levels or maintaining a steady supply of metabolic intermediates like glutathione and
NADPH necessary for optimum functioning of the primary antioxidant enzymes
(Vendemialeet al., 1999; Singh et al., 2003).
Glutathione, ascorbic acid, alpha-tocopherol, betacarotene, bilirubin, selenium,
NADPH, butylhydroxyanisole (BHA), mannitol, benzoate, histidine peptide, the
iron-bonding transferrin, dihydrolipoic acid, reduced CoQ10, melatonin, uric acid, and
plasma protein thiol, etc., as a whole play a homoeostatic or protective role against ROS produced during normal cellular metabolism and after active oxidation insult. Glutathione is the most significant component that directly quenches ROS such as lipid peroxides
(like HNE) and plays major role in xenobiotic metabolism. When an individual is exposed to high levels of xenobiotics, more glutathione is utilized for conjugation making it less available to serve as an antioxidant. It also maintains ascorbate (vitamin C) and alpha-tocopherol (vitamin E), in their reduced form, which also exert an antioxidant effect by quenching free radicals (Meister et al., 1994; Sies et al., 1995; Anderson et al.,
1996).
74 Exogenous Antioxidants: Contribution From the Diet
The most widely studied dietary antioxidants are vitamin C, vitamin E, and
beta-carotene. Vitamin C is considered the most important water-soluble antioxidant in
extracellular fluids, as it is capable of neutralizing ROS in the aqueous phase before lipid
peroxidation is initiated. Vitamin E is a major lipid-soluble antioxidant, and is the most
effective chain-breaking antioxidant within the cell membrane where it protects
membrane fatty acids from lipid peroxidation. Beta-carotene and other carotenoids also
provide antioxidant protection to lipid rich tissues. Fruits and vegetables are the major
sources of vitamin C and carotenoids, while whole grains, i.e., cereals and high quality
vegetable oils are the major sources of vitamin E (Block et al., 1992; Halliwell et al.,
1994; Jacobet al., 1995).
A number of other dietary antioxidants exist beyond the traditional vitamins
collectively known as phytonutrients or phytochemicals, which are being increasingly
appreciated for their antioxidant activity. One example is flavonoids, which are a group
of polyphenolic compounds. These are widely found in plants as glucosylated derivatives.
They are responsible for the different brilliant shades such as blue, scarlet, and orange.
They are found in leaves, flowers, fruits, seeds, nuts, grains, spices, different medicinal
plants, and beverages such as wine, tea, and beer (Harborne et al., 1994; Weisburger et al., 1997; Pietta et al., 2000; Gale et al., 2001).
Flavonoids exhibit several biological effects such as antitumoral, anti-ischaemic, anti-allergic, anti-hepatotoxic, antiulcerative, and anti-inflammatory activities. These are also known to inhibit the activities of several enzymes, including lipoxygenase, cyclooxygenase, monooxygenase, xanthine oxidase, glutathione-S-transferase,
75 mitochondrial succino-oxidase, NADH oxidase, phospholipase A2, and protein kinases.
Many of the biological activities of flavonoids are attributed to their antioxidant properties and free radical scavenging capabilities. The antioxidant activities of flavonoids vary considerably depending upon the different backbone structures and functional groups. A number of flavonoids efficiently chelate trace metals, which play an important role in oxygen metabolism. Free iron is a potential enhancer of ROS formation as it leads to reduction of H2O2 and generation of the highly aggressive hydroxyl radical.
Free copper mediates LDL oxidation and contributes to oxidative damage due to lipid peroxidation (Shu et al., 1998). Due to the inefficiency of our endogenous defense systems as well as the existence of some physiopathological situations, such as cigarette smoke, air pollutants, UV radiation, inflammation, ischaemia/reperfusion, etc., ROS can be produced in excess, and increasing amounts of dietary antioxidants will be needed for diminishing the cumulative effect of oxidative damage over an individual’s life span (Sun et al., 2002).
1.4.6 Oxidative Stress in The Nervous System
The oxidative stress is a shift towards the pro-oxidant in the pro-oxidant/antioxidant balance that can occur as a result of an increase in oxidative metabolism. Its increase at the cellular level can come as a consequence of several factors, including exposure to alcohol, aging, medications, trauma, infections, toxins, radiation, strenuous physical activity, and poor diet. Defense against all of these processes is dependent upon the adequacy of various antioxidants that are derived either directly or indirectly from the diet (Gotz et al., 1994).
The nervous system, including the brain, spinal cord, and peripheral nerves, is
76 rich in both unsaturated fatty acids and iron. The high lipid content of nervous tissue, coupled with its high aerobic metabolic activity, makes it particularly susceptible to oxidative damage. The high level of iron may be essential, particularly during brain development, but its presence also means that injury to brain cells may cause the release of iron ions, which leads to oxidative stress via the iron-catalyzed formation of ROS
(Bauer et al., 1999; Andorn et al., 1990). In addition, those brain regions that are rich in catecholamines are exceptionally vulnerable to free radical generation. The catecholamine adrenaline, noradrenaline, and dopamine can spontaneously break down
(auto-oxidise), or can be metabolized to radicals by the endogenous enzymes such as
MAO (monoamine oxidases) to form free radicals. One such region of the brain is the substantia nigra (SN), where a connection has been established between antioxidant depletion (including GSH) and tissue degeneration (Perry et al., 2002). A number of in vitro studies have shown that antioxidants, both endogenous and dietary, can protect nervous tissue from damage by oxidative stress (Contestabile et al., 2001). Uric acid, an endogenous antioxidant, was found to prevent neuronal damage in rats, both in vitro and in vivo, from the metabolic stresses of ischemia, oxidative stress as well as exposure to the excitatory amino acid glutamate and the toxic compound, cyanide. Vitamin E was found to prevent cell death (apoptosis) in rat neurons subjected to hypoxia followed by oxygen reperfusion (Yu et al., 1998). In the same study, it was shown that vitamin E prevented neuronal damage from reactive nitrogen species. Both vitamin E and beta-carotene were found to protect rat neurons against oxidative stress from exposure to ethanol (Copp et al., 1999). In an experimental model of diabetes-caused neurovascular dysfunction, beta-carotene was found to protect cells most effectively, followed by
77 vitamin E and vitamin C (Mitchell et al., 1999).
Most in vivo and clinical studies of the effects of lipid-soluble antioxidant
supplementation on neurological diseases have focused on vitamin E. A report in 1991
demonstrated that the rate at which Parkinson’s disease progressed to the point when the
patient required treatment with levodopa was slowed by 2.5 years in patients given large
doses of vitamin C and synthetic vitamin E (Prasad et al., 1999). Although one study
reported that high doses of vitamin E resulted in elevated plasma levels but failed to
increase vitamin E levels in cerebrospinal fluid (CSF), a later report demonstrated that
high doses of vitamin E did result in elevation of CSF vitamin E levels, and possibly
brain vitamin E levels (Pappert et al., 1996). Recently it was shown that the protein
responsible for the uptake of vitamin E is in fact present in brain cells of patients
suffering from vitamin E deficiency or diseases associated with oxidative stress (Jama et
al., 1996). In a Dutch study, it was found that the risk for Parkinson’s disease was lower for subjects who had higher dietary intakes of antioxidants, particularly vitamin E. The same group reported that a low dietary intake of beta-carotene was associated with impaired cognitive function in a group of persons aged 55-95; no such association was observed for either vitamins C or E (Hellenbrand et al., 1996). In an Austrian study, serum concentration of vitamin E was found to be significantly associated with cognitive function in adults aged 50 - 75 years measured by a standardized test (Schmidt et al.,
1998). In another study, it was found that patients suffering from Parkinson’s disease had consumed less of the small-molecule antioxidants betacarotene and vitamin C than did non-sufferers of the disease, implying that dietary antioxidants do play a protective role in this disease (Fahn et al., 1991). About 20% of FALS cases are associated with a
78 mutation in the gene for copper/ zinc superoxide dismutase, an important antioxidant
enzyme, and in vitro experiments demonstrated that expression of the mutant enzyme in neuronal cells caused cell death, which could be prevented by antioxidant small molecules such as glutathione and vitamin E (Ferrante et al., 1997).
There is substantial evidence that oxidative stress is a causative or at least ancillary factor in the pathogenesis of major neurodegenerative diseases, including
Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS, “Lou
Gehrig’s disease”) as well as in cases of stroke, trauma, and seizures (Ghadge et al.,
1997). Decreased levels of antioxidant enzyme activity have been reported in patients with Parkinson’s disease (Spina et al., 1989). Evidence of increase in lipid peroxidation and oxidation of DNA and proteins has indeed been seen in the substantia nigra of patients affected with Parkinson’s disease. Similar increase in markers of oxidative stress has also been seen in Alzheimer’s disease, Huntington’s disease, and in both familial
ALS (FALS) and sporadic ALS (SALS) patients (Saggu et al., 1989). It has also been reported that there are elevated lipid peroxide levels in the cerebrospinal fluid of patients maintained on neuroleptics and exhibiting symptoms of TD (Berger et al., 1997).
Weisburger et al. (1997) succeeded in decreasing the severity of TD using high doses of vitamin E, and called for further trials with combinations of various known antioxidants.
Schizophrenia (SCZ) is also believed to have a component of free-radical overload. Lipid peroxides have been found elevated in their blood and increased pentane gas, a marker for lipid peroxidation, in the breath of schizophrenics as compared with normal volunteers and with patients having other psychiatric illness (Phillips et al., 1993). The enzyme SOD was found increased, possibly as an adaptive response to free radical
79 overload in the SCZ patients (Abdalla et al., 1986). Studies of antioxidant treatment in schizophrenia have been very few; two recent studies that examined only vitamin C yielded conflicting results. As GSH peroxidase level was also found to be reduced in
SCZ patients, future trials with antioxidants in schizophrenia should include selenium and
GSH precursor nutrients (Levine et al., 1985).
1.4.7 HNE and HNE Modification in AD
In the past decade HNE (trans-4-hydroxy-2-nonenal, C9H16O2; Fig. 1.6) has been the most well-studied product of endogenous lipid peroxidation occurring in oxidative stress (Esterbauer et al., 1991). The x-6-family (linoleic and arachidonic acids) of polyunsaturated fatty acids may produce HNE as a result of free radical attack. HNE is a highly reactive electrophile and can form adducts with several nucleophilic groups of biomacromolecules. (See more details in Chapter 2, 3, 4)
1.4.8 Summary
Neurodegenerative disorders remain an important source of morbidity and suffering of the humankind. The role of free-radical-mediated oxidative injury in acute insults to the nervous system including stroke or trauma, as well as in chronic neurodegenerative disorders, is just being recognized. As we know, oxygen is an essential molecule for the survival of majority of living organisms. Oxidative stress is the harmful condition that occurs when there is an excess of free radicals and/or a decrease in antioxidant levels. There is evidence suggesting that the increase in energy metabolism via aerobic pathways enhances the intracellular concentration of free oxygen radicals, which in turn enhance the rate of the autocatalytic process of lipid peroxidation, inducing damage to brain structures, especially when physiological defenses become insufficient
80 or depleted. Antioxidants combat oxidative stress by neutralizing excess free radicals and
stopping them from setting off the chain reactions that contribute to various diseases and
premature aging. The evidence to date for oxidative stress in PD, TD, SCZ, AD and other
neurodegenerative diseases is strongly convincing. Clinical studies show that a number of
events associated with AD are capable of stimulating production of free radicals and depletion of antioxidant levels. Patients with Parkinson’s also have reduced glutathione levels and free radical damage is found in the form of increased lipid peroxidation and oxidation of DNA bases.
Tackling of the role of free radicals bring up a novel therapeutic target in those diseases. However, only when the mechanisms and involvement of free radicals in the pathogenesis of neurodegeneration as well as neurological or CNS diseases are understood, will antioxidant therapy be designed effectively and specifically targeted.
As pointed out, whether oxidative stress is eventually proven to be primary or secondary during the etiologic progression, the therapeutic rewards of antioxidants are likely to be substantial. The essential consideration is to deliver the proper scavenger to the affected site within the time frame of or prior to maximal tissue damage. Clearly, strategies aimed at limiting free radical production during oxidative stress and damage may slow the progression of neurodegenerative diseases. How can one combat the production of free radicals? Patients can replete their cellular and body stores with the body’s most important antioxidant,
81
Figure 1.6 The structure of HNE and its adducts with amino acids. A The structure of HNE and the formation of its Michael adducts with amino acids lysine histindine and cysteine. B The formation of 2 2-pentylpyrrole and fluorescent crosslink lysine adducts formed by HNE.
82 glutathione and its synergistic partner antioxidants, maintain their antioxidant defense
system, thus help to prevent or delay the progression of free radical related damages.
There are several natural and synthetic compounds already selected and extensively
studied. Preclinical experimental studies have proven that they can reduce acute
neurological disorders by preventing lipid peroxidation and diminishing free radical
generation. In fact, they appear to be highly beneficial in experimental models. Clinical
trials are therefore warranted with dietary GSH precursors, administered in combination
with other antioxidants, antioxidant cofactors, and non-antioxidant brain-trophic nutrients
such as phosphatidylserine.
1.5 Research Relevance
In the following chapters, we examined HNE modification of neurofilament
protein and Tau protein. In chapter 2, we found that neurofilament is a scavenger for
HNE and this adduction is a phosphorylation-regulated and sequence-specific modification may preserve very important function for neuron survival. In chapter 3, we further explore the protective function of neurofilament protein in cell culture. In chapter
4, we found that HNE not only is involved in NFT formation, but it also plays a major role in the formation and epitope production of NFT.
These works add significant new evidence for the critical role of Tau and neurofilament proteins in NFT formation in AD. In addition, these studies also strongly support the fact that HNE is a major player in oxidative modification and AD pathogenesis.
83
Chapter 2
Oxidative Modification of Neurofilament
Proteins in Alzheimer Disease
84 Introduction
HNE and HNE Modification in AD
In the past decade HNE (trans-4-hydroxy-2-nonenal, C9H16O2; see Figure 1.6 A in Chapter 1) has been the most well studied product of endogenous lipid peroxidation occurring in oxidative stress (Esterbauer et al., 1991). The ε-6-family of polyunsaturated fatty acids (linoleic acid and arachidonic acids) may produce HNE as a result of free radical attack. HNE is a highly reactive electrophile and can form adducts on several nucleophilic groups on biomacromolecules. HNE reacts with cysteine, histidine, and lysine residues of proteins to form Michael adducts that can be stabilized by cyclization to hemiacetals (see Figure 1.6 A in Chapter 1). Michael adducts of cysteine and histidine are irreversible and the lysine Michael adducts are formed reversibly (Nadkarni and
Sayre, 1995). HNE also forms Schiff base adducts with lysine, leading to a
2-pentylpyrrole (Sayre et al., 1993, 1996) (see Figure 1.6 B in Chapter 1) and other advanced lipoxidation end-product (ALE) adducts through condensative and oxidative evolution of initially- formed adducts (Sayre et al., 2001). The bifunctionality of HNE permits it to form a Schiff base Michael adduct crosslink between two protein-based lysines. Recently, a stable fluorescent HNE-derived lysine–lysine crosslink structure has been identified that contributes to the fluorescence seen in aged and/or diseased tissues
(Xu et al., 1999) (see Figure 1.6 B in Chapter 1). HNE-modified proteins may display altered biological functions due to conformational changes, hydrophobicity changes, and crosslink resulting from the adduction. An array of antibodies to specific HNE adducts are available to visualize the HNE adducts.
In last decade, HNE modification has been used intensively as a marker for the
85 studies related with lipid peroxidation. HNE modification has already been reported to be
significantly increased in AD brain comparing to controls (Sayre et al, 1997). Therefore, whether and how HNE is involving in the AD pathogenesis is an interesting topic that needs to be further explored.
Neurofilament Properties Related with This Study
Assembly
NF assembly is not clearly understood yet, though in vivo studies have demonstrated that NFs are obligate heteropolymers requiring NF-L to form a proper polymer with either NF-M or NF-H (Ching et al., 1991; Lee et al., 1993). NF formation is
believed to start with the dimerization of NF-L with either NF-M or NF-H subunits. The
highly conserved rod domains of the NF subunits are coiled together in a head-to-tail
fashion to form a dimer. Two coiled dimers overlap with each other in an anti-parallel,
half-staggered manner forming the tetramers. Finally, eight tetramers are packed laterally
and longitudinally together in a helical array, forming a ropelike 10 nm filament (see
Figure1.3 in Chapter 1) (Hein et al., 1994; Fuchs et al., 1998; Herrmann et al., 2000) In
the cross section of NF, there are ~32 molecules though the number may change in
different stages or conditions (Herrmann et al., 1999). The C-termini of NF-M and NF-H
are not in the coils but they form the side-arms of the NFs (Perrone et al., 2001).
These assembly characteristics of neurofilament make the C-termini of NF-H and
NF-M exposed in the cytoplasm and available for the post-assembly modifications, such
as oxidative modifications. However, this may be difficult or impossible for the
N-termini of all three subunits.
Transport
86 After their synthesis in the perikarya, neurofilament proteins (NFPs) are quickly translocated into the axons and assembled into filamentous structures. In radioisotopic labeling studies, neurofilament proteins move at a rate of 0.2–1 mm/day in the axon with the slow components whereas other organelles such as membrane vesicles can move at much faster rate of 200–400 mm/day (Okabe et al., 1990).
The mechanism by which NF proteins are transported has been a topic of debate for many years due to the lack of techniques in determining the form in which these proteins are transported. Polymer hypothesis suggests that NF subunits are assembled and transported mainly as polymeric structures (Roy et al., 2000; Wang et al., 2000), whereas the subunit hypothesis argues that the NF proteins are transported down the axon as individual subunits or small oligomers (Prahlad et al., 2000; Yabe et al., 1999; Hsieh et al., 1994; de Waegh et al., 1992). The photobleaching experiments with fluorescent-tagged proteins support the subunit hypothesis, in which the bleached axonal segment remained stationary with gradual recovery of its fluorescence without any obvious directionality (Cole et al., 1994). Recently, using GFP-tagged NF subunits and live cell imaging, two research groups have found a fast transport of NF polymers down the axon, at a rate up to 2 mm/s (Jaffe et al., 1998; Sternberger and Sternberger, 1983), which was interrupted by prolonged pauses and, resulted in a net slow velocity.
Therefore, the current understanding of NF transport is that they are transported bi-directionally in the axon along microtubules through common motors like dynein and kinesin. The average slow rate of NFs movement is because NF structures spend most of their time (>99%) pausing in the axon. The pauses might be the result of the lack of the transient interactions between motors and NF structures. Consistent with this model the
87 kinesin motor was found to co-localize with the motile NF pool in the squid giant axon
(Prahladet al., 2000). In addition, phosphorylation of NFPs (mainly on NF-H) resulted in
their dissociation from the kinesin motor, thereby decreasing their transport speed (Yabe
et al., 1999).
In the human sciatic nerves (~1m long), neurofilament may take 1-3 years to
reach the synapses. In this over long transport process, how the neurofilaments maintain
their physiological function is an interesting question.
Degradation
Many neurons extend their axons over great distances, up to 1 meter in human
sciatic nerves, to form synapses with appropriate receptor cells. To maintain the
physiological functions of the nerves, certain proteins need to have long lifetimes to span
the axon. NFPs are among them. During the transport process, degradation of NFPs is not
detected. Pulse-labeling experiments indicate that the cytoskeletal proteins are
metabolically stable during their passage through the axon, and essentially all of these proteins complete the trip to the axon terminus (Hoffman and Lasek 1975; Lasek and
Black 1977). So it is believed that the degradation of NFPs may only happen at synapses
(Lasek and Hoffman 1976; Roots, 1983), where dephosphorylation of NFs by the protein phosphatase 2A (PP2A) precedes NF degradation. This also indicates phosphorylation has a protective function for NFPs from degradation (Gong et al., 2003). At the axonal terminus, the half residence time of NF is around 2 days (Paggi et al., 1987). The disintegration of NFs during pathological conditions is accounted for by the presence of calcium-activated protease (calpains) in the axoplasm (Pant and Gainer, 1980; Buki et al.,
1999). Calpains can degrade many different proteins including important axonal
88 cytoskeletal proteins such as spectrin, microtubule-associated protein-1, tau, tubulin and
NFs, as well as several protein constituents of myelin (e.g. myelin basic protein, myelin
associated glycoprotein, proteolipid protein) (Kampfl et al., 1997). The breakdown and turnover of NFs could be accounted for by the activation of this protease in nerve termini.
It is possible that the recycling of NFs degradation products represent an important feedback mechanism regulating NFs production. The success or failure of axonal regeneration may be determined by the intrinsic neuronal factors that control the production of the axonal cytoskeleton (Buki et al., 1999).
In addition, less than 1% of total NFs synthesis was also detected in axons (Grant and Pant, 2000; Sotelo-Silveira et al., 2000), and the role of this small amount of newly synthesized NFs is unknown.
Summarizing the neurofilament transportation and degradation process, there is a very intriguing question is that how these proteins keep their integrity in such a long time and how can they deal with the intensive modifications in the cytoplasm. In this study, we provide some new findings for these questions.
Phosphorylation of Neurofilament Proteins
NFPs phosphorylation is topographically regulated within neurons. Recent studies show that phosphorylation of the NF subunits play a critical role in the regulation of the filament translocation, formation, and functions. It is also involved in the pathogenesis of some related neurodegenerative diseases.
Almost all (>99%) of the assembled NFs in myelinated internodal regions are known to be stoichiometrically phosphorylated in the Lys-Ser-Pro (KSP) repeat domains
(Hsieh et al., 1994). In contrast, the KSP repeats of NFPs in cell bodies, dendrites, and
89 nodes of Ranvier are less phosphorylated. The unphosphorylated NFs only account for
~1% of NFs in the neuron as judged by NF density and the relative volumes of the
respective compartments (de Waegh et al., 1992; Cole et al., 1994).
Although other Ser/Thr motifs are also phosphorylated, most phosphorylation
sites are in KSP motifs of the tail domains of NF-M and NF-H. It is consistent with the
studies done by Jaffe et al (1998) showing that 33 out of 38 phosphorylation sites in the
C-terminus of NFH are in KSP motifs.
Normally NF-M and NF-H undergo the post-translational hyperphosphorylation
upon entry into the axons. In vivo phosphorylation of NFPs in axons is a slow process
(Komiya et al., 1986). The explanation of this includes the slow transport of NF proteins
into the axon, delayed activation of local kinases, or more possibly the inaccessibility of
phosphate acceptor sites because of a “closed” conformation of the assembled polymer.
The topologically sequential phosphorylation may be due to the sequential opening of the
next accessible phosphorylation site after the pre-phosphorylation of the other sites (see
Figure 1.4 in Chapter 1).
NFPs phosphorylation plays multiple roles, such as axonal diameter, NF axonal
localization and assembly, C-terminal phosphorylation of NFPs, and NF transport.
The major function of the phosphorylation of NFPs is believed to be the regulation of
axonal radial growth. Extensive phosphorylation of KSP repeats in the tail domain of
NF-M and NF-H occurs primarily in axons (Sternberger and Sternberger 1983; Lee et al.,
1988; Nixon et al., 1992). This results in sidearm formation, increased
inter-neurofilament spacing, radial growth of axons, and increased conduction velocity
(de Waegh et al, 1992; Nakagawa et al., 1995; Yin et al., 1998). Phosphorylation of KSP
90 motifs triggered by a Schwann cell signal has been proposed as a key determinant of local control of NF accumulation, inter-filament spacing and radial growth of myelinated
axons (de Waegh et al., 1992; Nixon et al., 1994; Sanchez et al., 1996; 2000; Yin et al.,
1998). Recent studies using NF-H null mice, or mice transfected with a NF-H tailless mutant, suggest that neither NF-H nor its phosphorylated tail is essential for determining
neuronal caliber (Rao et al., 1998; Rao et al., 2003). These studies, together with those
using NF-M knock-out mice (Elder et al., 1999a,b; Kriz et al., 2000), indicate that NF-M
plays a more profound role in regulating the axonal diameter. Moreover, alternation of
NF-M and NF-H tail domain phosphorylation is associated with the pathology seen in
neurodegenerative disorders such as ALS and AD, in which tail domain phosphorylation and NF accumulation occur abnormally in perikarya (Sternberger et al., 1985; Manetto et al., 1988; Sobue et al., 1990; Cleveland et al., 2001).
It has been proposed that the heavily phosphorylated NF-H subunit detaches from
the transport carrier and resides in the axon for months, whereas less phosphorylated
subunits are transported at normal slow axonal transport rates and have a short residence time in the axon (Lewis et al., 1987; Nixon et al., 1991). Consistent with this hypothesis,
the studies of complete deletion of NF-H, NF-M, or both increase the rate of transport of the remaining neurofilament subunits in mouse sciatic nerves (Zhu et al., 1998)
Moreover, in cultured optic nerve axons, hypophosphorylated NF subunits have been interpreted to undergo axonal transport more rapidly than subunits more extensively
phosphorylated at their tail domains [90], and the C-terminal phosphorylation of the
NF-H subunit correlates with decreased NF axonal transport velocity (Jung et al., 2001;
Yabe et al., 2001). But Rao et al. (2002) constructed NF-H tailless mice by embryonic
91 stem cell mediated gene knock in approach and showed the NF-H tail is neither important for the axonal diameter or the transport rate. Ackerley et al. (2003) argued with these results by analyzing movement of GFP-tagged phosphorylation mutants of NF-H in neurons, which support a role for NF-H side arm phosphorylation as a regulator of NF transport. Most of the studies support phosphorylation of NF side arms regulating NF transport rates (Ackerley et al., 2000; Shea and Pant et al., 2003).
Research Relevance
As one of the most striking and earliest changes noted with the development of
Alzheimer’s disease (AD), the levels of oxidatively modified macromolecules are found increased in the brain of AD patients. These including oxidative modification of protein
(Good et al., 1996; Smith et al., 1996, 1997, 1998), nucleic acid (Nunomura et al., 1999), carbohydrate (Dong et al., 1993; Smith et al., 1994), and the adducts of lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE) (Montine et al., 1996; Sayre et al., 1997).
HNE adducts increased in neuronal cell bodies of AD cases and also presented in axons in the white matter of infants and aged controls (Smith et al., 1995; Sayre et al., 1997), which leads the question which specific axonal proteins are physiological targets of HNE modification.
In our previous studies, neurofilament heavy subunit (NFH), and to a less extent, neurofilament medium subunit (NFM), were identified to be the major targets of HNE adduction in axons (Wataya et al., 2002). This is consistent with the high lysine content in NFH and NFM, but unexpected results from studies of sciatic nerves of different animals and human brains showed that the levels of NFH-HNE adducts kept constant
92 along the axons and during the lifetime (Wataya et al., 2002). It is our great interest to find out what is (are) the mechanism(s) controlling the NFH-HNE level.
As a very unique character, NFH and NFM are almost the only proteins have multiple KSP repeats in their C-termini and more importantly these sites are also the targets of phosphorylation (Hsieh et al., 1990; de Waegh et al., 1992; Cole et al., 1994
Jaffe et al., 1998) and HNE modification (Wataya et al., 2002). In addition, we found that dephosphorylation can reduce the HNE antibody recognition, which let us to hypothesize that phosphorylation may control at least partially the level of NFH-HNE.
In this study, for the first time with in vitro analysis, we demonstrated that phosphorylation in the KSP repeats regulates the level of NFH-HNE, one type of oxidative modification on protein. It directly shows that NFH, as a cytoskeleton protein, can be a scavenger for at least one of the free radicals. With the studies of the neurofilament enriched sample, C-terminus of NFH, synthetic peptides, we show that
KSP repeats in NFH (maybe NFM) are the key motifs which used by neurons to regulate the level of HNE adducts through phosphorylation and dephosphorylation on serine residues in NFH. This is the first study that shows oxidative modification on protein can be a regulated cellular process through phosphorylation in neurons.
93 Materials and Methods
Antibodies:
The following antibodies were used throughout this study: (1) HNE-Michael antibody, rabbit antiserum to the products of HNE adduction to keyhole limpet hemocyanin
involving mainly Michael adducts of cysteine, histidine, and lysine (Uchida K et al.,
1993); (2) mouse monoclonal antibody to phosphorylated NFH (SMI-34; Sternberger
Monoclonals), (3) mouse monoclonal antibody to nonphosphorylated NFH (SMI-32;
Sternberger Monoclonals). The specificity of HNE-Michael antibody is confirmed with
these three methods. a) The genesis of the antibody is using HNE-KLH as immunogen. b)
This antibody can recognize HNE-Lys, HNE-His, HNE-Cys compounds and the
immunoreactivity is not diminished by immunoabsorption with the other two compounds.
c) Mass spectrometry data indicates that non-phosphorylated KSP peptides do not form
detectable HNE adducts. Specificity of the various antibodies was verified in all cases
by immunoabsorption for 16 h at 4 °C with the competing antigens or to adducts prepared from the reaction of HNE with lysine, cysteine, or histidine at a 1:1 concentration of
0.8 mM. Reaction of the latter two amino acids with HNE gives the corresponding
Michael adduct nearly stoichiometrically, whereas the reaction of HNE with lysine leads to a time-dependent mixture of mainly the reversibly formed Michael adduct but also
several additional adducts derived from initial Schiff base formation at the C1 carbonyl group of HNE (Nadkarni and Sayre ,1995). TG-4 recognizes phospho-serine 235 of Tau
(Vincent et al., 1996; Weaver et al., 2000) and MC15 recognizes phospho-Tau (Vincent
et al., 1996).
Enriched human neurofilament protein sample:
94 Enriched neurofilament protein fractions were obtained from frozen human tissue using the method of Shecket and Lasek (Shecket G et al., 1980).
Synthetic peptides:
The 20 amino acid-Peptides containing KSP repeats from both human and mouse
NFH and a random sequence were generous gift from Dr. Mervyn Monteiro (University
of Maryland, MA). Phospho/nonphospho-AKSPV and phospho/nonphospho-KSP were
synthesized from company (GeneMed Inc, CA). Phospho/nonphospho-KS were
synthesized in Dr. Sayre’s laboratory in CWRU. All the peptides are diluted to 2 mM in
PBS, TBS or water for the further studies.
HNE/dephosphorylation treatment and immunoblots:
HNE was synthesized (Nadkarni and Sayre, 1995) and initially dissolved in ethanol. Further dilutions were made in PBS, TBS, or water. NFH protein and peptide samples were incubated with 2mM HNE 37 ℃, 16 hrs.
For protein, ~20 µg/well of NFH protein were separated on 10% SDS PAGE and electrotransferred to Immobilon membrane (Millipore).
For peptides, 2 µl (2mM) peptide samples were applied on each dot on Immobilon membrane (Millipore), air dry, and rinse the membrane with methanol before usage.
Some samples were dephosphorylated (1 unit alkaline phosphatase type III
(Sigma)/50 µg protein or peptides in 0.1 M Tris, pH 8.0, with 0.01 M
phenylmethylsulfonyl fluoride for 16 h at room temperature or with 25% hydrofluoric
acid for 16 h at 4 °C after HNE treatment).
After all the pre-treatment, Membranes were blocked with 10% milk 1 hr, washed
with Tris-buffered saline-Tween 5X5 min, incubated with primary antibody 16 hrs 4 ℃,
95 after five washes of 5 min in Tris-buffered saline-Tween, peroxidase-labeled secondary antibodies were applied for 1 h at room temperature. After rinsing again as described before, the blots were developed together for the same length of time using ECL reagent
(Santa Cruz Biotechnology) and imaged by using image acquisition and analysis software
(Ultra-Violet Products Ltd.). The effective concentration of HNE and time for the
reaction were already determined in a previous study of tau protein (Takada et al., 2000).
CNBr cleavage:
cyanogen bromide (CNBr) cleaves the C-terminal of Methionine on human NFH
and gives a 135 Ka C-terminal fragment of NFH. It was performed as the method
described in literatures (Richard C et al., 1985; Elhanany E et al., 1994). 0.5 milligrams
of human NFH was incubated in the dark, at room temperature, and under argon in 50 ul of 70% formic acid containing 1 mg of CNBr (Sigma) for 3 hours. The reaction was stopped by dilution to 0.25 ml with water and dried by Speed Vacuum. The dry pellet was suspended in 0.25 ml of water and immediately separated by gel filtration with full-length NFH sample.
Chemical stabilization of HNE adducts:
KS Peptides were incubated with 2mM or 10mM HNE, pH 7.4, 37 0C in different
experiments. In the different time points, 100µl incubated samples was taken out and
quenched by 40µl, 100mM NaBH4 for 2 hours, then 20µl saturated NH4Cl was added for
1 hour. The final mixture was diluted 5 times and injected to HPLC for further analysis.
KSP repeats search in protein databases:
The website we used is http://us.expasy.org/tools/scanprosite/. We searched in
Swiss-Prot and TrEMBL protein databases for human proteins that have more than 10
96 KSP repeats. Only three proteins, NFH, NFM and human ovarian cancer related tumor marker CA125, were found .
Analysis of HNE modified AKSPV by mass spectrometry:
Both Matrix-assisted Laser Desorption/Ionization mass spectrometry (MALDI) and high performance liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (HPLC-ESI-MS/MS) techniques were applied in the experiments (Landry F. et al., 2000, Aebersold R, 2003). In MALDI-MS, 2 ul samples incubated as above were mixed with 2 ul matrix (a-cyanine, 3% TFA). And 2 ul mixed samples were put onto MS plate and dry in air. Program for small peptides were selected for the optimum detection. MS scan was performed at both positive and negative mode, at reflection mode, attention 60, 150 shots.
In HPLC-ESI-MS/MS, peptide samples were incubated as above and separated by
HPLC on a microbore column (C18aq, 5 BioResources) at 50 mL/min with a 0% acetonitrile containing 0.1% formic acid in first 10 minutes then gradient of 0 to 95% acetonitrile containing 0.1% formic acid for 60 min. These elutes were monitored by the
UV absorbance at 214 nm, and were analyzed by on-line ESI-MS. ESI-MS was performed with an ion trap LCQ mass spectrometer (LCQDUO, Finnigan). The mass spectrometer was equipped with an ESI needle with an ion spray voltage set at 3000 V and with nitrogen as the sheath gas. The capillary temperature and sheath gas pressure were set at 160 °C and 80 psi, respectively. MS scan (m/z 80-2000) was performed in the positive ion acquisition mode to detect intact molecular ions. The peptide AKSPV was detected as [M + H] + at m/z 501. The peptide phospho-AKSPV was detected as [M + H]
+ at m/z 581. The peptide HNE modified phospho-AKSPV was detected as [M + H] + at
97 m/z 737. The identification of the peptide AKSPV (m/z 501), the peptide AKSPV (m/z
581), and the peptide HNE modified phospho-AKSPV (m/z 737) were further confirmed
by collision-induced dissociation using the tandem MS/MS scan.
Data Analysis:
All immunoblots were processed following the same time and dilution parameters
to allow direct comparisons between various samples. Blots from all samples were
stained concurrently for each antibody and for exactly the same time of development. The
intensity of immunostained NFH bands was quantitated as reflectivity with an optically enhanced densitometry scanner (QS30; pdi, Inc.). Statistical analysis was performed with analysis of variance Fisher's post hoc protected least significant difference test.
98 Results
Enriched neurofilament protein samples were purified from normal human brain homogenize and electrophoresed then transferred on the Immobilon membranes. The membranes were incubated with 2mM HNE for 16 hours at 37 ℃. HNE adducts were detected as a major band around 205 kDa and dephosphorylation with alkaline phosphatase (AP) or hydrofluoric acid (HF) can reduce the adduct (Figure 2.1). When replace AP with BSA for the incubation, there is no reduction was detected (Figure 2.1).
Tau protein, also a lysine-rich and hyperphosphorylated protein, formed HNE adducts but did not show reduction of the adducts after dephosphorylation with either AP or HF (Figure 2.2).
Since the phosphorylation and probably HNE modification mostly happened in the C-terminus of NF-H, a 135 kDa C-terminal fragment of NF-H from amino acid 394 was obtained by cyanogen bromide (CNBr) cleavage which cleaves on the C-terminal side of Methionine at 9 more sites in the N-terminus of NF-H (Figure 2.3A). Treatment with HNE showed that HNE adducts were readily formed on the full length NF-H and the
C-terminus of NF-H at the similar level (Figure 2.3B) and dephosphorylation will reduce both of them (data not shown).
Because the phosphorylation sites on NF-H are mainly in the KSP repeats in the
C-terminus (Hsieh et al., 1990; de Waegh et al., 1992; Cole et al., 1994 Jaffe et al., 1998) and lysine residues are the major amino acid modified by HNE (Wataya et al.,
99
Figure 2.1 Phosphorylation state of NFH regulates level of HNE-NFH adduct.
NFH formed HNE-Michael adduct after HNE treatment and the adduct are reduced by dephosphorylation of NFH. Replacing AP by BSA, the sample did not show any effect.
AP: alkaline phosphatase.
100
Figure 2.2 Tau protein did not show the similar effect. Tau protein is a lysine rich and hyperphosphorylated protein. Tau formed HNE-Michael adduct after HNE treatment and the adduct is not reduced by dephosphorylation. HF: hydrofluoric acid, AP: alkaline phosphatases.
101
Figure 2.3 C-terminus of NFH is very reactive with HNE. A. CNBr cleavages the
C-terminal of Methanine and gives a 135 kDa C-terminal fragment of NFH that includes all the
KSP repeats. B Comparing with the full length NFH, the C-terminal fragment is also very reactive with HNE treatment.
102 2002), we hypothesized that KSP repeats maybe the most important motifs in NF-H that form the HNE adducts. To demonstrate this question, several steps were taken sequentially. First, three 20 amino acid peptides were obtained. Two of them are consensus sequences from human NF-H and mouse NF-H with three KSP repeats without phosphate groups, one of them is a random peptide without lysine. These peptides were treated with HNE. Because these peptides did not have phosphate groups, they did not show a strong modification after HNE treatment (Figure 2.4). Comparing with them, the
5 amino acid synthetic peptides—phospho-AKSPV, the most repeated KSP sequence in
NFH, showed strong HNE modifications after HNE treatment (Figure 2.5).
Nonphospho-AKSPV peptide did not form or form undetectable level of HNE adducts, whereas the phospho-AKSPV forms higher level of HNE adducts and these adducts are reduced by dephosphorylation (Figure 2.5). These data give us the most direct evidence showing that KSP repeat may play the key role in regulating HNE modification on NF-H.
To determine the minimal sequence requirement for this regulation, KSP peptides with and without phosphate groups were also synthesized. After HNE treatment and dephosphorylation, phospho-KSP peptide showed significant increase and decrease in
HNE adduct level and still preserves the regulatory properties. In the meaning time nonphospho-KSP failed to show significant changes in HNE adduct level (Figure 2.6).
Two shorter peptides, the KS peptides with or without phosphate group also were synthesized. both of these two formed very low level of HNE adduct and did not
103
Figure 2.4 Nonphospho-20 AA KSP peptides are not able to be intensively modified by HNE. 20 AA peptides are concensus sequences from human and mouse
NFH protein including 3 KSP repeats. These peptides can not be modified by HNE.
104
Figure 2.5 Phospho-AKSPV peptide is modified by HNE. Synthetic AKSPV peptides with and without phosphate group showed significant difference in forming
HNE adduct. Dephosphorylation phospho-AKSPV-HNE sample reduce the level of HNE adduct.
105
Figure 2.6 Synthetic K-S-P peptides with and without phosphation show different reactivity with HNE. Phospho-KSP peptide shows HNE-Michael adduct and this adduct is reduced by dephosphorylation. Nonphospho-KSP peptide does not show the intensive formation of HNE adduct.
106 show difference in level of HNE adduct at all (Figure 2.7). These results indicates that
flanking sequence like proline is required for preserving the phosphorylation dependent
regulation of HNE modification on NFH.
To confirm these results, the matrix assisted laser desorption/ionization (MALDI)
technique and high performance liquid chromatography-electrospray ionization-mass
spectrometry/mass spectrometry (HPLC-ESI-MS/MS) were used to study most of these
KSP peptides. Phospho-AKSPV and phospho-KSP all form higher and detectable level of
HNE-Michael adduct comparing to nonphospho-peptides and all the product peaks were
confirmed by fragmentation of the peptide peaks with MS/MS (Figure 2.8A,B,C). Almost
no detectable level of adducts were found with HNE treated nonphospho-peptides (Figure
2.8D). These data further confirm the conclusion that phosphor-peptide is easier to form
HNE adduct than non-phospho-peptides and dephosphorylation will remove not only the
phosphate groups but also HNE modification from these phosphorylated synthetic KSP peptides. The HNE adducts are not removed in control samples replacing AP with BSA
(data not shown).
HNE-Michael antibody is a polyclonal antibody that was used to detect HNE adducts in immunostainning analyses, which is generated from HNE treated Keyhole
Limpet Hemocyanin (KLH) as immunogen (Uchida et al., 1993). The specificity of the antibody was confirmed by using HNE-lysine, HNE-histidine, HNE-cysteine compounds
(Sweda et al., 1997). This polyclonal antibody recognized all three compounds with
different epitopes because the immunoreactivity of this antibody to
107
Figure 2.7 Synthetic K-S peptides with and without phosphorylation did not show significant difference in the formation of HNE adduct. Both these K-S peptides form very low level of HNE adduct and phosphor-KS peptide did not show higher level of HNE adduct formation.
108
Figure 2.8 HPLC-ESI-MS/MS confirms the removal of HNE from the HNE adduct after dephosphorylation of the sample. A. Phospho-AKSPV is detected in molecular weight around 581, and the peptide is confirmed by fragmentation. B
Phospho-AKSPV forms HNE-Michael adduct at molecular weight around 737, and this peak is confirmed by fragmentation. C HNE is removed from the Phospho-AKSPV-HNE adduct after dephosphorylation with alkaline phosphatases. Only nonphospho-AKSPV is detected as final product. D Nonphospho-AKSPV is detected in molecular weight around
501, and this peak is confirmed by fragmentation of the peak Nonphospho-AKSPV does not form HNE adduct.
109 each compound was not blocked by pre-absorption with the other two compounds (data not shown).
The phosphorylation states of neurofilament proteins are monitored by the antibody that recognizing phosphorylated neurofilament (SMI-34) and nonphosphorylated neurofilament (SMI-32).
The phosphorylation states of Tau proteins are monitored by the antibodies that recognizing phosphorylated Tau (TG-4 and MC15). TG-4 recognizes phosphoserine 235
(Vincent et al., 1996; Weaver et al., 2000) and MC15 recognizes phospho-Tau (Vincent et al., 1996).
Experiments were performed both in PBS, TBS (pH 7.4), or Millipored water, there were no significant difference in the formation and reduction of HNE adducts.
110 Discussion
This is the first study shows that NFH-HNE level is a phosphorylation-dependent and sequence-specific modification that is controlled directly by protein phosphorylation/dephosphorylation in the key motif-KSP sequence, which are extensively and uniquely repeated in the C-termini of NFH/M. With the study of the full length NFH, C-terminus of NFH, and different synthetic KSP peptides with and without phosphate group, we finally confirm that the regulative mechanism is preserved in the
KSP motifs. This finding gives a good explanation to the question why NFH-HNE level keep constant in axons during lifetime in mice, rats and human. It also may provide more evidence for the long time mystery why evolutionarily the multiple KSP repeats reside in
NFH/M uniquely. In addition, this finding may provide an explanation for those two
NFH transgenic mice studies mentioned above (Couillard-Després et al., 1998; Beaulieu et al., 2003).
In our studies with the phospho/nonphospho-KS peptides, we have not found the similar properties like NFH/M, the phospho/nonphospho-AKSPV peptides, and phospho/nonphospho-KSP peptides. Therefore, KSP, the three amino acid motif, is clearly the minimal sequence that preserves the phosphorylation-dependent regulation for
NFH/M-HNE adducts.
Even the glutathione (GSH) is considered the major scavenger for HNE in vivo, it
may only account for 30- 50% of total HNE (Siems et al., 1997 a, b; Forman et al., 2003)
and there are up to 10% of total HNE adducts to proteins (Siems et al., 1997 a, b). HNE modifications can cause dysfunction of the macromolecules and eventually insults in cell death (Nakashima et al., 2003). Therefore, the neurons as the most vulnerable cells in the
111 body must have certain defense systems to protect the cells from death.
HNE as a lipid peroxidation product from peroxidation of ω 6 polyunsaturated fatty acids (like arachidonic acid and linoleic acid) was consider the most toxic compounds in cellular oxidative stress (Uchida et al., 1993; Liu et al., 2003). HNE can be existed in a relatively longer time and diffused cellularly to the closed organelles or extracellularly to other cells to adduct with all the macromolecules (Reviewed in Eckl P.
M., 2003; Nakashima et al., 2004; Alary et al., 2004). The concentration of natural induced HNE can be high enough to induce apoptosis (Shackelford et al., 2000; Haynes et al., 2001; Anuradha et al., 2001; Mark et al., 1997; Choudhary et al., 2002; Cheng et al.,
2001; Lee et al., 2004). So the neurons as the most vulnerable cells in the body must have the defense system to protect the cells from death. Even the GSH is considered the major scavenger for HNE in vivo, it may only account for 50% of HNE (Forman et al., 2003) and it still has the possibility to have free HNE time to time (Uchida et al., 1993). If the
HNE adducts are accumulated in the cells, this will causes dysfunction of the macromolecules and eventually insults in cell death.
HNE reacts with proteins can form HNE-Michael adduct, HNE-pyrrole adduct, and protein crosslink (Liu et al., 2003). HNE-Michael adducts account for ~80% of HNE adducts and only Lysine-HNE-Michael adduct is reversible (Montine et al., 1996; Sayre et al., 1997). In neurofilament protein, the lysine enriched protein, lysine is the major amino acid that adducted by HNE, which provides the possibility for neurons to remove the adducts through cellular regulation. In Tau protein, lysine residues are not likely the major amino acid adducted by HNE, because Tau contains certain amount of cysteine and histidine residues, which will form irreversible HNE-Michael adducts easier than lysine
112 residues, so even Tau is phosphorylated and has two KSP in the protein, it did not show
the same regulation (Table 2.1). Only peptides with KSP and phosphorylation on serine
residue preserved the phosphorylation regulated HNE adduction. In most conditions, the
non-phospho-KSP peptides and peptides with lysine did not even show detectable level
of HNE adduct compared with phospho-KSP peptides (Table 2.1), which also suggests
that phosphorylation somehow promotes HNE adduction.
It has already been shown that HNE can induce the activation of MAPK,
PI3K/AKT pathways, which are serine/threonine kinases (Liu et al., 1999; Dozza et al.,
2004). This is reasonable link to the phosphorylation level of cellular proteins. HNE
induce GSH biosynthesis (Forman et al., 2003) which account for the clearance of around
50% of the free HNE. In concerning of the rest of the free HNE, we give a possible
clearance mechanism in neuronal cells.
NFH, with the multiple lysine residues, is highly susceptible to HNE adduction.
The high phosphorylation level in C-terminus, which is resided mainly in KSP repeats
(Jaffe et al., 1998), also made this protein interesting. As phosphorylation has already
been proposed and proven to play roles in interfilamental distance, axonal caliber,
transportation rate, and filament assembly, phosphorylation and translocation controlling
(Nakagawa et al., 1995, de Waegh et al., 1992, Yin et al., 1998，Lewis and Nixon 1988，
Ching et al., 1999，Zheng et al., 2003，Gibb et al., 1998, reviewed in Liu et al., 2004).
Moreover, here we proposed and proved another important and interesting function of
phosphorylation on NFH, which is to regulate the level of oxidative modification on NFH
by HNE. This provides the evidences that KSP repeats involved in a more important
113 cellular protective system. Evolutionally, as the most abundant protein in the most
essential cells, NFH could be applied some more important functions.
Protein database searches for small sequence repeats
(http://us.expasy.org/tools/scanprosite/), revealed that only NFH, NFM and ovarian
cancer related tumor marker CA125 proteins contain more than 10 KSP repeats (Table
2.2). Human ovarian cancer related tumor marker CA125 has ~30 KSP in a 22,152 amino
acid sequence. This protein is a serum antigen used as a marker for the screening of the
ovarian cancer and other tumors (Bast et al., 1983; Kato et al., 2004). Therefore, as far as
we know, human NFH is the only protein has 43/44 KSP repeats concentrated within
around 300 amino acids protein sequence in human protein database.
As we tested around 10 other peptides (Table 2.3), no other sequence showed
similar regulation to that obtained by phospho-KSP motif, suggesting a dual requirement
of KSP repeats and phosphorylation.
In axons, the possibility of proteolytic removal of the HNE adducts is not likely to occur, because degradation of neurofilament is only detected in the area close to synapses
(Lasek and Hoffman 1976; Roots, 1983). Our preliminary studies with the hypertonic
infusion of protease inhibitor leupeptin treatment of exposed sciatic nerve, did not show altered level of NFH-HNE adducts. However, we do not know whether leupeptin is the
appropriate inhibitor for the putative proteolytic activity or whether the infusion was an effective means of delivery (Perry’s lab unpublished data).
In this study, we proposed another possibility that phosphorylation may regulate the level of NFH-HNE and may provide protection for neurons from oxidative insults. This is supported by the studies showed that expressing human NFH can rescue vulnerable
114 neurons from death in two transgenic mice studies (Couillard-Després et al., 1998;
Beaulieu et al., 2003). More consistently, neuronal cells treated with HNE showed time
dependent increase of both level of NFH and phosphorylation level of NFH, which
accommodates the oxidative insults by HNE (Liu and Perry unpublished data). In
addition, cells overexpressing NFH showed significant protection from HNE cytotoxicity
in different neuronal cell lines (Liu and Perry unpublished data). These are also supported
by the recent studies about HNE functions, which showed that there is a narrow range for
HNE to promote cell proliferation (Cheng et al., 1999). Thus it appears that cells need to deliberately adjust the HNE level from time to time (Awasthi et al., 2003).
In this study, we explored the new function of these KSP repeats in human NFH, except for the functions from serine phosphorylation, KSP repeats in human NFH also acts as a scavenger for reactive oxygen species. Based on these new results we proposed the hypothetical model (Figure 2.9). When HNE level is increased in axons, neurons will response by tending to increase the phosphorylation level of NFH especially in KSP repeats, which can adduct more HNE reversibly; and when HNE levels decrease, neurons might signal the opposite response to trigger dephosphorylation, and HNE is released from NFH, then HNE is detoxified in the neurons. This function has never been shown before and it may be vitally important for neurons’ survival.
Even though we provide evidenceof the importantce of KSP repeats using synthetic peptides in this study, we still consider that the secondary (and tertiary) structure in which the KSP sequence lies to be important because there are 1-5 amino acids between most KSP repeats in NF-H and NFM. The role of these neighbouring sequences needs further investigation.
115
Table 2.1 Comparison of all the proteins and peptides in these studies.
116
Table 2.2 Protein database search for KSP repeats (>10 KSP in homosapiens)
117
Table 2.3 All the control peptides used in these studies did not show high level of
HNE adduct.
118 In summary, we demonstrate that KSP repeats in human NFH are the key motifs in modulating HNE-adduct levels on NFH, through regulation by phosphorylation and dephosphorylation on serine residues. This also indicates that NFH-HNE adduction is a regulated event in neurons through activation of kinases and phosphatases, which is directly regulated by signal transduction in neurons. This will be a major discovery that opens the door to the studies of regulation of oxidative modifications in cells and we proved that phosphorylation is directly involved in these regulations in this study.
119
Figure 2.9 Hypothetical model for the phosphorylation-regulated HNE modification in KSP motif.
120
Chapter 3
Cellular Protective Function of
Neurofilament Heavy Subunit
121 Introduction
Neurofilament Function
Neurofilament proteins display several characteristics in neurons, such as control of axonal elongation, axonal caliber, and axonal transportation. They are subject to pathological aggregation in neurodegenerative diseases, and exhibit a protective function in ALS transgenic mice.
The Basic Function of Neurofilament is Supporting Axonal Structure
As one of the members of the cytoskeletal system, NFs work together with microtubules and microfilaments to enhance structural integrity, cell shape, and cell and organelle motility. The major function of NFs is to control the axonal caliber, which is
directly related with phosphorylation state. This is important since the speed of
conductivity of an impulse down the axon is proportional to its caliber. NFs are
particularly abundant in neurons with large diameter axons (>5µM) such as those of
periphery motor neurons controlling skeletal muscle, where fast impulse conduction
velocities are crucial for proper functioning.
Detrimental Effects of NF Accumulations
It is well known that accumulation of NFs is a general hallmark for several
neurodegenerative diseases. These include ALS, AD, Lewy bodies in Parkinson’s
disease, progressive supranuclear palsy, Charcot-Marie-Tooth disease, diabetic
neuropathy, and giant axonal neuropathy (Hirano et al., 1984;Shepherd et al., 2002; Mori
et al., 1996; Bomont et al., 2000; Watson et al., 1994; Schmidt 1996; Schmidt et al.,
1997;).
122 The detrimental effect of these accumulations is seen when the protein inclusions in axons mechanically block the transportation of particles through the axon, which will eventually lead to neuronal death (Xu et al., 1993; Grant and Pant, 2000; Lariviere and
Julien, 2004). Studies show that abnormal accumulations of NFs in the perikaryon of motor neurons could be induced by overexpression of any of the three NF subunits individually. Increasing the levels of NF-H or NF-M not only impairs NF transport into the axon, but also inhibits dendritic arborization (Xu et al., 1993; Grant and Pant, 2000;
Lariviere and Julien, 2004). Ma et al. (1999) overexpressed human NF-L in mice and reported the severe loss of neurons in the parietal cortex and ventrobasal thalamus with age. Xu et al. (1993) showed transgenic mice overexpressing ~4 fold of mouse NF-L cause neuron degeneration and neuron loss, which resemble the pathology of ALS.
Recently, Sanelli et al. (Sanelli et al., 2004) using cells and mice overexpressing human
NF-L showed that the co-localization of copper/zinc superoxide dismutase (SOD1) and neuronal nitric oxide synthase (nNOS) with NF aggregates may cause sequestration of nNOS in NF aggregates, which leads to enhanced NMDA-mediated calcium influx, a potential cause of neuronal death in amyotrophic lateral sclerosis (ALS). This hypothesis sheds light on the mechanism of NF aggregations in causing neuron death. All these studies emphasize the importance of subunit stoichiometry for correct NFPs assembly and transport.
Protective Effects
Other studies have shown NFPs have a protective effect for neuronal survival.
Couillard-Després et al.(1998) showed that crossing transgenic mice overexpressing human NF-H and mice expressing a mutant superoxide dismutase (SOD1G37R) related to
123 human ALS increase the lifespan of the progeny by up to 65%. Another example of NFH
protection is found in the analysis of doubly transgenic mice that overexpress both
peripherin and human NF-H transgenes in NFL knockout mice (Beaulieu and Julien,
2003). The NF-H overexpression completely rescued the peripherin-mediated
degeneration of motor neurons in vivo. The overexpression of human NF-H shifted the
intracellular localization of peripherin from the axonal to the perikaryal compartment of spinal motor neurons, which suggests that the protective effect of extra human NF-H proteins in this situation partly comes from the sequestration of peripherin into the perikaryon of motor neurons thereby abolishing the development of axonal IF inclusions that might block transport. These findings illustrate again the importance of IF protein stoichiometry in formation, localization, and potential toxicity of neuronal inclusion bodies. This is further supported by studies showing NFs were not required for pathogenesis induced by mutant SOD1 because in the absence of NF-L, with depletion of
NFs in axons, life span was extended by 15% in SOD1 mutant mice (Williamson et al.,
1998). All of these studies lend support to a protective effect during condition of depleted axonal NFs or accumulated NFPs in the cell perikaryon.
The disruption in one allele of the NFL gene in mutant SOD1 mice resulted in a
40% decrease in axonal NF content and caliber of motor axons yet had no effect on disease severity and life span of SOD1 mutant mice (Nguyen et al., 2000), which suggests that axonal depletion of NF alone was not responsible for slowing disease in mutant SOD1 mice. The authors’ hypothesis is that perikaryal NFs can alleviate the toxicity of mutant SOD1 by sequestering p25/Cdk5 complex and by acting as a phosphorylation sink for deregulated kinase activity, thereby reducing the potential
124 toxicity of hyperphosphorylation of tau and of other Cdk substrates like Rb (Nguyen et
al., 2001, 2003).
Another possible explanation of NFPs protective function may relate to the
normal function of these proteins. In ALS, AD, and control brains, axonal NF displays
the most abundant carbonyl modifications in gray matter of the central nervous system
[146,147]. The 205 kDa NF-H and, to a lesser extent, the 160 kDa NF-M, displayed the
majority of the adducts of the lipid peroxidation aldehyde product—hydroxynonenal
(HNE) in SOD1G85R mutant mice and 1-33 months normal aged mice (Wataya et al.,
2002). The level of HNE modification showed no difference with age in any cases. These
results also support the NF sequestration hypothesis that NF sequester toxic components,
either p25/Cdk5 complex or toxic product of oxidation or others, consequently lead to protection of the cells and maintenance of the physiological condition (Liu et al., 2003)
(see Figure 1.5 in Chapter 1).
Many questions remain, such as how to reconcile apparently conflicting functions of NFs. Resolution of NFs role of toxicity and protection requires an understanding of why some NF knockout mice and NF transgenic mice are completely normal even though they have perykaryal inclusions for years, and what the initial cause(s) of the degeneration of neurons is (are). In addition to investigating the perykaryal inclusions, increased effort needs to be put into determine the initial causes leading to inclusion formation.
Research Relevance
Neurofilaments as the enriched proteins in neuronal axons are required for axonal extension and axonal transportation. Normally it is heavily phosphorylated in most of the
125 serine residues among the KSP repeats resided in the C-terminus of NFH. In addition,
this phosphorylation is hypothesized to have multiple functions related to neurofilament
aggregation and axonal transportation, but it is never been linked to the oxidative
modifications.
NFH and NFM are almost the only human proteins to have multiple KSP repeats
in their C-termini, and more importantly, these sites are also the targets of
phosphorylation (Hsieh et al., 1990; de Waegh et al., 1992; Cole et al., 1994 Jaffe et al.,
1998) and HNE modification (Wataya et al., 2002). In addition, we found that
dephosphorylation could reduce the HNE antibody recognition, which led us to determine
whether phosphorylation controls, at least partially, the level of NFH-HNE adduction. In
chapter 2, we have already determined that NFH-HNE modification is a
phosphorylation-dependent and sequence-specific modification that can be regulated by
phosphorylation and dephosphorylation of NFH.
Recently two transgenic mice studies have shown that human NFH presented a
protective function. Couillard-Després et al., (1998) showed that expressing human NFH
in superoxide dismutase mutant mice (SOD1G37R) increases the lifespan by up to 65%,
indicating human NFH has a protective function for oxidative damage. Picklo et al.,
(2003) showed that overexpressing human NFH can rescue the peripherin-mediated
motor neurons death in NFL knock-out mice. The mechanism(s) is (are) undiscovered,
but these studies open the question why and how NFH has the protective function.
In this chapter, using neuronal and other cell models overexpressing NFH, we showed that overexpressing NFH protects cells from HNE toxicity and that HNE treated cells adjust themselves to the HNE toxicity by increasing their cellular NFH level and
126 phosphorylation level. These cell self-protective functions act through activation of
kinases including MAPK and P38 kinase pathway. this data directly shows that NFH
protein not only performs the cytoskeletal function but also protects neurons from
oxidative attack through activation of different kinase pathways. In this study, for the first time using cell culture models, we demonstrated that phosphorylation and increased levels of NFH could protect neuronal cells from HNE toxicity. This function is most likely through the activation of kinases induced by HNE treatment. It directly shows that
NFH, as a cytoskeleton protein, can be a scavenger for at least one of the free radicals.
127 Materials and Methods
Antibodies:
SMI-34, monoclonal antibody specific to phospho epitope on human NFH;
SMI-32, monoclonal antibody specific to nonphospho epitope on human NFH; NF200, monoclonal antibody specific to total NFH (Sternberg Monoclonal Inc); phospho-MAPK
, monoclonal antibody specific to phospho-ERK1/2 (Biolab); monoclonal antibody specific to Actin (Chemicon International, Inc.); HNE-Michael antibody, rabbit polyclonal antibody specific to HNE-Michael adduct (Alexis Biochemicals).
Chemicals and inhibitors:
HNE is synthesized in Dr. Lawrence M. Sayre’s laboratory as the method in the
literature (Nadkarni and Sayre, 1995).U0126 is the specific inhibitor for MAPK and
SB203580 is the specific inhibitor for p38 kinase (Calbiochem). 10 µM of inhibitors were
used in the cell culture 30 minutes before HNE treatment.
Mouse NFH cDNA:
Mouse NFH cDNA can is a generous gift from Dr. Don Cleveland (San Diego,
CA) (Lee et al., 1993). pMSV-NFH is a cDNA with full length NFH and MSV promoter was already cloned in pUC19 vector. Ampicillin was used for the cDNA amplification
process.
Cell culture:
Human M17 neuroblastoma cells and mouse N2A neuroblastoma cells are commonly used cell lines for neuronal cell studies, kindly provided by Dr. Robert B.
Petersen (Institute of Pathology, CWRU). Cells were cultured in Opti-MEM media (Life
Technologies) with 5% donor calf serum and 1% penicillin/streptomycin with fungizone
128 (Life Technologies). 10 ml 1X105 or 2 ml 1X105 cells/ml cells were put in 10 cm dish or
each well in the six-well plates. After overnight incubation, media was changed and the
cells were used for all the treatment.
M17 cells were differentiated by adding 10 µM retinoic acid in the media for 5-6
days before HNE treatment..
In HNE experiments, 50 µM HNE final concentration was applied in the cell
culture for different times (0-4 hours), After pipeting the cells off and centrifuging for 10
minutes at 4,000 rpm RT (IEC, CentraMP4R centrifuge), the cells were lysed by using 50
µl 1X cell lysis buffer (Cell Signaling). Protein concentration was determined by using
BCA protein assay (Pierce) and 20 µg total protein/lane was loaded for SDS PAGE.
In cell viability experiments, M17 cells were plated in six-well plates. After overnight incubation, both different concentrations of HNE and different incubation times were used.
In transfection experiments, cells were transfected with mouse NFH cDNA with
DOTAP liposomal transfection reagent (Roche) as the procedure suggested in the product sheet. After 24 hours transfection, final concentration of 50 µM HNE was put in the M17 cell culture medium and final concentration of 0.5 mM HNE was put in N2A cell culture medium for different time (0-4 hours, for >50% cell death in 4 hours). Cell death was determined using LDH assay (Invitrogen) and trypan blue staining.
In the MAPK kinase activity experiments, cells were first treated with 10 µM of the specific inhibitors for 30 minutes before adding final concentration of 50 µM HNE.
Cells were collected after a 4-hour treatment with HNE. Protein concentration was
129 determined by using BCA protein assay (Pierce) and 20 µg total protein/lane was loaded for electrophoresis.
Cell viability assay: LDH assay and Trypan blue staining:
LDH cell viability assay (Roche) is a colorimetric assay for the quantification of
cell death and cell lysis It is based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant. The experiments were performed in 96-well plate as the protocol.
Trypan Blue was diluted at 0.8 mM in PBS. The stock will be stable for 1 month
at room temperature. After all the pretreatment, cells were trypsinized and mix cells with
1:1 with trypan blue solution, then count cells on hemocytometer. Viable cells exclude trypan blue, while dead cells stain blue due to trypan blue uptake.
Protein Assay
Protein concentration was determined by using BCA protein assay (Pierce) and 20
µg total protein was loaded for electrophoresis.
Immunoblotting:
After all the pre-treatment, Membranes were blocked with 10% milk 1 hr, washed with Tris-buffered saline-Tween (50 mM Tris, 150 mM NaCl, 0.1% Tween20) 5X5 min, incubated with primary antibody 16 hrs 4 ℃, after five washes of 5 min in Tris-buffered saline-Tween, peroxidase-labeled secondary antibodies were applied for 1 h at room
temperature. After rinsing again as described before, the blots were developed together for the same length of time using enhanced chemiluminescence (ECL) reagent (Santa
Cruz Biotechnology) and imaged by using image acquisition and analysis software
(Ultra-Violet Products Ltd.).
130 Quantification of the intensity of the blots was performed by using image-analysis software (KS300, Zeiss).
Statistical Analysis:
All the experiments were repeated for at least three times. The data were analyzed using the students’ t-test.
131 Results
M17 cells are human neuroblastoma cells and differentiate under the treatment of retinoic acid forming long axons. 50 µM HNE treatment of M17 differentiated cells induced higher levels of NFH expression in 4 hours, including the both phosphorylated and non-phosphorylated forms of NFH (Fig 3.1). An ~5-fold increase of total NFH,
~10-fold increase of phosphorylated NFH, and ~3-fold increase of non-phosphorylated
NFH were detected using western blot analysis of cell lysate from M17 differentiated cells after 4-hour 50 µM HNE treatment.
NFH phosphorylation level was measured over a time course of HNE treatment. It was found that phosphorylation of NFH protein increased and peaked within 1 hour and
then decreased (Fig 3.2).
The activity of MAPK (including Erk1 and 2) was determined by using
anti-active-MAPK monoclonal antibody on western blot of the cell lysate. The result
showed that MAPK were activated in M17 differentiated cells following HNE treatment
(Fig 3.3). Inhibition of Erk1/2 by specific inhibitor U0126 showed total inhibition of
Erk1/2 activity (Fig 3.3). Total level of MAPK was re-probed by using anti-total MAPK
monoclonal antibody (Fig 3.3), and it showed same level of total MAPK.
M17 differentiated cells had higher level of NFH than non-differentiated M17
cells (Fig 3.4B). When M17 differentiated and undifferentiated cells were treated with
various concentrations of HNE for 4 hours, the differentiated cells were found to be more
resistant to HNE toxicity than the non-differentiated cells (Fig 3.4A). Since HNE can
132
Figure 3.1 HNE treatment induced higher level of NFH in M17 cells. 4 hour 50
µM HNE treatment induced around 5 fold in crease of total NFH level (NF 200). The included around 3 fold increase in nonphosphorylated NFH (SMI-32) and 10 fold increase in phosphorylated NFH (SMI-34).
133
Figure 3.2 HNE treatment induced higher phosphorylation level of NFH. 50
µM HNE induced higher phosphorylation level of NFH in different time, which peaked in samples of HNE treated M17 differentiated cells for 2 hour.
134
Figure 3.3 MAPK were activated in M17 cells after HNE treatment. 50 µM 4 hour
HNE treatment induced the activation of MAPK. Specific inhibitor U0126 totally inhibited MAPK activity.
135 induce increased protein and phosphorylation levels of NFH within 4 hours (Fig 4.1, Fig
4.2), one possibility is that increased NFH and its phosphorylation may play a role in the protection. However, retinoic acid can induce a broad range of gene expression in cells, so this is not an ideal cell model to investigate this point.
To further determine the relationship between increase of NFH /phospho-NFH and protection from HNE toxicity in neuronal cells, a mouse cDNA was obtained and transfected into cells to overexpress NFH. M17 differentiated cells were transiently transfected with the mouse NFH cDNA and exhibited a more than 5-fold increase of total
NFH and around 3-fold increase of phosphorylated NFH after 24 hours (Fig 3.5B). When the cells were treated with HNE, the transfected M17 cells showed significant protection from HNE cytotoxicity than the control M17 cells transfected with empty vector (Fig
3.5A).
This result was also confirmed in another mouse neuronal cell line—N2A cells.
After transfection, the N2A cells produced more than 10 fold of total NFH and more than
5 fold of phosphorylated NFH. NFH (Fig 3.6B). When treated with HNE, the transfected
N2A cells showed significant protection from HNE treatment compared to the control
N2A cells transfected with empty vector (Fig 3.6A).
Both types of cells overexpressing NFH did not show any morphological abnormalities even after 7 days under converse phase microscope (data not shown), which indicate the overexpression of mouse NFH even 10 fold than basal level may be not toxic for these cells in a short time.
136
Figure 3.4 M17 differentiated cells with higher level of NFH showed significant
protection form HNE cytotoxicity with LDH cytotoxicity assay. M17 cells were
differentiated by incubating with 10 µM retinoic acid. Differentiated M17 showed around
4 fold increases in NFH level. M17 differentiated cells showed more protection than M17 undifferentiated cells in different HNE concentration treatment in 2 hours. RA: retinoic acid. “*”=P<0.05.
137
Figure 3.5 Overexpressing NFH in M17 cells resulted in significant protection from HNE cytotoxicity using trypan blue staining. After transfection with mouse NFH,
M17 cells showed significant increases in both total NFH and phospho-NFH level.
Transfected M17 cells were more resistant to HNE toxicity than empty vector transfected
M17 cells.
138
Figure 3.6 N2A cells showed significant protection with overexpressing NFH using trypan blue staining. N2A cells are mouse neuroblasdoma cells. Transfected N2A
cells showed significant increases in both total NFH level and phospho-NFH level.
Overexpressing mouse NFH in N2A cells showed more protection than empty vector
transfected N2A cells.
139 Discussion
In this study, we found that HNE treatment induced increases in both total NFH and phosphorylated NFH protein levels. In addition, increased levels of NFH and phosphorylated NFH are coincident with highergreater protection from HNE cytotoxicity in cultured neuronal cell lines. When mouse NFH is overexpressed in two different neuronal cell lines, the transfected cells show significant protection from HNE toxicity as compared to control cells transfected with empty vector. Also further studies showed that
MAPK kinases were activated by HNE treatment in M17 cells, which already been shown MAPK can phosphorylate NFH. Combining all these results, this study directly demonstrated that NFH protein and its phosphorylation protect neuronal cells from oxidative stress.
Lipoxidation adducts are increased in neuronal cell bodies in AD cases and are present in axons in the white matter of infants and aged controls (Wataya et al., 2002).
These findings propose the questions of how these lipid peroxidation products and specific axonal proteins interact and whether neurons have compensatory mechanisms. In this study, we focused our studies on HNE adducts because they are the most readily detected and best characterized of the aldehyde-derived adducts. HNE is highly reactive and considered one of the most neurotoxic aldehydes produced in vivo (Sayre et al.,
1997). In addition, whereas HNE adducts are among the best characterized chemically, it is likely that HNE can be used as a model for the other reactive aldehydes produced by lipoxidation or glycoxidation because they have common features.
It has already been shown that HNE can induce the activation of MAPK,
PI3K/AKT pathways, which are serine/threonine kinases (Liu et al., 1999; Dozza et al.,
140 2004), and MAPK is a major kinase for neurofilament proteins phosphorylation (Liu et
al., 1999; Dozza et al., 2004). Therefore, NFH is probably phosphorylated by MAPK
after the kinases are activated by HNE treatment.
HNE also induce glutathione (GSH) biosynthesis, the major cellular molecule for
clearance of up to 50% free HNE (Forman et al., 2003). The remaining free HNE will at
least partially attack other biomolecules, such as enzymes in neurons, therefore, neurons
should have a compensatatory system for the extra free HNE.
It has already been reported that lower levels of HNE have physiological
functions in promoting cell proliferation (<10 µM), and higher level of HNE cause apoptotic pathway activation, and even higher level of HNE can directly cause necrosis of the cells (Mattson,2002). In this study, the cells are most likely dying through the apoptotic pathway.
Consistent with the cell culture studies, two mouse studies already demonstrated that overexpression NFH has a protective function (Couillard-Després et al., 1998; Picklo et al., 2002). The mechanism(s) is (are) unknown, but one hypothesis is that NFH may act as sink for oxidative stress. This is consistent with the property of the protein and also reasonable for neuronal survivable. NFH is a lysine-rich protein with the unique characteristic of multiple KSP repeats resided in the C-terminus of NFH. Phosphorylation is most prominent in the serine residues among the KSP repeats. In addition, the free amino groups from the side-chain of lysine provide a huge reactive pool for the reactive oxygen species (ROS), providing a possible molecular basis for a regulative mechanism for HNE modification.
141 Our recent studies have already proven this point (See chapter 2). In that study, we showed that NFH can act as a scavenger for HNE and this process is regulated by phosphorylation and dephosphorylation of NFH in the KSP motifs. This amazing regulative mechanism is arranged in the unique KSP repeats residing in the C-terminal of
NFH protein. NFH can adduct with more HNE molecules under phosphorylation or reverse the HNE adduct under dephosphorylation. This mechanism provides strong evidence for NFH acting as a scavenger in vivo.
In this study, only short-term protection of NFH was studied. In conditions of high concentrations of HNE, many cells are dying after four-hour HNE treatment presumably through apoptosis (Lee et al., 2004). Whether the overexpression of NFH can provide long-term protection in lower HNE concentration, more like the cellular condition, is another interesting question worthwhile to be determined in the future.
M17 cells are human neuroblastoma cell line, which can be differentiated by retinoic acid treatment and form long axons, enriched in neurofilament proteins. In addition, M17 is an efficient working cell line for transfection experiments that has already been used intensively in our lab. For further studies, primary neurons could be a good model for this type of studies with an efficient transfection method. N2A cells are also commonly used in transfection and neuronal cell studies.
In this study, mouse NFH was overexpressed in the neuronal cells, so whether the
NFH is in the free subunit form or assembled form is unknown. Since neurofilament has both free form and assembled form in vivo, it is worthwhile to ask which form is the functional form. Therefore, it will be helpful to study the NFH subunit and the filament separately.
142 In summary, neurofilaments as the most abundant proteins in the neurons, not only provide a cytoskeletal function but also provide further physiological function in adjusting the homeostasis of the free radicals and protect cells from oxidative attack.
Combined with the transgenic mice studies, it becomes clearer that NFH protective functions is at least partially an anti oxidative stress function. On the other side, this HNE modification on NFH can cause protein crosslinking or other changes promoting the NF aggregation in term of decades. The aggregation of neurofilament and other cellular proteins in neurons cause neuronal degeneration in patients.
143
Chapter 4
Oxidative Modification of Tau Protein
Is Involved in NFT Formation
144 Introduction
Tau Protein Structure
Tau is a hydrophilic protein that has been widely characterized in solution. It
appears as a random coiled protein by analysis of the circular dichroism spectra
(Cleveland et al., 1977). In addition, tau has been suggested to be a highly asymmetric
protein, compatible with the long rod structure observed by electron microscopy
(Hiokawa et al., 1988).
Several different tau polypeptides exist in the brain protein extract showed by gel electrophoresis. These polypeptides are generated by both alternative RNA splicing
(Goedert et al., 1989; Himmleret al., 1989a, b; Kosiket al., 1989) and posttranslational modifications including phosphorylation (Goedert et al., 1992). In NFT, there are six CNS
tau isoforms in their phosphorylated form. The six tau isoforms are as follows: 1) containing exons 2, 3, and 10, plus all the constitutive exons (Baker et al., 1999); 2) having exons 2 and 3; 3) containing exons 2 and 10; 4) having only exon 2; 5) with only
exon 10; and 6) only containing the constitutive exons. This combination of
hyperphosphorylated tau isoforms results in the appearance of three major electrophoretic bands with a mobility corresponding to that of proteins with a relative molecular weight of 68,000, 64,000, and 60,000 (Delacourte et al., 1999; Goedertet al., 1991, Greenberg et
al., 1992; Lee et al., 1991). Furthermore, AD brains contain higher quantities of tau than unaffected controls (Khatoon et al., 1992’ 1994).
Tau Phosphorylation
In the 1980s, tau was found as a phosphoprotein (Baudier et al., 1987,
Grundke-Iqbal et al., 1986, Iharaet al., 1986), and all these studies focused on the
145 serine/threonine phosphorylation of the tau protein. Recently, one study has focused on
its phosphorylation on tyrosine (Ihara et al., 2002).
The phosphorylation of tau is developmentally regulated; it is higher in fetal
neurons and decreases with age during the development. Hyperphosphorylation of tau is already been related to pathological situations (tauopathies) (Lovestone et al., 1997, Ihara et al., 1995). Interestingly, different tau isoforms has different phosphorylation sites
(Hernandez et al., 2001). This could be due to the different cellular localization or
subcellular compartmentalization of the different tau isoforms, or the fact that different kinases or phosphatases can modulate tau phosphorylation in a different way.
Hyperphosphorylated tau has been described in extracts of both normal and AD tissue
(Kopke et al., 1993).
There are 79 putative serine or threonine phosphorylation sites on the longest
CNS tau isoform, which contains 441 residues. And there are even more in PNS tau,
although its phosphorylation has not been widely studied. The kinases involved in the phosphorylation of Tau protein have been divided into two main groups: proline-directed
kinases and non-proline-directed kinases. The first group includes tau protein kinase I
(glycogen synthase kinase 3, GSK3), tau protein kinase II (cdk5), MAP kinase (p38),
JNK, and other stress kinases or cdc2. The second groups includes protein kinase A
(PKA), protein kinase C (PKC), calmodulin (CaM) kinase II, MARK kinases (Goedert et al.,1997, Hanger et al., 1992, Imahori et al., 1997, Lucas et al., 2001), or CKII that modifies residues close to acidic residues mainly in exons 2 and 3 (Correas et al., 1992).
In many cases, phosphorylation regulates the binding of tau to microtubules or to the
146 membrane (Brandt et al., 1995). Thus, phosphorylation appears to play a predominant role in regulating tau function.
One example is GSK3, which may play a major role in the phosphorylation of
Tau. Two types of GSK3 phosphorylation have been proposed: primed (following prior phosphorylation of the substrate by another kinase) or unprimed phosphorylation (Frame et al., 2001). Primed phosphorylation occurs at threonine-231 and affects microtubule
binding, while unprimed phosphorylation occurs at serine-396 or -404 and does not affect microtubule binding (Cho et al., 2002). These sites can be identified by the AT180 and
PHF-1 antibodies, respectively (Cho et al., 2002).
GSK3 phosphorylation of tau facilitates its aggregation into filamentous polymers
(Jackson et al., 2002). The β-amyloid peptide (Aβ) is also involved in augmenting the phosphorylation of tau by GSK3 (Yankner et al., 1996), probably by increasing the
enzymatic activity of the kinase (Alvarez et al., 1999), which only proves to be toxic for neurons in which tau protein is present (Rapoport et al., 2002).
The phosphorylation of tau may promote a conformational change that can be
functionally reversed in the presence of trimethylamine N-oxide (TMAO), a natural occurring osmolyte (Tseng et al., 1999). This conformational change can also be reversed
by the chaperon protein Pin-1 (Lu et al., 1999), a molecule that upon tau binding facilitates the posterior action of PP2A in dephosphorylating the protein (Zhou et al.
2000). These conformational changes could facilitate tau aggregation, and recently, it has been suggested that such conformational changes (or others) may result in an increase of
α-helices in the secondary structure of tau, since the content of α-helices is greater in tau from PHF (Jicha et al., 1999, Minoura et al., 2002, Sadqi et al., 2002). However, it has
147 also been suggested that filament formation is also partially dependent on the presence of certain β-sheet structures within tau (Von Bergen et al., 2000). It would be of interest to know whether the tau present in other aggregates such as Pick's bodies adopts a different conformation. Nevertheless, it does seem that a conformational change is undertaken
when tau polymerizes into PHF (Abraha et al., 2000, Ghoshal et al., 2002). Furthermore, it appears that this conformational change could involve the binding of the
amino-terminal region of the tau molecule to its microtubule binding region (Carmel et al., 1996).
Several phosphatases like protein phosphatase (PP) 1, PP2A, PP2B (calcineurin),
and PP2C (Geddes et al., 1993, Goedert et al., 1992, Szucs et al., 1994, Yamamoto et al.,
1995) have been implicated in reversing the phosphorylation of tau. However, only PP1,
PP2A, and PP2B (Gong et al., 1994) have been shown to dephosphorylate abnormally hyperphosphorylated tau. Although PP2C can dephosphorylate tau when it is
phosphorylated by PKA in vitro, it is not capable of dephosphorylating the abnormally hyperphosphorylated tau isolated from AD brain tissue (Gong et al., 1994).
Tau Polymerization
Montejo et al. (1986) described that purified tau protein can form fibrillar
polymers resembling the PHF found in the brain of AD patients and that deamidation
could facilitate this polymerization (Montejo et al., 1987, 1988) which was found in PHF
(Watanabe et al., 1999). In vitro, tau assembly has been further studied (Troncoso et al.,
1993, Wilson et al., 1997). The requirement of a high concentration of protein for tau to polymerize suggests that other factors could be needed to facilitate tau assembly in vivo.
The sulfoglycosaminoglycans (sGAGs), which are present along with tau in NFT, were
148 the first molecules tested and found to facilitate tau polymerization in vitro independently of the phosphorylation state of tau (Goedert et al., 1996, Perez et al., 1996). sGAGs are polyanions, and other polyanions such as the glutamic acid-rich region present at the carboxy-terminal region of tubulin can also facilitate aggregation, the third tubulin
binding motif of the tau molecule are required for this aggregation (Perez et al., 1996).
Oxidation could play a role in tau aggregation. Oxidation of cysteine to produce
disulfide cross-linking favors tau self-assembly in tau 3R molecules, where a single
cysteine is present, but not in tau 4R molecules, where the presence of two cysteines may
permit the formation of intramolecular disulfide bonds (Barghorn et al., 2002). In vitro
studies have shown that tau can be assembled through oxidation processes (Fenton's reaction) in the presence of iron (Troncoso et al., 1993). Also, fatty acids like arachidonic acid can induce tau polymerization in vitro (Abraha et al., 2000, Wilson et al., 1997). This type of polymerization could be related to the possible interaction of tau with plasma membrane components (Arrasate et al., 1997, 2000). Another lipid peroxidation molecule-HNE has been shown facilitating tau aggregation in vitro and in cells (Perez et al., 2000, 2002).
It has been shown that anionic micelles and vesicles induce tau fibrillation in vitro
(Chirita et al., 2003). It has been suggested that tau could play a role in the activation of phospholipase C (PLC) -γ to generate arachidonic acid through the hydrolysis of phosphoacetylcholine (Hwang et al., 1996). The arachidonic acid generated could
facilitate tau aggregation (Abraha et al., 2000), possibly due to the arachidonic acid micelles can act as polyanions since their negative charged carboxyl groups are exposed
at the surface. A compound produced from oxidation of arachidonic acid-HNE has been
149 shown to facilitate tau assembly, but only if tau is hyperphosphorylated (Perez et al.,
2000, 2002), which is in part due to GSK3 phosphorylation. On the other hand, it has been proposed that carnosine can quench the effect of HNE (Aldini et al., 2001), and antioxidants like N-5 butyl hydroxylamine could be used as neuroprotectors (Atamna et al., 2001). Finally, α-synuclein has also been seen to facilitate tau assembly (Giasson et
al., 2003).
A Phosphorylation Before or After Aggregation
A long time argument is whether tau phosphorylation occurs before or after PHF assembly. The antibody 12E8 recognizes a phospho-serine (S262) located in one of the
tubulin binding motifs present in tau molecule. The binding of this antibody to tau is
augmented in AD extracts; however, this antibody does not react with assembled PHF
(Hernandez et al., 2002). This suggests that tau phosphorylation occurs before its
assembly, and this is supported by other studies (Gordon-Krajcer et al., 2000). Indeed, strong evidence exists that the phosphorylation of tau promotes its self-assembly (Alonso
et al., 2001).
B. Toxic Effects of Tau
In neurodegenerative diseases, the cytotoxicity mediated by tau could be due to its
hyperphosphorylation or to the formation of aberrant aggregates. In the first case, it has been observed that expression of pseudo-hyperphosphorylated tau promotes toxicity
associated with the induction of apoptotic cell death (Fath et al., 2002). This result is in agreement with observations in GSK3 transgenic mice (Hernandez et al., 2002).
There is an inverse correlation between the number of extracellular tangles and the number of surviving neurons in AD, suggesting that neurons developing
150 neurofibrillary lesions could degenerate (Goedert et al., 2002). On the other hand, the
appearance of extracellular NFT occurs in those regions that contain neurons with intracellular NFT. This suggests that intracellular inclusions precede cell death and that extracellular NFT form as a result of cell lysis, perhaps due to the binding of tau to extracellular matrix components like sGAGs (Perez et al., 1996). Thus, the presence of
tau aggregates could be postulated as toxic and result from the fact that tau aggregates are
sticky structures. This tau form could bind to proteins needed for normal metabolism and
therefore deprive the cell of these proteins, like ferritin (Hernandez et al., 2001) or Pin-1
(Lu et al., 1999). It has also been proposed that abnormally hyperphosphorylated
cytosolic tau might sequester other MAPs like MAP1B or MAP2. Such sequestering may result in the destabilization and disassembly of microtubules, and in the appearance of
morphological changes that could promote the disruption of synaptic contacts (Iqbal et
al., 1994).
C. PHF Morphology
It has been proposed that PHF morphology may depend on the proportion of tau
isoforms containing three or four tubulin binding motifs (Goedert et al., 1996), on the
association with sGAGs (Arrasate et al., 1997), or on the presence of specific residues present in tau molecule (DeTure et al., 2002). Since the pioneer paper of Kidd (Kidd et
al., 1963), different techniques have been applied to characterize the structure of PHF, including X-ray diffraction (Wisniewski et al., 1976), electron microscopy,
high-resolution transmission electron microscopy (TEM), atomic force microscopy, or cryoelectron microscopy (Ruben et al., 1991, Wischik et al., 1985). The results suggest that the structure of PHF is compatible with a helical ribbon made up of two parallel
151 strands. The formation of NFT from PHF appears to be facilitated by glycation (Ruben et
al., 1995), oxidation (Perez et al., 2000, 2004).
The Pathological Relevance Between Tau Protein and AD
The best-known tauopathy is AD, in which two main pathological structures form in the brains of patients: senile plaques (composed of the β-amyloid peptide), and neurofibrillary tangles (NFT). These NFT are made up of paired PHF comprised of
hyperphosphorylated tau (Grundke-Iqbal et al., 1986). The number of NFT has been
correlated with the degree of dementia in this disease (Arriagada et al., 1992), and thus
there is great interest to know how these structures form.
NFT are composed of bundles of paired helical filaments (PHF), the major component of which is the microtubule-associated protein τ (Grundke-Iqbal and Iqbal,
1989; Lee et al., 1991). In PHF, τ is abnormally hyperphosphorylated (Grundke-Iqbal et al., 1986; Trojanowski and Lee, 1995; Lee et al., 2001), ubiquitinated (Iqbal and
Grundke-Iqbal, 1997; Mori et al., 1987; Perry et al., 1987), oxidized (Mattson et al.,
1995; Takeda et al., 2000a), proteolytically processed and aggregated into filaments (Lee et al., 2001; Lovestone and McLoughlin, 2002; Perez et al., 2002) when compared to that in normal neurons. Hyperphosphorylation of τ renders it unable to bind to microtubules and therefore unable to promote or maintain microtubule assembly (Baudier and Cole,
1987). Moreover, hyperphosphorylation makes τ more resistant to proteolytic degradation, which may play a key role in neurofibrillary degeneration in AD patients
(Iqbal et al., 1998; Eidenmuller et al., 2000). Hyperphosphorylation of τ most likely reflects a combination of increased kinase and decreased phosphatase activities (Gong et
152 al., 2000). Many kinases such as GSK3, p38, ERK, and PKC can phosphorylate τ, although which kinase is critical in producing hyperphosphorylated τ in AD is unknown.
In previous studies, we found a clear mechanistic relationship between τ
phosphorylation and oxidative stress, a key pathogenic modulator of disease (Perry et al.,
1998; Smith et al., 2002a; Gomez-Ramos et al., 2003). Data from the literature shows
that oxidative stress activates several kinases such as ERK, p38, and JNK, which have
been shown to be activated in AD (Zhu et al., 2000, 2001, 2002) and are capable of
phosphorylating τ (Reynolds et al., 2000). Additionally, once phosphorylated, τ and
other cytoskeletal proteins are vulnerable to modification by carbonyl products of
oxidative stress (Perez et al., 2000, 2002; Wataya et al., 2002) and consequent
aggregation into fibrils (Perez et al., 2000).
In previous studies, we found a clear mechanistic relationship between τ
phosphorylation and oxidative stress, a key pathogenic modulator of disease (Perry et al.,
1998; Smith et al., 2002a; Gomez-Ramos et al., 2003). Data from the literature shows
that oxidative stress activates several kinases such as ERK, p38, and JNK, which have
been shown to be activated in AD (Zhu et al., 2000, 2001, 2002) and are capable of
phosphorylating τ (Reynolds et al., 2000). Additionally, once phosphorylated, τ and
other cytoskeletal proteins are vulnerable to modification by carbonyl products of
oxidative stress (Perez et al., 2000, 2002; Wataya et al., 2002) and consequent
aggregation into fibrils (Perez et al., 2000).
In vitro analysis of the assembly of τ into fibrillar polymers has indicated that
recombinant, unmodified, or weakly phosphorylated τ (Montejo et al., 1986; Crowther et
al., 1992; Wille et al., 1992) is able to polymerize, while hyperphosphorylated τ has a
153 much lower capacity for polymerization (Schneider et al., 1999), which indicates that
there must be an alternative mechanism to facilitate the assembly of carbonyl-modified
phosphorylated τ into fibrillar polymers in vivo. Anionic compounds like sulfated
glycosaminoglycans (sGAG), RNA, and heparin can facilitate τ polymerization (Siedlak
et al., 1991; Goedert et al., 1996; Kampers et al., 1996; Perez et al., 1996). Proteolysis
(Wischik et al., 1988), glycation, or direct oxidation (Grundke-Iqbal et al., 1986;
Troncoso et al., 1993; Yan et al., 1994; Schweers et al., 1995; Ledesma et al., 1998) have
also been implicated in τ aggregation. Fatty acids such as arachidonic acid (Wilson et al., 1995, 1997; Abraha et al., 2000; Gamblin et al., 2000) and products of lipid oxidation
(Uchida et al., 1993; Perez et al., 2000) have also been demonstrated to promote τ aggregation. The relative role of each of these contributors in vivo is unknown. We previously showed that phosphorylated τ, but not other τ forms, polymerize both in vitro
(Perez et al., 2000) and in cells (Perez et al., 2002) by modifications induced by the lipid peroxidation product, 4-hydroxynonenal (HNE), a naturally occurring product of lipid peroxidation (Uchida et al., 1993) that forms protein adducts which are increased in AD
(Montine et al., 1997, 2002; Sayre et al., 1997). These data are consistent with other studies showing that τ conformational change, as detected with the well-studied monoclonal antibody Alz50, can be induced by HNE (Takeda et al., 2000a). To investigate whether HNE-modified τ forms the major epitopes in NFT, we examined seven distinct monoclonal antibodies raised against NFT. The recognition of all seven antibodies is significantly enhanced by HNE treatment, which may indicate that HNE is a major effector for NFT formation.
154 MATERIAL AND METHODS
Antibodies:
Mouse monoclonal antibodies raised against NFT: PHF-1 recognizing phosphoserine 396/404 (Otvos et al., 1994); TG-4 recognizing phosphoserine 235
(Vincent et al., 1996; Weaver et al., 2000); MC15 recognizing phospho-τ (Vincent et al.,
1996); TG-5 recognizing 220-240 of htau40 (Jicha et al., 1997a); MC-1 and Alz50
(Carmel et al., 1996; Jicha et al., 1997a), both recognizing an intramolecular conformation epitope of residues 7-9 and 312-342; TG-3 recognizing conformational epitope involving phosphothreonine 231 (Jicha et al., 1997b). Mouse monoclonal antibodies raised against normal τ: τ-1 recognizing serine 199/202 without phosphates
(Papasozomenos et al., 1987) and 5E2, a τ antibody independent of the phosphorylation state (Joachim et al., 1987; Kosik et al., 1989). Monoclonal antibodies raised against neurofilaments: SMI-32 and SMI-34 (Sternberger Monoclonals, Inc). Polyclonal antisera to ubiquitin (Perry et al., 1989).
Immunocytochemistry:
Hippocampal tissue from AD cases (ages 65,82,90, all with 4 hour postmortem
intervals) as well as age-matched controls (ages 65,72,84) was fixed in methacarn
(methanol:chloroform:acetic acid, 6:3:1), dehydrated and embedded in paraffin.
Methacarn was used since it does not artifactually create an “oxidative damage-like state”
in the proteins caused by carbonyl adduction, in contrast to formalin or
glutaraldehyde-based fixatives. Sections (6µm) were placed on silane-coated slides.
After rehydration through xylene, graded ethanols, water, and treatment with 3% H2O2, during 30 min to reduce endogenous peroxidase activity, sections were blocked with 10%
155 normal goat serum (Sigma) in Tris-buffered saline (TBS) for 30 min. The sections were
stained with the peroxidase anti-peroxidase method (Sternberger, 1986) using
3,3’-diaminobenzidine (DAB) and 0.015% H2O2 as cosubstrates.
Adjacent serial sections were used to directly compare pathological structures
recognized by different antibodies. The number of NFT immunostained by the
antibodies was determined by image analysis (KS300, Zeiss). The number of NFT and
density of immunolabeled structures were quantified using a computer-assisted imaging
of the same five consecutive fields, defined by landmarks, in the CA1 and CA2 regions in
adjacent serial sections.
Preparation of τ:
Human τ was prepared from cortex of cases showing no neuropathological or clinical neurological signs of AD and with a postmortem interval less than 8 hr. τ protein was isolated by a modification of the procedure previously described by Lindwall and
Cole (1984). Tissue was homogenized in five volumes of 0.1M PIPES, pH 6.8, 2mM
EDTA with a Polytron® homogenizer (Brinkman Instruments), then centrifuged at
12,000g for 20 min. The supernatant was brought to 0.75M NaCl and 2%
β-mercaptoethanol (BME), boiled for 4 min and rapidly cooled on ice. The supernatant was centrifuged again at 10,000g for 30 min at 4°C, brought to 2.5 % perchloric acid and left on ice for 15 min followed by centrifugation at 12,000g for 20 min. τ was precipitated from the supernatant with 20% trichloroacetic acid (TCA), and the pellet was washed with ethanol repeatedly. The purified τ was resuspended in phosphate-buffered saline (PBS) or TBS.
156 PHF-τ, which is highly phosphorylated and stable to postmortem dephosphorylation, was prepared as previously described (Greenberg et al., 1992) from
AD cases. Gray matter was homogenized in 10 vol “H buffer” (10mM Tris, 1mM
EGTA, 0.8 M NaCl and 10% sucrose) by using the Polytron® and centrifuged at 27,200g for 20 min at 4˚C. The supernatant was saved, and the pellet was rehomogenized in 10 vol “H buffer”, spun as described above, and combined with the previous supernatant.
The final supernatant was brought to 1% sarcosine and 1% BME and incubated for 2 hr at
RT. After, the supernatant was centrifuged at 22,000g for 1 hr at RT. The pellet was resuspended in 3-4 ml of H buffer, layered over a discontinuous sucrose gradient of 35% and 50% sucrose in 10mM Tris, 1mM EGTA and 0.1% β-mercaptoethanol, and spun for
2 hr at 40,000g in a Beckman ultracentrifuge (SW50 rotor). PHF-τ, layered at the
35/50% interface, was precipitated by trichloroacetic acid and resuspended in PBS.
Recombinant τ (full-length) was prepared as previously described (Perez et al.,
1998).
The phosphorylation state of τ was reduced by treating human τ samples with
20U/ml of alkaline phosphatase type III (Sigma) for 16 hr at 37°C in 0.1M Tris-HCl, pH
8.0, in the presence of 0.01 M phenylmethylsulfonyl fluoride (PMSF) to reduce proteolysis or, alternatively, τ was dephosphorylated by treatment in 50% hydrofluoric acid for 16 hr at 4°C (Smith et al., 1996; Takeda et al., 2000a).
Immunodots:
Samples were treated with 1 mM HNE (Takeda et al., 2000a) for 2 hr before application of protein to the membrane. Dot blots were prepared by applying 4 µg of τ
protein per dot directly onto Immobilon (Millipore) membrane and air drying. The
157 efficacy of the assay we use was validated in a previous study where the Alz50 epitope was examined (Takeda et al., 2000a). In the Takeda study, we observed that
HNE-induced modifications are time- and molecular weight polymer-dependent, i.e., τ is continuously modified by HNE and the Alz50 epitope decreases with the formation of high molecular weight polymers. The amount of protein used per dot coupled with the short antibody incubation times, allowed the assay to be performed to optimize the recognition of HNE-induced modifications. In the same study (Takeda et al., 2000a), using several protein concentrations and Coomassie blue staining, we observed that the assay proceeds in a linear range for the conditions used here. The membrane was then blocked with 10% nonfat dry milk for 15-30 min at RT and incubated for 1 hr with the primary antibodies. After five washes of 5 min each with 0.1% Tween in TBS, secondary horseradish peroxidase labeled antibodies were applied for 1 hr at RT. After extensive washing in Tween-TBS, dot blots were developed using enhanced chemiluminescence (ECL) (Amersham).
Quantitation of the intensity of binding was performed by using image-analysis software (KS300, Zeiss). Experiments were performed at least in triplicate and control dots of untreated proteins were included and used as background readings for each experiment.
Statistical Analysis:
The data were analyzed using the students’ t-test.
158 RESULTS
The specificity of seven unique NFT monoclonal antibodies are summarized
(Figure 4.1).
To compare relative recognition of each of the antibodies against different τ forms, we made comparative studies with recombinant τ, normal τ, and PHF-τ (Figure
4.2). Recombinant τ was poorly recognized by most of these antibodies, except TG-5, probably due to lack of phosphorylation and conformations (Fig 4.2A). Normal τ, which is phosphorylated in vivo, can be recognized by TG-5 and PHF-1 and, to a lesser extent, by TG-4 (Fig 4.2B). PHF-τ was strongly recognized by PHF-1, TG-4, MC15, and TG-5.
Surprisingly, three conformation-dependent antibodies, MC1, Alz50, and TG-3, showed little recognition (Fig 4.2C). This may be due to the disruption of specific τ conformations caused by the purification process, since these antibodies recognized NFT by immunocytochemistry staining of sections from the same AD brain samples (See Fig
4.4 in discussion). Furthermore, we observed that dephosphorylation reduced recognition by the phospho-dependent NFT antibodies (PHF-1, TG4, and MC15) (Figure
4.2B,C), especially in PHF-τ. TG-5 antibody is least affected in all the treatments, and the reason why TG-5 showed lower binding capacity to recombinant τ is unknown.
To determine whether the above described antibody recognition to τ was dependent on or promoted by oxidation-related modifications, we treated the three forms of τ, with or without prior dephosphorylation, with HNE. We found that HNE treatment of normal τ greatly enhanced the antibody recognition from 4 to 34 fold (Figure 4.3B).
Such carbonyl-enhancement was dependent on τ phosphorylation state since dephosphorylation with either phosphatase or hydrofluoric acid prior to HNE treatment
159 greatly reduced the enhancement by HNE. This effect was statistically significant for
all the antibodies tested except TG-5 and PHF-1. PHF-1 antibody is usually considered as
a strong NFT antibody despite of dilution, which may explain the less significant
reduction of PHF-1 after dephosphorylation (Reduction is also apparent). Confirming
the phosphate-dependent nature of the effect of HNE treatment, antibody recognition of
recombinant τ is not enhanced by HNE (Figure 4.3A). Further, HNE had no effect on
the recognition capacity of any of the antibodies against PHF-τ irrespective of the phosphorylation state (Figure 4.3C) suggesting the possibility that PHF-τ has already been modified by endogenous aldehydes to an extent that the conformational change recognized has been fully induced or PHF-τ has acquired some specific structures to prevent further HNE modifications.
In contrast to τ, antibodies against neurofilaments (SMI 32 and SMI 34), phosphorylated or not, presented a lower recognition capacity for neurofilament proteins after treated with HNE (data not shown). Also, treatment of purified ubiquitin with
HNE did not alter the recognition capacity of ubiquitin antibodies (data not shown).
These results exclude the possibility that HNE treatment has a universal enhancement effect for all antibodies.
τ-1 (dephosphorylated τ) and a monoclonal antibody specific to phospho-serine/threonine were used to examine the efficiency of the dephosphorylation process by alkaline phosphatases or hydrofluoric acid. In a previous study, dephosphorylation by HF lead to the complete removal of organic phosphate from τ [61].
5E2 (phosphate independent) was used to control for the maintenance of τ protein levels throughout the treatments (data not shown).
160
Figure 4.1 Summary of the specificity of seven NFT antibodies. (A) They belong
to three groups: PHF-1, TG-4, and MC15 recognize phospho-epitopes; TG-5 recognizes
primary sequence; and MC1, Alz-50, and TG-3 recognize conformational epitopes. (B) A
diagram shows the recognition sites of these antibodies on τ protein (MC15 is not included).
161
Figure 4.2 Recognition of various τ forms with and without dephosphorylation by antibodies to NFT. Three phospho-dependent antibodies, PHF-1, TG-4, and MC15 recognized PHF-τ and normal τ (weaker), dephosphorylation showed significant reduced recognition on PHF-τ but not on normal τ (even also reduced) (B,C). TG-5 is the least influenced antibody, because it recognizes primary sequence (weaker on recombinant τ).
Conformational dependent antibodies MC1, Alz-50, and TG-3 did not recognize all three forms of τ. Relative intensities were compared on the same blots. (*) = p < 0.01.
162
Figure 4.3 Enhancement (fold increase) of antibody recognition by HNE modification. HNE treatment of normal τ enhances antibody recognition capacity by
4-34 fold, an effect was reduced significantly by prior dephosphorylation (B).
Recombinant τ, poorly phosphorylated, shows no HNE enhancement (A). Similarly, hyperphosphorylated τ, PHF-τ, shows no HNE enhancement in the presence or absence of phosphate. Relative intensities were compared on the same blots and with self-comparison for each antibody. (**) = p < 0.05, (*) = p < 0.01 (C).
163
Figure 4.4 Immunocytochemistry on adjacent serial sections (landmark, senile plaque (large arrows)) with the antibodies to NFT. All these antibodies recognized NFT, dystrophic neuritis of senile plaques, and neuropil threads. Scale bar: 50µm.
164 DISCUSSION
In this study, we analyzed seven antibodies against PHF-τ that recognize τ
phospho-epitopes, τ primary sequence, or τ conformational changes. They share the property of enhancement of recognition capacity by HNE modifications suggesting that carbonyl-modified τ may be a major antigenic determinant of NFT. Furthermore, the apparent requirement of phosphorylation for the reaction of HNE with τ is consistent with previous cytological and biochemical studies showing that increased phosphorylation precedes ubiquitination of τ and NFT formation (Bancher et al., 1989, 1991; Augustinack et al., 2002). The data support the idea that phosphorylation of τ is required for its modification by carbonyls (Figures 4.2 and Figure 4.3).
τ in NFT (PHF-τ) is abnormally hyperphosphorylated (Grundke-Iqbal et al., 1986;
Trojanowski and Lee, 1995; Lee et al., 2001), ubiquitinated (Iqbal and Grundke-Iqbal,
1997; Mori et al 1987; Perry et al., 1987), proteolytically-processed, aggregated
(Lovestone and McLoughlin, 2002; Perez et al., 2002), and modified by products of oxidative stress (Mattson et al., 1995; Takeda et al., 2000a). However, how the τ isomers aggregate into filament is still an open question. In a prior study, our group showed that Alz50, a well known NFT epitope, is induced by HNE modification of normally phosphorylated τ (Takeda et al., 2000a). Additionally, HNE, a carbonyl formed from lipid peroxidation, can facilitate phosphorylated but not non-phosphorylated or less phosphorylated τ isomer, to polymerize into PHF-like filaments both in vitro
(Perez et al., 2000) and in cells (Perez et al., 2002).
In a previous study, Jicha and colleagues (1997a) showed that Alz50 and MC-1 epitopes can be produced by the immunoblotting process for normal τ. Our group
165 showed the same result (Takeda et al., 2000a), so, in this study, we used dotblots instead
of immunoblots.
HNE can modify the nucleophilic side chains of the amino acids cysteine,
histidine, and lysine, through formation of Michael adducts, lysine-derived pyrroles, and
crosslinks (Xu et al., 1999; Liu et al., 2003). These structures influence the properties of
proteins through different mechanisms, such as by crosslinking, changing hydrophobicity
or altering protein structure, which will also influence the related antigen reactions with
antibodies.
From this point, one possible explanation for the HNE enhancement of antibody
recognition is that NFT-like epitopes exist at a very low level in normal τ but that the
modifications induced by HNE can stabilize these epitopes in a specific way. This
HNE-modified phospho-τ form can preserve higher levels of these specific epitopes,
which also represent the major NFT epitopes. If this is true, HNE-induced
modifications of phospho-τ may represent the major cause of NFT formation.
Consistent with the hypothesis that τ might exist in an equilibrium of conformational states that can be perturbed, is that 2,2,2-trifluoroethanol, which can stabilize certain local conformations of protein (Lang et al., 1994) includingτ (Hoffmann et al., 1997), promotes formation of the TG-3 epitope in τ (Jicha et al., 1997b).
All the phosphorylation sites of PHF-τ can also be found in normal τ. The difference is that normal τ has around 3 moles of phosphate while PHF-τ has about 11 moles (Ksiezak-Reding et al., 1992). This may explain why the phospho-dependent
NFT antibody recognition capacity to normal τ is lower than to PHF-τ. (Fig 4.2 B,C).
And the same reason can be applied to the dephosphorylation process, which showed that
166 reduction of antibody recognition of PHF-1, TG-4, and MC15 in normal τ is less significant than in PHF-τ after dephosphorylation (Fig 4.2 B,C).
Recombinant τ did not show HNE-induced enhancement, consistent with phosphorylation being required for the formation of NFT epitopes (Fig 4.3A). PHF-τ did not show the HNE-induced enhancement, and the possibilities are that PHF-τ is already extensively modified by carbonyls or has acquired some specific structure to prevent further HNE modification (Fig 4.3C) ..In the prior study with Alz50, we found that the epitope is lost with high molecular weight polymer formation like that found in
NFT. In fact, isolated SDS-insoluble NFT are not recognized by Alz50 (unpublished observation). Interestingly, our purified PHF-τ was not recognized by three conformational dependent NFT antibodies (MC1, Alz50, and TG-3), but all the antibodies recognition were enhanced in normal τ after HNE treatment (Fig 3B), which strongly indicates that all these specific NFT epitopes can be produced directly from
HNE modifications of τ. These results are also supported by the data showing that these antibodies stained NFT in brain samples form the same cases of AD (Fig 4). PHF-τ and normal τ should become indistinguishable from recombinant τ after dephosphorylation and linearization, but dephosphorylation of PHF-τ or normal τ did not render them exactly the same as recombinant τ (Can be noticed in Fig 2 and 3), which suggests that additional changes exist in normal τ and PHF-τ forms compared with recombinant τ.
TG-5 antibody reactivity is the least affected, possibly due to its mapping primarily to the primary sequence of τ.
Our findings raise the very real possibility that the lipoxidation-induced modifications of τ protein are a regulated and vital process of the phosphorylation
167 mediated stress response of neurons prior to formation of NFT. Studies concerning the
link between τ phosphorylation modification and the antioxidant enzyme heme oxygenase-1 show that these processes co-exist, and in vitro transfection studies also show that they are interrelated processes (Takeda et al., 2000b). This evidence suggests that hyperphosphorylation and carbonyl adduction of τ may be a critical response to oxidative insults, which play important homeostatic functions. This idea is supported by data indicating that neurons with hyperphosphorylated τ exhibit a reduction in oxidative stress (Nunomura et al., 2001). Therefore, therapeutic strategies envisaging the reduction of τ hyperphosphorylation may be counterproductive since they potentially may interfere with a normal stress response associated with τ phosphorylation (Smith et al., 2002b).
168
Chapter 5
Discussion and Future Directions
169 In this dissertation, we studied two critical proteins involved in Alzheimer disease and other neurodegenerative diseases—neurofilament heavy subunit (NFH) and Tau protein. They both involve in NFT formation in AD and individually they involves in many other types of neurodegenerative diseases, like ALS, CML, diabetes. As oxidative stress is a major concern related with neurodegenerative diseases in our lab, we further studied one of the lipoxidation markers---HNE modification of these two proteins.
In chapter 1, detailed background introduction about AD, neurofilament protein, tau protein, and oxidative stress were presented. Based on the previous finding in our lab, new evidence are presented that are supportive for the involvement of oxidative stress and these two proteins in neurodegenerative dieseaese.
In chapter 2, the molecular mechanism that regulates NFH-HNE adduct is defined. Our previous study reported that levels of NFH-HNE adducts did not change with age or transport in neuronal axons of human, mice, rats. The question is that what the mechanism is, and the result demonstrated that NFH-HNE modification is a phosphorylation-dependent and sequence-specific modification controlled by neuronal signals. This study provided new evidence that support the NFH protective function reported in other two transgenic mice studies (Couillard-Després et al. 1998; Picklo et al.,
2002). In their studies, they had already concerned about the oxidative stress that may play a role in these transgenic mice models, but they did not provide any further evidences than a hypothesis. In this study, we used HNE as a representative for oxidative stress showed that NFH can act as a scavenger for oxidative stress and the consequential adducts are reversibly controlled at least partially by neurons.
170 As showed in chapter 2, NFH is a very special protein in the neuronal axons, it
not only provides many cytoskeletal functions but also provide protective function. As
we know, NFH involves in neurodegenerative diseases and other diseases, it is necessary
for clearer study with this protein. Because AD is a disease developed over decades and
its correlation with age is more than any other factors. Therefore, as the major aging
theory, free radical modifications should be a direct cause for what happened in AD. Here
we studied HNE modification of neurofilament and we clearly showed an amazing
regulatory mechanism for control of NFH-HNE level in neurons. Therefore, there are
some other possibilities that other similar or different regulations exist in neurons.
Further explore these mechanisms will help us to understand the neuronal regulations and
hopefully we can define the cause of neurodegenerative disease.
HNE as a lipid –peroxidation product is well studied in the last two decades, the
chemical structure of the HNE adduct give us the opportunity to further explore the
cellular changes of the modifications. As the major and only reversible HNE
adduct-lysine HNE adduct. There is a possibility for the neuronal removable of this
modification. Other free radicals like MDA, acrolin, should be studied more as HNE,
then these studies will be able to provide strong support for the cellular studies.
Moreover, these will benefit for deciphering the removal of this adduct in cells. In addition, these studies will help people to understand the free radical damage more clearly and it will facilitate the production of antioxidant drugs and elongate human being lifespan. To understand the oxidative stress better, it will fundamentally improve the understanding of aging process and many related disease. The major killer disease like cardiovascular diseases, cancer, and neurodegenerative diseases, all related with
171 oxidative stress. The oxidative modified biomolecules are the major pathophysiological changes and we need to know how to deal with them.
HNE modification reversibility provides the opportunity for us to understand how the cell can manipulate the oxidative modifications. So many oxidative modification are presented in cells and some will make real damage some may not some harmful.
As opposite to the HNE-Lys-Michael adduct, other HNE adducts are not chemically reversible. In case of formation of those modifications, two possibilities exist.
One is these adducts will accumulate and degraded by proteasome pathway. Another is cellular enzymatically removal of these adducts. How powerful the cells are, we do know yet.
In chapter 3, we studied the effect of HNE treatment in neuronal cells and the effect of overexpression of NFH in neuronal cells. The results showed that HNE can induce increased level of total NFH and phosphorylated NFH, which provide the evidence that NFH may play a major role in the axons against oxidative stress. The activation of different kinases by HNE treatment also showed by many studies (Lee et al.,
2004). We showed that HNE treatment induced the activation of MAPK, which has already been reported as a neurofilament kinase and caused the phosphorylation of neurofilament (Pant et al., 2001). Therefore, signal transduction pathway may have already involved in the neuronal control of HNE modification.
The physiological function of HNE is still need more studies and the clear difine the phyisologocal concentration and pathological concentration of HNE will give a further demonstration about HNE functions in cells. HNE modifications give us the opportunity to further explore the oxidative modifications in cellular level, whether it is
172 possible for us to understand more about the oxidation in cellular level and find a way to
utilize the knowledge we get is still a time consuming question..
Now neurofilament as the most abundant protein in axons performed to maintain
the homeostasis of HNE concentration, this is a example for other similar modifications
or mechanisms, so as regulations are the major cellular characteristics, we need to study
the things more carefully and thoughtfully. That how the cells accommodate most of the
oxidative attacks in the daily bases needs to be found out.
In chapter 4, we studied the HNE modification in the NFT formation and NFT
epitope formation. Here we proved that HNE promote the major epitope formation in
NFT. HNE modification has already been reported to facilitate tau aggregation forming
PHF-like filaments in vitro or in cells (Perez et al., 2000, 2002). No study has shown how
important the HNE modification in NFT formation. Through this study with seven
distinct NFT antibodies, we provide new evidence that HNE can produce most of the
epitopes of NFH, which indicates that HNE may play a major role in the formation of
NFT at least NFT epitopes.
Aggregation of tau protein is a topic that has been intensively studied, but the
conditions that promote tau aggregation is still ambiguous. As the hyperphosphorylated
tau has lower potential to aggaregate in vitro, there must be some actor facilitating tau
polymerized. Several compounds have been reported in facilitating tau aggregation. One
is HNE molecule. As HNE adduct level was found increased in neurofibrillary tangles
and HNE is produced in cells commonly, it is a close connection to propose that HNE may play a role or play a major role in NFT formation. As demonstrated in this studies, distinct NFT antibodies covered all kinds of epitopes in NFT showed universally
173 increased immunoreactivity after HNE treatment, which suggests a more important role
for HNE in NFT formation or at least in NFT epitopes formation.
All the functions of these protein aggregations in neurodegenerative diseases are unknown, and whether they are the cause or the consequences are still in argument. In the point of cell self-protection, it is impossible for a huge aggregation exist in cells. One possibility is that these aggregates are the results of incapable removal of the modifications of these proteins in cells, which leads to the formation of these protein aggregates. Another possibility is that the protein aggregates are actually part of the cellular protection mechanisms. The dilemma should be solved by the extensive research works as soon as possible.
Concerning with the research in this dissertation, the following experiments should be finished in the future. This will include the concerns below:
1. Study the similar peptide sequences that could have the phosphorylation-dependent regulation.
2. Exame the effect of interruption of phosphorylation and dephosphorylation process with different inhibitors, such as okdiatic acid in cell culture.
3. Find out the signaling pathways that can induce neuronal protection from oxidative insults, such as testing different chemicals and biomolecules in cell cultures.
4. Determine the HNE midifcaton in NFT formation and the effect of removing phosphorylation in these aggregates.
5. Mice models that overexpress NFH can be tested for the HNE toxicity in some careful ways.
6. Determine whether NFH aggregation will affect tau aggregation or vise
174 versa.
More cellular or animal studies will provide precise data for the NFH protection in neurons. Moreover, the neuronal situation is so special that need more studies and broad coverage of the knowledge we got. I hope that base on the understanding of these specific characteristics in neurons, some more valuable data can be produced in the recent future.
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234
APPENDIX
Publications, Abstracts, and Conferences
235 Publications:
Published:
1. Bishop GM, Robinson SR, Liu Q, Perry G, Atwood CS, Smith MA. Iron: a
pathological mediator of Alzheimer disease? Dev Neurosci. 2002;24(2-3):184-7.
2. Liu Q, Raina AK, Smith MA, Sayre LM, Perry G. Hydroxynonenal, toxic carbonyls,
and Alzheimer disease. Mol Aspects Med. 2003, 24(4-5):305-13.
3. Perry G, Taddeo MA, Petersen RB, Castellani RJ, Harris PL, Siedlak SL, Cash AD,
Liu Q, Nunomura A, Atwood CS, Smith MA. Adventiously-bound redox active iron
and copper are at the center of oxidative damage in Alzheimer disease. Biometals.
2003;16(1):77-81.
4. Taddeo MA, Smith MA, Liu Q, Atwood CS, Sayre LM, Perry G. Metal-catalyzed
redox activity in neurodegenerative disease. In: Metal Ions and neurodegenerative
Disorders, Zatta P, Ed, World Scientific Publishing Co., Singapore, 2003,pp1-14.
5. Liu Q, Xie F, Siedlak LS, Smith MA, Perry G. High Molecular Weight
Neurofilament Proteins in neurodegenerative diseases. Cell Mol Life Scie, in press
2004.
6. Liu Q, Siedlak SL, Harris PLR, Lee HG, Zhu X, Avila J, Takeda A, Smith MA,
Perry G. Tau modifiers as therapeutic targets for Alzheimer disease. Biochem
Biophys Acta, in press. 2004.
7. Moreira PI, Nunomura A, Honda K, Liu Q, Aliev G, Oliveira CR, Santos MS, Zhu
X, Smith MA, Perry G. Stress and homeostatic regulation of oxidative damage in
neurodegenerative disease. Proceedings of the XII Biennial Meeting of the Society
for Free Radical Research, in press. 2004
236 8. Liu Q, Smith MA, Avilá J, Bernardis JD, Kansal M, Takeda A, Zhu X, Nunomura
A, Honda K, Moreira1 PI, Oliveira CR, Santos MS, Shimohama S, Aliev G, de la
Torre J, Ghanbari HA, Siedlak SL, Harris PLR, Sayre LM, Perry G.
Alzheimer-specific epitopes of tau represent lipid peroxidation induced
conformations. Free Radical biology and medicine In press 2004
Comments
9. Liu Q, Honda K, Perry G, Smith M. on NEWS: Radical Development: Reactive
Oxygen Species Critical in Development, Is Redox Regulation of Development in
Caenorhabditis elegans a Critical... 2 Mar 2004. Alzheimer Research Forum, 2004,
10. Liu Q, Honda K, Moreira P, Perry G, Smith M, Zhu X. on PAPER: Palacino JJ. et
al., 2004, Parkin and Mitochondria: Are They Allies in the War Against
Parkinson’s... 30 Apr 2004. Alzheimer Research Forum, 2004.
11. Liu Q, Honda K, Moreira P, Perry G, Smith M. on PAPER: Zhang Q. et al., 2004,
Embalming Amyloid-β: The Role for Aldehyde Stress in Alzheimer's Disease...
2 May 2004. Alzheimer Research Forum, 2004,
12. Liu Q, Honda K, Moreira P, Perry G, Smith M, Zhu X. on PAPER: Coskun PE. et
al., 2004, Mitochondria and Alzheimer’s Disease: A Complex Interrelationship
Recently, Coskun... 27 Aug 2004. Alzheimer Research Forum, 2004,
Abstracts:
13. Perry G, Liu Q, Wataya T, Shimohama, S, Nunomura A, Siedlak SL, Sayre LM,
Smith MA. Neurofilament proteins are major targets of oxidative damage in the
nervous system. J Neuropathol Exp Neurol 61:456, 2002.
14. Liu Q, Smith MA, Siedlak SL, Harris PLR, Sayre LM, Perry G.
237 Phosphorylation-dependent regulation of neurofilament protein oxidation in
Alzheimer disease. 25th Graduate Student Symposium, p. 25, 2002.
15. Liu Q, Smith MA, Siedlak SL, Harris PLR, Sayre LM, Perry G.
Phosphorylation-dependent regulation of neurofilament protein oxidation. Neurobiol
Aging 23 (Suppl 1):S511-S512, 2002.
16. Liu Q, Smith MA, Sayre LM, Perry G. Neurofilament protein oxidation in
Alzheimer’s disease. 2003 Biomedical Graduate Student Symposium, P.46, 2003.
17. Liu Q, Smith MA, Siedlak SL, Harris PLR, Maccioni RB, Sayre LM, Perry G.
Phosphorylation and sequence dependency of neurofilament protein oxidation in
Alzheimer’s disease. Soc Neurosci Abstr, Program No.628.14, 2003.
18. Liu Q, Sayre LM, Siedlak SL, Harris PLR, Maccioni RB, Smith MA, Perry G.
Phosphorylation and sequence dependency of neurofilament protein oxidation.
Research ShowCASE 2004.
19. Liu Q, Smith MA, Siedlak SL, Harris PLR, Maccioni RB, Sayre LM, Perry G.
Phosphorylation modulated oxidative modification of neurofilament protein. J
Neuropathol Exp Neurol 63:523, 2004
20. Liu Q, Smith MA, Siedlak SL, Harris PLR, Maccioni RB, Sayre LM, Perry G.
Phosphorylation dependent control of oxidative modification in Alzheimer’s disease.
Soc Neurosci Abstr, 2004
Submitted:
21. Moreira PI, Honda K, Liu Q, Aliev G, Oliveira CR, Santos MS, Zhu X, Smith MA,
Perry G. Alzheimer’s disease and oxidative stress: the old problem remains unsolved.
Curr Med Chem-Central Nervous System Agents (Submitted)
238 In preparation:
22. Quan Liu, Mark A. Smith, Sandra L. Siedlak, Ricardo B. Maccioni†, Don W.
Cleveland, Lawrence M. Sayre, George Perry. Overexpression of neurofilament
heavy subunit protects neurons from oxidative stress. (Prepared to be submitted to
JBC).
23. Quan Liu, Mark A. Smith, Dandan Wang, De Lin, Sandra L. Siedlak, Peggy L.R.
Harris, Ricardo B. Maccioni†, Robert G. Salomon, Shu G. Chen, Lawrence M. Sayre,
George Perry. Phosphorylation and sequence dependency of neurofilament protein
oxidation in Alzheimer disease. (Prepared to be submitted to JBC or higher journals).
24. Quan Liu, Peggy L.R. Harris, Mark A. Smith, George Perry. High molecular weight
neurofilament subunits are the major pathophysiological targets of HNE modification
in brain. (Prepared to be submitted to journal of neurochemistry).
Conferences / Meetings Attended and Presented:
The 25th Graduate Student Symposium in CWRU, 2002
The 33th annual meeting of Society for Neuroscience in New Oreland 2003
The 26th Graduate Student Symposium in CWRU, 2003
The 2nd Research Showcase in CWRU 2004
The 80th American Association of Neuropathologists annual meeting in
Cleveland, Ohio 2004
The Society for Neuroscience's 34th Annual Meeting in San Diego, California
2004
239