WO1999036082A2 - The use of increased neurofilament protein levels as protection and therapy against neurodegeneration with oxidative stress involvement - Google Patents

Abstract

The present invention describes the use of increased levels of NF proteins to provide protection against reactive oxygen species and neurodegeneration associated with oxidative stress. The neurodegeneration associated with oxidative stress may be due to aging, injury, or neurodegenerative disorders, such as Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Giant Axonal Neuropathy, neuropathies associated with diabetes, toxic neuropathies, Lewy body Dementia, or Guam-Parkinsonism.

Description

THE USE OF INCREASED NEUROFILAMENT PROTEIN LEVELS

AS PROTECTION AND THERAPY AGAINST NEURODEGENERATION WITH

OXIDATIVE STRESS INVOLVEMENT

FIELD OF THE INVENTION

The present invention relates to the use ofincreased neurofilament protein levels as protection against reactive oxygen species in general, and in particular, neurodegeneration with oxidative stress involvement.

BACKGROUND OF THE INVENTION

Neurons are important cells in the nervous system, being involved in receiving, organizing, and transmitting information. Each neuron contains a cell body, anaxon(athin, tube-like process that arises from the cell body and travels some distance before terminating), and dendrites (neuronal processes of the cell body that are shorter and thicker than axons). The cytoskeleton of the neuron provides mechanical strength to the axons and dendrites and a track for transport of materials between the cell body and the nerve terminal. The cytoskeleton is a system of interconnected macromolecular filaments. Three polymeric structures form the basis of this cytoskeleton: actin filaments (microfilaments), microtubules, and intermediate filaments.

Intermediate Filaments:

Intermediate filaments (IFs) are 10 nm filaments found in most eukary otic cells . There are six classes of IFs recognized according to sequence homology and gene structure: type I and II IFs include the acidic, neutral, and basic keratins; type HI IFs include vimentin, desmin, the glial fibrillary acidic protein

(GFAP), peripherin, and plasticin; type IN IFs include neurofilament proteins and α-internexin; type V IFs include the nuclear lamins; and type Nt IFs include nestin expressed in neuroepithelial cells.

Neuronal Intermediate Filaments: Neuronal intermediate filaments (NIFs) include neurofilaments, peripherin, α-internexin, vimentin, and nestin. The NIF proteins are encoded by a large multigene family displaying cell and tissue-specific expression patterns throughout development. There is a sequential appearance of the NIFs in developing neurons. Nestin is expressed during embryonic development of neuroectodermal cells (Lendahl etal, (1990) Cell 60:585-595). This is followed by the co-expression ofvimentin and α- internexin(Cochard andPaulin(1984)J. NeuroscL 4:2080-2094; Tapscottetα/., (1981)Dev. Biol 86:40-45). These NIFs are gradually replaced by NF proteins in maturing CNS neurons (Carden et al, (1987) J. NeuroscL 7:3489-3504; Kaplanetα/., (1990) J NeuroscL 10:2735-2748;Pachterand Liem (1984) Dev. Biol. 103 :200-210).

The NIF proteins are made up of an assembly of protein subunits. The current model ofNIF assembly involves 1 ) the bonding of two subunits to form a dimer; 2) the aggregation of two antiparallel dimers to form a tetramer, called a protofilament (Steinert and Roop ( 1988) Annu. Rev. Biochem. 57:593- 625); 3) the joining of about eight protofilaments end on end; and 4) the association of these joined protofilaments to other joined protofilaments by staggered overlaps to form a 10 nm filament. The cytoplasmic NEF proteins share a homologous central region of similar size (approximately 310 amino acids) flanked by amino- and carboxy-terminal domains varying greatly in sequence and in length. The central region of NTF proteins forms an extended α-helical rod domain that plays a critical role in protein assembly into 10 nm filaments.

Neurofilaments: Of all the NEF proteins that participate in the formation of the neuronal cytoskeleton, the neurofilament triplet proteins are the most abundant. These neurofilaments (NFs) are expressed exclusively in neurons. NFs are found predominantly in axons, where they run longitudinally and parallel to each other. While NFs are present in most populations of neurons in the nervous system, they are particularly abundant in large myelinated axons of peripheral nerves that originate from motor and sensory neurons.

NFs provide mechanical support to the neuron and also play a role in modulating the caliber of large myelinated axons. Axonal caliber is a determinant of conduction velocity. NFs are formed by the copolymerization of three NF protein subunits : light (61 kDa) (NF-L), medium (90 kDa) (NF-M), and heavy (110 kDa) (NF-H) (Hoffman and Lasek (1975) J. Cell Biol. 66:351 - 366). NF-L subunits form the core of the NF and are essential forNF assembly: NF-L is required with either NF-M or NF-H for polymer formation (Ching and Lien ( 1993 ) J. Cell Biol. 122:1323- 1335; Lee et al, (1993) J. Cell Biol. 122:1337-1350). NF-H and NF-M subunits cannot form polymers by themselves; there is an absolute requirement for NF-L subunits in order to form IFs (Gardneretα/., (1984)J. Newro5'ct. i?e5. l l:145-155;HisanagaandHirokawa(1990)J.M?/.5tø/. 211:871-882; Hirokawa (1991) "Molecular architecture and dynamics of the neuronal cytoskeleton" In Burgoyne (ed.) 7be Neuronal Cytoskeleton (New York: Wiley-Liss) 5-74).

NF-M and NF-H subunits have long C-terminal tail domains that form side-arm proj ections in the NF structure, cross-linking NFs and other neuronal structures into a three-dimensional IF matrix. The tail domain of NF-H is rich in charged amino acids and has multiple repeats of Lys-Ser-Pro (KSP) that account for unusually high content of phosphoserine residues in this protein ( Julien and Mushynski (1982)J. 5/σ/. Chem. 257: 10467 '-10470; julien etal, (1988) Gene 68:307-314; and Lees etal, (1988)EMB0J. 7: 1947-1955). The presence of charged amino acids in a domain ofNF-H that forms side-arm projections led to the suggestion that local changes in phosphorylation regulate the spacing between neurofilaments and axonal caliber (Dewaeghetα/., (1992) Ce//68:451-463; Cole et al, (1994) J. NeuroscL 14:6956-6966).

The three different NF subunits are encoded by three different genes, NF-L, NF-M, and NF-H, each of which is under separate developmental control. During neurogenesis, there is differential expression of the three subunits: the NF-L and NF-M proteins are coexpressed during early embryonic development, while the activation ofNF-H expression is delayed to the postnatal period (Shaw and Weber (1982)Nαtwre 298:277-279; Mienetal, (1986) Mol Brain Res. 1 :243-250; Carden etal, (1987) J. NeuroscL 7:3489-3504).

Neurofilaments and Neurodegenerative Diseases:

Neurofilaments have been linked to a number of neurodegenerative diseases. Large motor neurons are particularly vulnerable to NF abnormalities because of their high NF content and their long axons. Abnormal depositions ofNFs (often called spheroids or Lewy bodies) is a phenomenon observed in many neurodegenerative diseases (Table 1).

Table 1. Human Diseases with Abnormal NF Accumulations Disease Abnormalities Prevalence

ALS NF depositions in motor neurons 70% of cases

• Decline of 60% in NF-L mRNA

Parkinson's disease Lewy bodies in substantia nigra and locus coreuleus 100% of cases

• Declines of 30% NF-L mRNA and 70% NF-H mRNA Alzheimer's disease Cortical Lewy bodies 20% of cases

• Decline of 70% in NF-L mRNA Lewy body Dementia Cortical Lewy bodies Guam-Parkinsonism NF depositions in motor neurons 100% of cases

Giant Axonal Neuropathy NF accumulations in peripheral axons Peripheral Neuropathies NF accumulations in peripheral axons that can be induced by various toxic agents, such as IDPN, hexanedione, acrylamide

As an example, there is evidence that NFs play a central role in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). ALS is an adult-onset and heterogeneous neurological disorder that affects primarily motor neurons in the brain and spinal cord. The degeneration of motor neurons in the brain and spinal cord leads to denervation atrophy of skeletal muscles and, ultimately, to paralysis and death. Although multiple genetic and environmental factors may be implicated in ALS, the striking similarities in the clinical and pathological features of sporadic ALS and familial ALS suggest that similar mechanisms of disease may occur.

A characteristic pathological finding in ALS patients is the presence of abnormal NF accumulations in the cell body and proximal axon of surviving motor neurons. These NF accumulations have been viewed as a marker of neuronal dysfunction, perhaps reflecting defects in axonal transport. Recent evidence suggests that NFs may also play a causative role in ALS and other motor neuron diseases. Aberrant neuronal swellings that are highly reminiscent of those found in ALS have been reported in transgenic mice overexpressing either the human NF-H or the mouse NF-L. Overexpression ofhuman NF-H in mice provoked a late-onset motor neuron disease characterized by the presence of aberrant NF accumulations in spinal motor neurons (Cote etal, (1993) Cell 73:35-46; Collardetor/., (1995) Nature 375:61 -64). Neuronal atrophy of skeletal muscle in these mice occurred as a result of axonal degeneration.

High-level expression of the wild-type mouse NF-L gene in mice induced an early-onset motor neuron disease accompanied by massive accumulation ofNFs in spinal motor neurons and muscle atrophy (Xu etal, (1993) Cell 73:23-33). Similarly, expression of a mutant assembly-disrupting NF-L gene provoked massive accumulation ofNFs in spinal motor neurons, selective death of motor neurons, neuronophagia, and severe denervation atrophy of skeletal muscle (Lee etal, (1994) Neuron 13:975- 988).

As additional evidence for NF involvement in ALS, a recent report has revealed that there is a 60% decrease in levels ofNF-L mRNAin the motor neurons of patients with ALS (Bergeron et al. , ( 1994) Brain Res. 659:272-276). As well, mutant NF-H alleles have been found in some ALS patients

(Figlewicz et al, (1994) Hum. Molec. Genet. 3:1757-1761).

A mutant form of the human copper-zinc superoxide dismutase (SOD) gene is responsible for 2% of ALS cases (Gurney et al, (\ 994) Science 264 : 1772- 1775). Transgenic mice expressing the human SOD 1 mutation develop a motor neuron disease similar to ALS, in which neuronal swellings occur. These swellings are rich in NFs (Tu et al, (1996) Proc. Natl. Acad ScL USA 93(7):3155-3160).

NFs are also implicated in Parkinson' s disease. The pathological hallmark of idiopathic Parkinson' s disease is the presence ofLewy bodies (LB s), cytoplasmic inclusions made up of altered NF proteins. These LBs are located in neurons of the substantia nigra. A subset of demented elderly patients also exhibit LB-like inclusions in their cortical neurons. The mechanisms involved in the abnormal aggregation ofNF proteins to form LBs are still unknown. It has been found that levels ofNF-L and

NF-H mRNAs in substantia nigra neurons are reduced in Parkinsonian patients as compared to age- matched controls. There is also reduced NF synthesis in LB-containing neurons.

In patients with Alzheimer's disease, cortical LBs are present in approximately 20% of cases. It has also been discovered that there is a 70% decrease in NF-L mRNA expression in these patients (Crapper McLachlan et α/., (1988) >/ec. Brain Res. 3:255-262).

Abnormal accumulations ofNFs in distinct regions of the neuron also occur in a variety of other disorders, including an inherited giant axonal neuropathy (Carpenter etal, (1974)). Giant axonal neuropathy (GAN) is a comparatively rare neurologic disorder affecting humans and dogs. Neurofilaments collect in focal accumulations at the distal ends of nerves in the peripheral and central nervous systems. These accumulations, which are found multifocally along a single axon, consist of massive collections of abnormally oriented and whorled NFs.

Abnormal accumulations of NFs are also present in toxic neuropathies induced by β,β'- iminodipropionitrile (IDPN), 2,5-hexanedione, acrylamide, and aluminum.

The mechanisms underlying the abnormal aggregation ofNF proteins in neurodegenerative diseases are still unknown. It is very interesting, however, that a decreased level ofNF-L mRNA is associated with degenerative neurons in ALS, Parkinson' s disease, Alzheimer ' s disease, and other neurodegenerative diseases.

Neurofilaments and Aging:

Aging is a factor that may contribute to axonal atrophy. There is a normal decline (50-60%) in NF mRNA expression with aging (Parhadetα/., (1995)J. NeuroscL Res. 41:355-366). Theresulting decrease in NFs may be linked to axonal atrophy and a reduced capacity for compensatory axonal outgrowth during aging. Methods of enhancing neuronal regeneration could attenuate the aging process.

Oxidative Stress and Disease:

Although neurons depend on oxidative metabolism for survival, a consequence of this process is the production of reactive compounds such as hydrogen peroxide and oxyradicals (Cohen and Werner, 1994). Unchecked, these reactive species can lead to RNA damage, peroxidation of membrane lipids, and neuronal death. Several mechanisms serve to limit this oxidative stress, including the presence of reducing compounds such as ascorbate and glutathione and enzymatic mechanisms such as superoxide dismitase, which catalyzes the reduction of superoxide radicles. Oxidative stress may also be relived by aminosteroid reagents that serve as free radical scavengers. Recent genetic evidence has linked genetic disturbances in the metabolism of oxyradicals by superoxide dismutase to the etiology of ALS . In Parkinson' s disease, attention has been focused on the possibility that oxidative stress induced by the metabolism of dopamine may underlie the selective vulnerability of dopaminergic neurons observed in Parkinson's disease. The preliminary catabolic pathway of dopamine to 3,4-dihydroxyphenylacetic acid (DOPAC) ia catalyzed by monoamine oxidase and generates hydrogen peroxide. Hydrogen peroxide, in the ferrous form, which is relatively abundant in the basal ganglia, may generate hydroxyl free radicals. If the protective mechanisms are inadequate because of inherited or acquired deficiency, the oxyradicals could cause degeneration of dopaminergic neurons. Supporting this proposal is the observation of increased lipid hydroperoxides in the substantia nigra in PD.

Reactive oxygen species (ROS), including hydroxy radicals, superoxide anions, and hydrogen peroxide, are highly reactive substances that can cause tissue injury. ROS are produced in cells by enzymatic, spontaneous, and photochemical oxidation reactions resulting in oxidative stress (H. Sies, Oxidative Stress, 1985). ROS are produced as by-products of oxidative damage to a wide variety of macromolecules and cellular components (Fridovich in Eichhorn, and Marzilli, ed., Advances in Inorganic Biochemistry (New York: Elsevier/North Holland, 1979) 67-90; Freeman and Crapo

(1982) Laboratory Investigation 47: 412-26). For example, ROS can be generated by the cytotoxic effects ofionizing radiation (Petkau (1980) Acta. Physiol. Scand. Suppl.492:81-90; Biaglowetα/., (19 3) Radial Res. 95 :437-455), by various chemotherapeutic agents (Tomasz ( 1976) Chem. Biol. Interact. U:S9-91; ownand Sim(l911)Biochem. Biophys. Res. Commun. 77: 1150-1157;Borek and Troll (1983) Proc. Natl Acad. ScL USA 80: 1304-1307), and by a variety of biological processes, including aging (DiGuiseppi and Fridovich (1984) CRC Crit. Rev. Toxicol 12 : 315-342; Slater (1984) Biochem. J. 222: 1-15).

ROS are highly reactive and can damage biological molecules. Examples of disorders associated with the generation ofROS include synovial inflammation induced by bacterial Upopolysaccharide endotoxin (LPS), inflammation caused by adjuvant-induced arthritis, bleomycin-induced lung fibrosis, reperfusion injury, transplantation rejection, hyperoxia, and diseases caused by oxygen and light It has been suggested that ROS may be involved in hyperthermic cell injury as well (Omar etal. , ( 1987) Cancer 5 Res. 47 3473)

ROS have been implicated in neurodegeneration (Bowling andBeal (1995) //e Sci. 56 1151-1171), particularly in Alzheimer's Disease (Smithetα/., (1995) Trends Neurosci 18 172-176, Smith etal, (\996)Nature 382:120-121, Good etal, (1996) Am. J. Pathol. 149 21-28, Sayre etal, (1997) J. Neurochem. 68(5) 2092-2097) These studies have identified markers of oxidative stress in l o neurofibrillary tangles and senile plaques of Alzheimer ' s patients, including advanced glycation end products, nitrotyrosine, free carbonyls, heme oxygenase-1, and advanced lipid peroxidation end products Other evidence implicating oxidative stress in neurodegenerative diseases and aging is listed in Table 2

Table 2. Evidence of Oxidative Stress in Neurodegenerative Diseases

15 Disease Evidence References

ALS Mutations in SOD1

-Tyrosine nitration via peroxyrutπte Bruijn et al (1997) PNAS 94 7606-7611 Wiedau- -Increased peroxidase activity Pazos e/ α/, (1996) Science 271 515- 518

Alzheimer's Mutations coding for cytochrome c Davis et al , (1997) PNAS 94 4526-4531 oxidases in mitochondrial DNA Increased lipid peroxidation Sayre et al (1997) Neurochem 8 2092-2097

Advanced glycosylation end products Smithed/, (1995) TINS 18 172-176

Increase of ROS by amyloid-β Behl et al , (1994) Cell 77 817-827

Carbonyl modification of NF-H Smith et al , (1997) J Neurochem 64 2660- 266

Parkinson's Increased iron in substantia nigra Olanow (1993) Trends Neurosci 16 439-444 Glycoxidation Castellani et al, (1996) Brain Res. 737: 195-200

The exact mechanism by which RO S cause damage to neurons is yet unknown. During aging and in neurodegenerative disease, such as ALS, Alzheimer' s Disease, and Parkinson' s Disease, there is a dramatic reduction in the levels of neurofilament mRNAs. This could contribute to increased vulnerability of neurons to oxidative stress.

One study suggests that the ROS peroxynitrite causes tyrosine nitration in NFs of ALS patients, which interrupts NF phosphorylation and assembly. It is suggested that this impaired phosphorylation ofNF subunits may affect axonal transport, causing NF accumulation and motoneuron death (Chou etal, (1996) J. Neurological Sciences 139(Suppl.)16-26).

Protective cellular mechanisms against ROS damage are provided by anti-oxidants and radical scavengers such as β-carotene, glutathione, cysteine, and ascorbic acid, as well as by enzymes such as superoxide dismutase and catalase. For example, when cells are exposed to oxygen-generating compounds or other oxidative stresses, glutathione (GSH) and related cellular sulfhydryl compounds become oxidized (Adams et al, (1983) J. Pharmacol. Exp. Ther. 227:749-754).

Superoxide dismutases (SODs) are a group of metalloproteins that provide a defense mechanism against oxygen toxicity: SODs catalyze the conversion of the superoxide anion to hydrogen peroxide, which can then be detoxified to water and oxygen by catalase and glutathione peroxidase. There are several known forms of SOD containing different metals and different proteins. Eukaryotic cells contain copper-zinc SOD and manganese SOD.

Mutations in the human copper-zinc SOD (SOD 1 ) gene located on chromosome 21 have been found in approximately 20% offamilial ALS cases. To date, more than 40 different SOD1 mutations have been identified (Brown (1995) Cell 80:687-692). The presence of abnormal NF accumulations in motor neurons of some familial ALS cases caused by S OD 1 mutations (Rouleau et al. , ( 1996) Annals of Neurology 39: 128-31) and the finding of similar NF inclusions in transgenic mice expressing SOD 1 mutants (Tu etal, (1996) Proc. Natl. Acad. ScL USA 93(7):3155-3160) suggest that NF proteins, which are abundant proteins with long half-lives, are favored targets of oxidative damage by SOD 1 mutants.

Many lines of evidence suggest that SOD 1 mutations cause ALS through mechanisms involving a gain of adverse function rather than a loss of SOD activity (Borchelt et al, (1995) J. Biol Chem.

270 :3234-3238). For example, transgenic mice overexpressing SOD 1 mutations developed motor neuron disease even though the SOD activities in mice were not reduced (Gurney etal. , ( 1994) ScL

264:1772-1775; Wong etal, (\995)Neuron 14:1105-1116;Rippsetα/., (1995)Prøc. Natl Acad

ScL USA 92:689-693). Moreover, mice homozygous for the targeted disruption of the SOD 1 gene do not develop motor neuron disease (Reaume etα/., (1996) Nat. Genet. 13:43-47). Anumber of mechanisms have been proposed. One mechanism suggests that SOD 1 mutations render the copper in the active site of SOD 1 more accessible to peroxynitrite, allowing the formation ofnitronium-like intermediates that can nitrate proteins at tyro sine residues (Beckman et al. , ( 1994) Prog. Brain Res.

103:372-380;Beckmanetα/., (1993)Nαtwre364:584-585). The nitration of tyrosine residues inΝF- L to nitrotyrosine inhibits normal ΝF-L phosphorylation, which is required for assembly ofΝF subunits

(Chou et al, (1996) J. Neurological Sciences 139(Suppl.)16-26).

Reactive nitrogen species such as peroxynitrite might also create crosslinks by the formation of dityrosine and thereby induce ΝF aggregation (Julien (1997) Trends Cell Biol. 7:243-249). Oxidative modification ofΝF proteins by altered SOD 1 activity could result in the formation of protein crosslinks, for example, through a copper-mediated oxidation of sulfhydryl groups or a production of carbonyls on lysine residues. Carbonyl-related modifications ofΝF-H have been reported in the neurofibrillary pathology of Alzheimer's disease (Smith et al, (1996) Nature 382: 120-121).

Another proposed mechanism suggests that SOD 1 mutants increase peroxidase activity, leadingto the formation of an increased number ofhydroxyl radicals from hydrogen peroxide than the wild-type SOD (Wideau-Pazos et al, (1996) Science 271 :515-518). This increase in ROS levels then causes increased cellular damage. Recently, it has been discovered that normal SODl protects calcineurin, a phosphatase, from inactivation by preventing oxidation of the iron at its catalytic site, whereas SOD 1 mutations can alter the activity of calcineurin (Wang etal, (1996) Nature 383:434-437). These results suggest that SOD 1 and oxidation could play a role in signal-transduction phosphorylation cascades, with SOD 1 5 mutations leading to anomalous phosphorylation of NF proteins.

Previous reports have indicated that NF proteins have multiple calcium binding sites, including high affinity sites, suggesting NF involvement in calcium homeostasis (Lefebvre and Mushynski (1988) Biochemistry 27:8503-8508; Lefebvre and Mushynski (1987) Biochem. Biophys. Res. Comm. 145: 1006- 1011). From these studies, it is conceivable that increasing the NF protein levels might confer l o protection against rises in intracellular calcium resulting from oxidative stress and mitochondrial damage reported in mutant SODl transgenic mice (Wong etal, (1995) Neuron 14: 1105-1116;Dal Canto and Gurney (1995) Brain Res. 676 : 25-40). A calcium involvement in ALS has been strongly supported by the selective vulnerability of motor neurons lacking typical calcium-binding proteins, parvalbumin and calbindin as shown in studies on ALS patients and monkeys (Reiner et al, (1995) Exp. Neurol

15 131 :239-250; Elliott and Snider (1995) Neuroreport 6:449-452), as well as in a line of SODl transgenic mice (Morrison et al. , ( 1996) J. Comp. Neurol. 373 : 619-31 ). Indeed, ifNF proteins act as calcium chelators, the dramatic declines in NF mRNA levels occurring during aging (Parhad et al. , (1995) J. NeuroscL Res. 41 :355-66) and, to agreater extent, in neurodegenerative diseases including ALS (Bergeron et al, (1994) J. Neuropathol Exp. Neurol. 53:221-230; Kuchel et al, (1997)

20 Neuroreport 8 :799-805) may contribute to increase the susceptibility of specific neuronal populations to oxidative stress and calcium-mediated death.

Efforts to slow down SOD 1 -induced disease in transgenic mice by various therapeutics, including vitamin E, riluzole, gabapentin and D-penicillamine, produced little benefits (Gurney etal, (1996) Annals of Neurology 39 : 147- 157; Hottinger et al. , ( 1997) Eur. J. Neurosci. 9 : 1548- 1551 ), while 25 bcl-2 overexpression increased the longevity of SODl miceby~15% (Kosticetα/., (1997) Science

277:559-562).

This review ofNFs, neurodegeneration, and oxidative stress indicates that oxidative stress is involved in neurodegenerative diseases and aging. As is evident, there still remains a need for a means of protecting neurons against such neurodegeneration involving oxidative stresses.

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Publications referred to throughout the specification are hereby incorporated by reference in their entireties in this application.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a means of protection against reactive oxygen species (ROS) in general, and in particular, neurodegeneration associated with oxidative stress.

The present invention describes the use ofincreased levels ofNF proteins to provide protection against ROS and/or neurodegeneration associated with oxidative stress in a subject. The NF proteins (or fragments thereof) can be (from) NF-H, NF-M, or NF-L, or any combination thereof. The subject can be any animal, including mammals. Preferably, the subject is human.

The neurodegeneration associated with oxidative stress can be due to neurodegenerative disorders, aging, or injury. Neurodegenerative disorders include Amyotrophic lateral sclerosis (ALS), Alzheimer' s disease, Parkinson' s disease, Giant Axonal Neuropathy, neuropathies associated with diabetes, toxic neuropathies such as those induced by β,β'-iminodipropionitrile (IDPN), 2,5-hexanedione, acrylamide, and aluminum, Lewy body Dementia, or Guam-Parkinsonism.

Any strategy to up-regulate the levels ofNF proteins in a subject can be used. Strategies include the administration of purified or synthetic NF proteins, or fragments thereof, gene therapy, including the administration of viral vectors encoding NF proteins, and the administration of chemical compounds to increase NF protein expression. Various other obj ects and advantages of the present invention will become apparent from the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The genomic structure of the Ad5-hNF-L viral vector. In a), the genome ofthe vector is displayed in map units (m.u.) with lOOm.u. corresponding to the complete genome. The region containing the human NF-L expression cassette covers m.u. 1.25 to 9.25, and is shown by hatched lines. TR means terminal repeat. In b), the detailed structure ofthe expression cassette is shown. From right to left, it includes: "Promoter" being the human NF-L gene minimal promoter (hatched); "Exon 1 , Exon 2, Exon 3 , Exon 4" being the four coding exons ofthe hNF-L gene (flecked) separated by introns (in black); and "pA" being the hNF-L polyadenylation signal. The direction of transcription is given by an arrow above the cassette.

Figure 2. Adenoviruses to direct β-gal and hNF-L expression to spinal motor neurons.

Adenoviral recombinants containing a CMV-lacZ expression cassette were injected into the right tibialis muscle of 2 month old hNF-H+/+ mice. The lacZ expression in spinal motor neurons sending their axons into the L5 ventral roots was detected in β-gal stained / neutral red counterstained sections at

(a) 7 days and (b) 14 days post-inj ection. The neurofilamentous swellings in hNF-H+/+ mice were not affected by the viral-mediated β-gal expression, (c), hNF-L expression 48h post-infection by Ad5- hNFL in a dissociated culture ofE 13 hNF-H+/+ spinal cord. The hNF-L promoter provides neuronal specific expression in this mixed culture, (d), Detection of hNF-L expression in hNF-H+/+ mice spinal cord ipsilateral to the injection, 21 days after injection of Ad5-hNFL into the right tibialis muscle, (e),

No hNF-L expression could be detected in the spinal cord contralateral to the injection side, (f), Toluidine blue stained Epon sections show a reduction in the number of perikaryal swellings in the ipsilateral spinal cord (arrows) when compared to (g) the non-injected contralateral side. The hNF-L proteins in spinal motor neurons could be detected 9 months after muscular viruses injection. At this time point, the motor neurons expressing hNF-L had no neurofilamentous swelling (d, insert), unlike motor neurons on the contralateral side (e, insert).

Figure 3. Northern and western blot analysis of double transgenic mice.

(A) Detection ofNF-L and hNF-H mRNAs in spinal cord of transgenic mice. The probe used for the detection ofNF-L hybridizes with both human and mouse NF-L transcripts. An actin cDNA probe was used for standardization. Lane 1, hNF-H+/+; lane 2, hNF-H+/-; lane3, hNF-L+/+; lane4, hNF-

L+/-; lane 5, hNF-L+/+;hNF-H+/+; lane 6, hNF-L+/-;hNF-H+/-; lane 7, normal; lane 8, hNF-L+/- ;hNF-H+/+ and lane 9, hNF-L+/+;hNF-H+/- mice. (B) Coomassie stained SDS-PAGE and western blot analysis ofTriton-insoluble spinal cord protein extracts from 6 month old mice. Western blots were carried out on replica filters. Lanes identification is identical to figure 1A.

Figure 4. Reduction of perikaryal swellings by extra hNF-L.

Light micrographs of spinal motor neurons in the lumbar region, (a), normal; (b), hNF-L+/+; (c), KNF- H+/+; (d), hNF-L+/+;hNF-H+/+; (e), hNF-H+/-; (f), hNF-L+/-;hNF-H+/-; (g), hNF-L+/-;hNF- H+/+; (h), hNF-L+/+;hNF-H+/-. NF swellings appear as lightly stain areas. Thin arrows point to normal appearing motor neurons, large arrows in (h) point to a giant axon found in hNF-L+/+;hNF- H+/- mice.

Figure 5. Restoration of axon calibers by hNF-L co-expression.

Light micrographs show transverse sections ofL5 ventral roots ofthe various transgenic mice at 6 months of age. (a), normal; (b), hNF-L+/+; (c), hNF-H+/+; (d), hNF-L+/+;hNF-H+/+; (e), hNF- H+/-; (f), hNF-L+/-;hNF-H+/-; (g), hNF-L+/-;hNF-H+/+; (h), hNF-L+/+;hNF-H+/-. Mice overexpressing the hNF-H transgene alone show dramatic decrease in axonal caliber and some degeneration profile © and e). Co-expression of hNF-L restores axonal diameters (d and f).

Figure 6. Caliber distribution of ventral root axons in 6 month old mice.

Cross-section areas of L5 ventral root axons were expressed into diameters of circles with corresponding surface areas. The histograms compare (a), normal versus hNF-L+/+; (b), hNF-H+/+ versus hNF-L+/+;hNF-H+/+; (c), hNF-H+/- versus hNF-L+/-;hNF-H+/-; (d), hNF-L+/+;hNF-H+/- versus hNF-L+/-;hNF-H+/+. Figure 7. Restoration ofNF network in axons of doubly transgenic mice.

Electron micrographs ofL5 ventral root ultrathin sections of 6 month old mice: (a), normal; (b), hNF- L+/+; (c), hNF-H+/+; (d), hNF-L+/+;hNF-H+/+; (e), hNF-H+/-; (f), hNF-L+/-;hNF-H+/-; (g), hNF-L+/-;hNF-H+/+; (h), hNF-L+/+_;hNF-H+/-. Note the difference ofinter-NF spacing between mice overexpressing hNF-H alone © and e) and those overexpressing both hNF-L and hNF-H at different levels (d, f, g and h).

Figure 8. Restoration of slow axonal transport by hNF-L co-expression.

³⁵S-methionine was injected into the spinal cord of 3 month old animals at the entry point ofthe L5 ventral root. After 28 days, the L5 ventral roots, L5 DRGs and 8 successive 3 mm segments of sciatic nerves were isolated. The pooled ventral roots and DRGs, lane vr, represent 12 mm of axonal length.

As previously reported, the rate of axonal transport is diminished in hNF-H+/+ mice. The axonal transport of cytoskeletal components in doubly transgenic mice is significantly enhanced as compared to hNF-H+/+ mice. Note the transport rates for mouse NF-M (arrow) and tubulin (asterisk) which are restored in the doubly transgenic mice.

Figure 9. Rescue of abnormal limb contraction.

(a), Normal mice extend their legs when lifted by their tail whereas hNF-H overexpressing mice contract their hindlimbs. (b), In contrast, mice overexpressing both human NF transgenes extend their limbs like normal mice.

Figure 10. Expression of human NF-H and SODl^G37R proteins in transgenic mice. A) Immunodetection of human NF-H protein in spinal cord extracts from normal mice (lane 1), transgenic mice bearing the SODl^G37R(lane2), doubly S0D1^G37R;NF-H⁴⁴ transgenic mice (lane 3), and transgenic mice expressing the human NF-H⁴⁴ protein (lane 4) . Protein extracts were obtained by homogenization of spinal cord in SUB (0.5% SDS, 8M Urea and 2% β-mercaptoethanol). Samples were electrophoresed on 7.5% polyacrylamide SDS-PAGE and transferred on nitrocellulose filter. The transgene expression product was detected with a human NF-H specific primary antibody (provided by Dr. V. M.-Y. Lee) and ECL (Amersham) chemoluminescence kit for Western blotting. B) Immunodetection ofhuman mutant and mouse SOD 1 proteins in spinal cord extracts from normal mice (lane 1 ), transgenic mice for SOD 1 ^G37R(lane 2), SOD l∞^NF-H⁴⁴ transgenic mice (lane 3), and transgenic mice expressing the human NF-H⁴⁴ protein (lane 4). Protein extracts were obtained by homogenization of spinal cord in SUB (0.5% SDS, 8M Urea and 2% β-mercaptoethanol), electrophoresed on 15% polyacrylamide SDS-PAGE, and incubated with a primary antibody directed against the SODl protein (Biodesign inc).

Figure 11. Increased life-span of S0D1^G37R transgenic mice by NF-H overexpression.

Survival curves of transgenic mice expressing S0D1^G37R alone or together with NF-H transgenes coding for the NF-H⁴³ or NF-H^proteins. The survival probability of transgenic mice is plotted as a function of their age in months. It is remarkable that expression ofthe human NF-H⁴⁴ transgene increased the mean longevity of S0D1^G37R by approximately 6 months.

Figure 12. Decreased neurodegeneration in SODl^G37Rtransgenic mice co-expressing human NF-H proteins.

Light micrographs show the lumbar (L5) ventral root axons, dorsal root axons and motor neurons from a normal mouse (A, B, C), a singly SODl ^G37R transgenic mouse (D, E, F), a doubly SODl ^G37R;NF-

H⁴⁴ transgenic mouse (G, H, I) and a doubly S0D1^G37R;NF-H¹³ transgenic mouse (J, K, L). Neurofilamentous swellings are indicated by arrows in (L). Tissues were embedded in Epon and 1 mm sections were stained with toluidine blue. Bar = 50 mm

Figure 13. Increased lifespan of SODl^G37R transgenic mice by NF-L overexpression. Survival curves of transgenic mice expressing S OD 1 ^G37R alone or together with the NF-L transgene.

The survival probability of transgenic mice is plotted as a function of their ages in weeks. It is remarkable that NF-L overexpression increased the mean longevity of SOD 1 ^G37R transgenic mice by approximately eight weeks.

DETAILED DESCRIPTION OF THE INVENTION The present invention resides in the discovery that increased levels ofNF proteins or fragments thereof provide protection against neurodegeneration associated with oxidative stress.

The present invention describes the use ofincreased levels ofNF proteins to provide protection against neurodegeneration associated with oxidative stress in a subject.

NF Proteins

The NF proteins can be NF-H, NF-M, or NF-L, or any fragment thereof, or any combination thereof.

Subject

The subject can be any animal, including mammals. Preferably, the subject is human.

Oxidative Stress The cellular damage may be caused by oxygen-derived free radical formation. The three most important are superoxide (0₂ ^"), hydrogen peroxide (H₂0₂), and hydroxyl ions; these are produced during normal metabolic processes as well as in reaction to cell injury. The extent of their damaging potential can be decreased by antioxidants.

Neurodegeneration The neurodegeneration associated with oxidative stress can be due to neurodegenerative disorders, aging, or injury. Neurodegenerative disorders include Amyotrophic lateral sclerosis (ALS), Alzheimer' s disease, Parkinson' s disease, Giant Axonal Neuropathy, neuropathies associated with diabetes, toxic neuropathies such as those induced by β,β'-iminodipropionitrile (IDPN), 2,5-hexanedione, acrylamide, and aluminum, Lewy body Dementia, or Guam-Parkinsonism.

Increasing NF Protein Levels

Any strategy to up-regulate the levels ofNF proteins in a subject can be used. Strategies include the administration of purified or synthetic NF proteins, or fragments thereof, gene therapy, and the administration of chemical compounds to increase NF protein expression. Administration of NF proteins

Purified or synthetic NF proteins may be administered to increase the NF levels in a subject. The proteins utilized may be obtained by methods known by those skilled in the art. For example, they may be purified from a natural source. Alternatively, they may be made by recombinant means utilizing available sequence data.

The NF proteins may be delivered alone or in combination, and may be delivered along with a pharmaceutically acceptable vehicle. Ideally, such a vehicle would enhance the stability and/or delivery properties. The invention also provides for pharmaceutical compositions containing the active factor or fragment or derivative thereof, which can be administered using a suitable vehicle such as liposomes, microparticles or microcapsules. In various embodiments ofthe invention, it may be useful to use such compositions to achieve sustained release ofthe active component.

The amount ofNF protein that will be effective in the treatment of a particular disorder or condition will depend upon the nature ofthe disorder or condition and can be determined by standard clinical techniques.

Gene Therapy

Gene therapy can also be used to increase NF protein levels. Gene therapy methods include the use of recombinant viral vectors encoding NF proteins.

Generally any virus capable of infection and gene transfer can be employed. Suitable viruses for this invention include adeno viruses, adeno-associated virus, herpes simplex viruses, the AID S virus, and retroviruses well known to those skilled in the art.

The viral vector employed may, in one embodiment, be an adeno viral vector that includes essentially thecompleteadenoviralgenome(Shenketα/., (1984)Cwrr. Topics Microbiol Immun. l l l(3):l-39). Alternatively, the viral vector may be a modified adenoviral vector in which at least a portion ofthe adenoviral genome has been deleted. Preferably, the viruses used in the construction of viral vectors are rendered replication-defective to remove the effects of viral replication on the target cells. Generally, any mammalian NF protein can be employed in the present invention. Preferably, the NF protein is human. The DNA sequences can be either cDNA or genomic DNA. DNA encoding the entire NF protein, or any portion thereof, may be used. Due to the degeneracy ofthe genetic code, other DNA sequences that encode substantially the same NF protein or a functional equivalent can also be used. Multiple gene copies may also be used.

The sequence ofthe human NF-L gene has been published (Julien et al. , ( 1987) Biochim. Biophys. Acta 909: 10-20). Other NF-L sequences are also published, including the mouse sequence (Lewis and Cowan (1986) >/. Cell. Biol. 6: 1529-1534), and the rat sequence (Chin and Lien (1989) Europ. J. CellBiol. 50:475-490). Any other NF-L sequences published in the future can also be used in this invention.

The sequence ofthe human NF-H gene has been published (Lees etal, (\9 %)EMBOJ. 1: 1947- 1955). Other NF-H sequences are also published, including the mouse sequence (Julien etal, (1988) Gene 68:307-314). Any other NF-H sequences published in the future can also be used in this invention.

The sequence ofthe human NF-M gene has been published (Myers etal, (\981)EMBOJ. 6:1617-

1626). Other NF-M sequences are also published, including the mouse sequence (Levy etal, (1987) Eur. J. Biochem. 166:71-77). Any other NF-M sequences published in the future can also be used in this invention.

The DNA sequences encoding the NF-L protein are under the control of a suitable promoter. Any suitable promoter or any portion thereof may be employed to mediate expression, including an NF-L gene' s own promoter, other neuron-specific promoters such as NF-H, NSE, Thy- 1 , or priori, or a viral promoter such as the CMV or S V40 promoter. APP, PDGF, and α-tubulin can also be used. The NF gene's own promoter is preferred when neuron-specific expression ofthe NF is desired.

In order to produce the gene constructs used in the invention, recombinant DNA and cloning methods, which are well known to those skilled in the art, may be utilized (see Sambrook et al, Molecular Cloning, A Laboratory Manual, 2d ed. (New York: Cold Spring Harbor Laboratory Press, 1989), including the use of restriction enzymes, site directed mutagenesis, ligation, homologous recombination, and transfection techniques.

It will be appreciated that administration ofthe viral vectors ofthe present invention will be by procedures well established in the pharmaceutical arts, e.g. by direct delivery to the target organ, tissue or site, intranasally, intravenously, intramuscularly, subcutaneously, intradermally and through oral administration, either alone or in combination.

Preferably, the viral vectors are administered by injecting vector suspension into various locations of the nervous system, or by injection into nerves, or injection into peripheral tissues such as skin or muscles, which are innervated by neurons. In the latter case, the vector enters the neurons via the axons or axon terminals, and the vector genome is transported retrogradely in the axon to the nucleus.

The dosages administered will vary from subject to subject and will be determined by the level of enhancement of neurofilament function balanced against any risk or deleterious side effects. Monitoring levels of transduction, neurofilament expression and/or the presence or levels of normal neurofilament will assist in selecting and adjusting the dosages administered.

Administration of Chemical Compounds

Chemical compounds may be administered to increase the NF protein levels in a subject by affecting endogenous gene expression. The expression of genes is a highly regulated process: different genes are selectively expressed in different cells at different times. There are many steps in the pathway leading from DNA to protein, all of which can be regulated : ( 1 ) transcriptional control wherein it is determined when and how often a gene is transcribed into primary RNA transcripts; (2) RNA processing control, wherein RNA transcripts are modified by posttranscriptional processing and splicing into mRNA; (3 ) RNA transport control which selects which copies of mRNA in the nucleus are to be exported to the cytoplasm; (4) translational control whereby mRNAs in the cytoplasm are selected for translation by ribosomes into protein; (5) mRNA degradation control wherein certain mRNA molecules in the cytoplasm are selectively destabilized; and (6) protein activity control whereby specific protein molecules can be selectively activated, inactivate, or compartmentalized.

Many ofthe known mechanisms of gene regulation act at the initial stage of transcription. The transcriptional regulation of eukaryotic genes is thought to occur via regulatory elements at several locations: promoter sequences, located immediately upstream ofthe transcription start site; and

5 regulatory elements such as enhancer or repressor sequences located upstream ofthe promoter, within intron sequences (non-coding sequences located between exons or coding sequence), and within 3 ' sequences located downstream from the coding region. Promoter sequences are required for the accurate and efficient initiation of gene transcription. Enhancers and/or repressors regulate promoter activity to affect the level and cell specificity of gene transcription. Enhancers and repressors can effect l o their regulatory influences over long distances and in a position and orientation-independent manner.

For many genes, it is clear that multiple regulatory elements are involved in a complex series of interactions.

Any chemical compounds that work at any of steps in the pathway from DNA to protein to increase NF protein levels can be used in this invention. For example, a chemical may act as aDNA-binding 15 protein to turn on the expression of a NF protein. Another chemical may act to inhibit other negative

DNA-binding proteins from acting as repressors. In both cases, the endogenous NF proteins levels will be increased.

Improvement over Current Tools:

Presently, efforts to slow down SOD 1 -induced disease in transgenic mice by various therapeutics have 20 not been very effective. A number of agents, including vitamin E, riluzole, gabapentin and D- penicillamine, have produced little benefit (Gurney etal, (1996) Annals of Neurology 39: 147-157;

Hottinger et /., (1997) Eur. J. Neurosci. 9: 1548-1551). One technique, bcl-2 overexpression, increased the longevity of SODl mice by only 15% (Kostic etα/., (1997) Science 211.559-562).

The present invention involving the overexpression ofNF-H protein has been able to extend the lifespan 25 of SODl mice by up to 65%. This is an enormous improvement over currently available methods.

The present invention is described in further detail in the following non-limiting examples. It is to be understood that the examples described below are not meant to limit the scope ofthe present invention. It is expected that numerous variants will be obvious to the person skilled in the art to which the present invention pertains, without any departure from the spirit ofthe present invention. The appended claims, properly construed, form the only limitation upon the scope ofthe present invention.

5 EXAMPLE I

CONSTRUCTION OF THE Ad5-hNF-L VECTOR

A defective adenovirus vector was created containing an expression cassette for the human neurofilament light gene (Ad5-hNF-L). The adenovirus vector was derived from the human adenovirus serotype 5 (Ad5) mutants/309 (Jones and Shenk(1979) Cell 17:683-689), by creating a deletion in l o the early region 1 (E 1 ) from nucleotides 452 to 3328 (based on the wild-type Ad5 sequence); this deletion rendered the virus replication defective. A stretch ofDNA containing the coding sequence of the human NF-L gene with its promoter and polyadenylation signal (hNF-L) was first inserted into a pXCJL.1 shuttle plasmid, having sequences homologous to the Ad5 genome. This generated the plasmid pXCJLhNF-L (LW), which was used to insert the hNF-L fragment into the adenovirus by

15 homologous recombination. The resulting Ad5-hNF-L plasmid is shown in Figure 1.

Materials:

All restriction enzymes and T4 DNA ligase were obtained from Bethesda Research Laboratories (BRL) (Burlington, ON, Canada). The pXCJL.1 shuttle plasmid, pJM 17 plasmid, and 293N3 S cells were obtained from Dr. Frank Graham (McMaster University, Hamilton, ON, Canada). 293 Cells

20 were obtained from Dr. Philip Branton (McGill University, Montreal, PQ, Canada) . Molecular weight markers, agarose, and low-melting agarose were purchased from BRL. Glassmilk kits were obtained fromBio 101 (Vista, CA). Dulbecco'smodifiedEagleMedium(DMEM), fetal calf serum, and horse serum were purchased from Gibco. Jocklik-modified DMEM was obtained from Gibco . Yeast extract was obtained from Difco. The Qiagen maxiprep kit was obtained from Qiagen (Chatsworth, CA).

25 Geneclean was obtained from Bio-Can. Preparation ofthe hNF-L gene fragment:

A 2959 bp DNA fragment containing a genomic clone for the human NF-L gene with its minimal promoter and a polyadenylation signal was excised from plasmid pSKB AM-XB A-hNF-L (J. P. Julien) by digestion with Xbal and BamHl . 10 μl of pSKBA-XBA-hNF-L (approximately 2-3 μg) was digested with 10 units of Xba 1 and 10 units ofB amH 1 in Medium S alt Buffer (MSB) in a volume of

20 μl at 37 ° C for at least one hour. Following digestion, the sample was run in a horizontal agarose gel according to standard techniques (Sambrook et al. , ( 1989) Molecular Cloning: a laboratory manual (Cold SpringHarbor Laboratory: Cold Spring Harbor, NY)). Molecular weight markers and undigested DNA were run in the same gel as controls. Following ethidium-bromide staining, the gel was photographed under UV illumination using a polaroid camera. The 2959 bp Xbal-BamHl fragment of pSKBAM-XBA-hNF-L was purified using the Glassmilk kit according to the manufacturer's instructions.

Preparation of Shuttle Plasmid:

5 μlpXCJL. l (approximately 2.5 μg) was digested with lOunitsofXbal in React 2 buffer in a volume of20 μlat37°C. After2hours, 10unitsofBamHl, 3 μlNaC10.5M, 1 μl ofReact2, and 6 μl of water were added. The digestion pursued for 2 hours more. Following digestion, the sample was run in a horizontal agarose gel according to standard techniques (Sambrooketα/., (1989) upra). Molecular weight markers and undigested DNA were run in the same gel as controls. Following ethidium- bromide staining, the gel was photographed under UV illumination using a polaroid camera. The 6.64 kb Xbal-BamHl fragment of pXCJL. l was purified using the Glassmilk kit according to the manufacturer's instructions.

The 2959 bp DNA fragment from plasmid pSKBAM-XBA-hNF-L was ligated with the 6.64 kb Xbal- BamHl fragment ofthe pXCJLl plasmid. 1 μl (approximately 1 ng) ofthe 2959 bp hNF-L fragment was ligated with 1 μl (approximately 1 ng) ofthe 6.64 kb pXCJL.1 fragment in a reaction mix containing 1 unit ofT4 DNA ligase and 2 μl 5Xligationbufferinafinalvolumeof 10 μl; the reaction was performed at 4 ° C overnight. 5 μl ofthe ligation product was transformed into E. coli strain DH5 bacteria and plated. Ten ampicillin-resistant colonies were selected and grown in Luria-Broth medium according to standard techniques (Sambrook et al, (1989) supra). Small scale extractions of plasmid DNA were performed using an alkaline lysis protocol (Sambrook etal, ( 1989) swprα). Plasmid DNA was analyzed by restriction endonuclease digestion using 10.5 μl ofplasmid DNA with each ofthe following: A) 10 units ofBamHl and lOunitsofXbal inMSB;B) 10 units ofEcoRl and lO units ofXbal in High Salt Buffer; or C) lOunits ofEcoRl and lOunitsof Sphl in High Salt Buffer. Each digestion was performed in a final volume of 15 μl at 37°C for a minimum of 1 hour. The digested samples were analyzed by electrophoresis in an agarose gel, ethidium-bromide staining, and photographing under UN illumination using a polaroid camera. Desired recombinants generated the following fragments: digestion A two fragments of 11.4 kb and 6.6 kb; digestion B three fragments of 500bp, lkb, and 16.5 kb; and digestion C six fragments of 500 bp, 900 bp, 1.3 kb, 1.7 kb, 2 kb, and 11.4 kb. Three plasmid preparations showed the desired fragments. One of them, designated pXCJLhΝF-L (LW) was selected for subsequent use. Large scale extraction of pXCJLhΝF-L (LW) was performed using the Qiagen maxiprep kit according to the manufacturer's instructions.

Construction ofthe Ad5-hNF-L vector:

The pJMl 7 vector allows insertion ofDΝA into the adenovirus genome by homologous recombination, since it contains the genome of Ad5 dl309 along with the pBRX plasmid at map unit 3.7 ofthe genome (McGrory et al, (1988) Virology 163 :614-617). The methods for homologous recombination- mediated insertion ofDΝA into the adenovirus genome using the pJM 17 plasmid have been described (McGrory et al, (1988) supra).

pJM17 was grown in Terrific Broth medium (Sambrook et al, (1989) supra), and a large-scale extraction was performed using the Qiagen maxiprep kit according to the manufacturer's directions.

Ten petri dishes (60mm) were plated with the 293 cells in DMEM plus 10% fetal calf serum.

The pXCJLhΝF-L (LW) plasmid and the pJMl 7 plasmid were cotransfected into 293 cells using the calcium-phosphate precipitation technique. 4 μg of pJM 17 DΝA was mixed with 6 μg of pXCJLhΝF-

L (LW) DΝA in Hepes-buffered saline (140 MΝaCl, 5 mMKCl, 1 mM Νa₂HP0₄, 0.1% dextrose, 20 mMHepes pH 7.05) and 125 mM CaCl₂ in a final volume of 1 ml. After 20 minutes, the θ.5 ml ofthe precipitate was added slowly to each of two 293 plated petri dishes. The dishes were then incubated at 37 ° C for 4 hours. The medium containing the precipitate was then removed and the cells in each dish were covered with 5 ml of DMEM containing 5% fetal calf serum, 1% low-melting 5 agarose, and 2% yeast extract. The cells were then returned to the incubator until viral plaques appeared. A total of 10 petri dishes of 293 cells were transfected.

Homologous recombination between pXCJLhNF-L and pJMl 7 led to the appearance of viral plaques in transfected cells. One viral plaque, termed Ad5-hNF-L, was isolated from one ofthe petri dishes. The plaque was collected with a pipette and resuspended in 400 μl ofPB S . 200 μl ofthe suspension l o was used to infect a 100 mm petri dish containing 293 cells; the remaining 200 μl was frozen. After cytopathic effects had developed (3 days), the contents ofthe petri dish was centrifuged at 2000 rpm for 10 minutes, and the supernatant collection and frozen. The cells were then incubated overnight in 0.5 ml of 0.5 mg/ml pronase in 0.01M Tris, 0.01M EDTA, 0.5% SDS at 37°C. The DNA was extracted with phenol-chloroform and resuspended in 50 μl of TE buffer.

15 The structure ofthe virus was confirmed by Southern hybridization. For this purpose, 5 μl ofthe isolated DNA was digested with 2 μl ofBamHl and2 μl ofXbal in BSB buffer in a final volume of 40 μl at 37 °C for at least one hour. As a positive control, 1.5 μg ofthe pXCJL-hNF-L was also digested with Xbal in BSB buffer at 37°C for at least one hour. As a positive control, 1.2 μg of pXCJL-BZRGό (containing no NF sequence) was digested with Sal 1 in OPA buffer at 37 ° C for at

20 least one hour. After digestion, the DNA fragments and molecular weight markers were separated in an agarose gel and blotted onto a nitrocellulose membrane (Sambrook et al, (1989) supra).

A hNF-L DNA probe was prepared by digesting 1 μg of pXCJL-hNF-L DNA with Bglll in React3 buffer at 37 ° C for at least one hour. Following digestion, the sample was run in a horizontal agarose gel according to standard techniques (Sambrook etα/., (1989) supra). Molecular weight markers and 25 undigested DNA were run in the same gel as controls. Following ethidium-bromide staining, the gel was photographed under UN illumination using a polaroid camera. A 1 kb fragment was extracted from the agarose gel and purified using Geneclean according to the manufacturer' s instructions. This fragment was nick-translated with ³²P. Probe synthesis was done as described in Sambrook et al, (1989) supra.

The nitrocellulose membrane was hybridized with the hNF-L probe, washed, and exposed to X-ray film according to Sambrook etal, (\989)supra. A positive band ofabout 4.5 kb was seen in both the DNA extracted from cells infected with the recombinant plaque and the pXCJL-hNF-L plasmid, but not in the pXC JL-BZRG6 DNA, confirming insertion ofthe hNF-L expression cassette into the recombinant virus.

A purified stock ofthe Ad5-hNF-L vector was prepared as follows: the Ad5-hNF-L supernatant collected from the 100 mm petri dish was used to infect three 150 mm petri dishes containing a confluent layer of 293 cells, whichwereincubatedat37°C. Upon development of complete cytopathic effects, the cells were harvested and freeze-thawed three times in a dry ice-ethanol bath. Large debris was removed by centrifugation at 2000g for 10 minutes. The supernatant was combined with the supernatant saved from the petri dish. This solution was then used to infect 3 x 10⁸293N3 S cells, a subclone of 293 cells that has been selected for its ability to grow in suspension cultures. The cells were grown in spinner culture in Jocklik-modified DMEM containing 5% horse serum. Once complete cytopathic effect was obtained, the cells were harvested by centrifugation at 2000g for 10 minutes, freeze-thawed three times, and cleared by low-speed centrifugation. The viral vector was then purified by two rounds of cesium chloride gradient ultracentrifugation, followed by dialysis in a solution of 10% glycerol in O.OlMTris pH 7.6, and titered by plaque assay on293 cells (Graham and Prevec (1991) Manipulation of Adenovirus Vectors.

EXAMPLE II EXPRESSION OF hNF-L IN NEURONS IN VITRO

Mixed Spinal Cord Cultures

Cultures of dissociated spinal cord-DRG were prepared from E 13 hNF-H overexpressors or normal mouse embryos as described by Durham et al, (1996) Exp. Neur. 140: 14-29. Briefly, the spinal cords and DRGs were removed from E13 embryos, finely minced, and dissociated by trituration following 30 minutes incubation in 0.25% trypsin (Gibco BRL). Cells were plated at a density of 200,000 cells/well on round glass coverslips ( 13 mm) and incubated for 24 hours in a glucose-enriched minimal essential medium (EMEM, 10% FCS and horse serum). Thereafter, cultures were maintained inmodifiedN3 medium with 2% horse serum. On day 5, cultures were treated for 48 hourswith 1.4 μg/ml cytosine-b-arabinoside (Calbiochem) to reduce proliferation of non-neuronal cells. Spinal cord-

DRG cultures were used in infection experiments with adenoviral vectors 3 weeks after dissociation.

In Vitro Infection of Cultures Motor Neurons

In vitro infection studies were carried out using dissociated spinal cord-DRG cultures from hNF-H transgenic or normal mice. Replication-incompetent adeno viruses were added to the cultures at a final titer of 5 x l0⁶PFU/ml of Ad5 -hNF-L virus (equivalent to lOviral particles/cell; m.o.i.). Cellswere exposed to adenoviral vectors for 5 hours at 37 ° C and were then washed in culture medium and further cultured in modified N3 medium.

Immunohistochemistry

Twenty-four, 48, or 72 hours following exposure to adenoviral vectors, cells were fixed in freshly prepared phosphate buffered 4% paraformaldehyde solution and incubated with 0.1 % Triton X- 100 in Tris-buffered saline for 10 minutes at room temperature. The cells were blocked for 3 hours at room temperature in IF buffer, 20 mM Hepes pH 7.9, 250 mM KC1, 1% BSA, 0.2% fish skin gelatin (Sigma), 0.1% Triton X-100, then treated with the primary antibody overnight at 4° C in a humid chamber. The primary antibodies were diluted in gelatin-buffer (20mM Tris-HCl pH 7.3, 150 mM NaCl, 1% fish skin gelatine (Sigma), and 0.1% Tween 20) at the following titers: monoclonal mouse anti-human NF-L DP5- 1121 :2000 (N.T.L. France); monoclonal mouse anti-NF-LRPN.1105 1 : 1000 (Amersham); monoclonal rat anti-human NF-H (OC95) 1 :200 (kindly provided by V. M.-Y. Lee); polyclonal rabbit anti-NF-H 1 : 1000 (Sigma); monoclonal mouse anti-NF-M NN18 1: 1000 (Boehringer Mannheim); monoclonal mouse anti-β-tubulinKMX-1 -:200 (Boehringer Mannheim); and monoclonal mouse anti-actin C4 1 : 1000 (Boehringer Mannheim). Following several washes with gelatin buffer, the cells were incubated with secondary antibodies. For indirect fluorescence detection, a fluorescein-conjugated secondary antibody was used (Jackson ImmunoResearch Laboratories, Inc.). Cells were mounted using Slow Fade (Molecular Probes Inc.). Alternatively, we used secondary biotin-conjugated antibodies and streptavidin-peroxidase system (Vectastain Vector Laboratories) to obatin a brown precipitate from DAB (3,3'-diaminobenzidine, Sigma).

Results

Cultures of dissociated spinal cord from hNF-H+/+ embryos were used to demonstrate the cell- specificity ofthe Ad5-hNFL virus. In this in vitro assay, expression ofthe hNF-L gene was observed only in neuronal cells (Fig.2c). In contrast, another viral vector encoding for the β-galactosidase (β-gal) under the control ofthe cytomegalovirus promoter (Ad5-CMV-LacZ) was expressed in the various cell types present (data not shown).

EXAMPLE III PROTECTIVE EFFECT OF ELEVATING LEVELS OF NF-L PROTEINS IN MOUSE

MODEL OF MOTOR NEURONOPATHY: ADENOVIRUS TRANSFECTION

The Ad5-hNF-L construct was injected into transgenic mice expressing the human NF-H gene, a mouse model of motor neuronopathy. The results show that increased levels of hNF-L proteins can suppress motor neuron disease.

Generation of Human NF-H Transgenic Mouse

The mouse model of motor neuronopathy was generated as described in Cote et al, (1993) Cell 73:35-46. Briefly, a39kb fragment of the genomic human NF-H gene, including the complete NF-H transcriptionalunitflankedwith9.6kb of5' sequencesand 13.4kbof3' sequences, wasmicroinjected into fertilized mouse eggs. Integration ofthe human transgene into the mouse genome was assessed by Southern blot analysis of genomic DNA isolated from the mouse tail. Transgene copy number was estimated by densitometric analysis. Human NF-H transgene expression was assessed by Northern blot analysis; human NF-H mRNA was detected only in the brain, cerebellum, and spinal cord of transgenic mice, not in liver, kidney, lung, spleen, muscle, or heart. The expression ofthe transgene was limited to nervous tissue. Production of human NF-H protein, determined by SDS gels and immunoblotting, was increased up to 2-fold as compared to the levels of endogenous mouse NF-H protein. The hNF-H transgenic mice appeared normal during the first few weeks of postnatal development. Then, progressively, the mice began to manifest signs of neurological abnormality: they developed fine tremors, had abnormal limb flexions, and developed signs of weakness. By three to four months of age, the NF-H transgenics showed striking abnormalities in motor neurons ofthe anterior horn and dorsal root ganglia. Many neurons showed prominent swellings ofthe perikarya and proximal axons, consisting of densely packed 10 nm neurofilaments. These filaments are composed by heteropolymerization of multiple NF subunits. Abnormalities indicative of axonal atrophy are also detected. The abnormal accumulation ofNFs plays a central role in motor neuron degeneration by disrupting the intracellular supply of components required for axonal integrity (Collard etal. , ( 1995) Nature 375:61-64). This disruption of axonal transport by NF disorganization is a pathological mechanism consistent with several aspects of ALS. The progressive axonopathy in the NF-H transgenics is accompanied by secondary atrophy of skeletal muscle fibers. In summary, the modest overexpression ofhNF-H proteins in these transgenic mice provokes a progressive neuronopathy with pathological features that resemble those observed in ALS.

Delivery of Recombinant Adenoviruses to Transgenic Mice

The Ad5-hNF-L construct was injected into two month old hNF-H +/+ transgenic and normal mice. All surgical procedures were carried out under general anaesthesia and in accordance with The Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The recombinant adenoviruses were in a 10 mM Tris-HCl buffer solution pH 7.6 at a concentration of 3 x l0⁹ PFU/mL. Ten injections of2μL each were performed in the right tibialis muscle. Control inj ection were done using a solution of 5 mg/mL bovine serum albumin (Sigma) in 10 mM Tris-HCl pH 7.6. The mice were killed at 7, 14, or21 days post-injection and analyzed for β-galactosidase activity or for human NF-L immunodetection.

Detection of β-galactosidase Activity Anaesthetized mice were perfused with 50 mL of PBS pH 7.4 followed by 50 mL of phosphate buffered 2% paraformaldehyde pH 7.4. Tissues were dissected and further fixed for 45 minutes at room temperature in fresh fixative. After 2 washes in PBS pH 7.4, the samples were incubated overnight in a staining solution ( 1 mg/mL X-Gal (Sigma), 5 mM K₃Fe(CH)₆, 5 mMK₄Fe(CH)₆-3H₂0, 2 mMMgCl₂, 0.01% sodium deoxycholate, 0.02% NP40 inPBS) at 37°C. After 3 brief washes with 3% DMSO in PBS, the samples were immersed in 15 sucrose in PB S for cryosectioning. Cryosections of 15μm were mounted onto gelatin-coated slides, dehydrated, and counterstained with neutral red.

Morphometric Analysis In order to measure axonal calibers, thin sections ( 1 μm) ofEpon-embedded (Marivac) L5 ventral roots from transgenic and normal mice were stained with 2% Toluidine blue (JBS). The images of sections were then digitalized using aHamamatsu CCD camera mounted on a Leitz Diaplan microscope with a 100X objective. The digitalized images were subsequently analyzed using a morphometric software (Image 1, Universal Imaging Corp., USA).

Results

Injections of Ad5-CMV-LacZ viruses (controls) into the right tibialis muscle of hNF-H+/+ mice, as shown in Figure 2, resulted in a robust and specific expression of β-gal in motor neurons enervating the injected muscle. No obvious cytotoxic effect due to viral infection could be detected. These experiments also revealed that neither the viral infection nor the CMV-driven expression of lacZ reduced the neurofilamentous swellings in hNF-H+/+ mice (Fig.2a and b). Seven days post-injection, it was noticed that normal mice showed prominent β-gal staining of motor neuron perikarya, whereas the hNF-H+/+ mice had a limited number of positive cells with staining restricted to a spotty pattern. The poor β-gal staining in the hNF-H+/+ mice observed at a relatively short time interval after viral injection reflects an impairment of retrograde axonal transport, as a consequence ofNF accumulations.

The Ad5-hNFL viral vector was injected into the right tibialis muscle ofNF-H+/+ mice, and the spinal cord of these animals was examined 21 days post-infection. As shown in Figure 2, the immunodetection ofhNF-L proteins using a specific anti-human NF-L antibody (DP5- 112) occurred only in spinal motor neurons ipsilaterally to the injected side (Fig.2d and e). Moreover, no perikaryal swellings occurred in the hNF-L-positive motor neurons. Thin sections of Epon-embedded spinal cord further demonstrated that the number of neurofilamentous swellings in the perikarya of motor neurons were reduced in the spinal cord ipsilateral to the Ad5-hNFL injected side, as compared to the non-injected contralateral side (Fig.2f and g). Moreover, it was demonstrated that the Ad5-hNFL vector is suitable to direct a long term expression of hNF-L protein. The insert in Figure 2d shows detection of hNF-L proteins in a motor neuron of hNF-H+/+ mice 9 months after muscular injection ofthe viruses.

These results indicate that increased levels of hNF-L proteins can suppress motor neuron disease caused by the overexpression of hNF-H proteins.

5 EXAMPLE IV

PROTECTIVE EFFECT OF ELEVATING LEVELS OF NF-L PROTEINS IN MOUSE MODEL OF MOTOR NEURONOPATHY: MATING TRANSGENIC MICE

The protective effect ofincreased levels ofNF-L protein against neurodegeneration was also shown by overexpressing human NF-L in a mouse model of motor neuronopathy. To this end, transgenic mice l o overexpressing the hNF-L gene were mated with transgenic mice expressing the human NF-H gene, a mouse model of motor neuronopathy.

The NF-H transgenic mice were generated as described above. The transgenic mice overexpressing hNF-L were generated as described in Julien et al, (1987) Genes & Development 1 : 1085-1097. Briefly, a 21.5 kb DNA fragment containing the human NF-L gene, including all exon sequences, 5 ' -

15 flanking sequences, and sequences downstream ofthe first polyadenylation site, was microinjected into the male pronucleus of fertilized mouse eggs. Injected eggs were transferred to the oviduct of pseudopregnant females. The presence of hNF-L DNA in offspring of founder mice was determined by Southern blot analysis ofDNA extracted from the tails. Transgenic mice were then examined for the presence of hNF-L transcripts in their brain RNA. The hNF-L protein was identified using a

20 monoclonal antibody raised against bovine NF-L, which recognizes the human but not the mouse NF-L protein. Human NF-L protein was identified in both rain homogenates and in assembled neurofilaments prepared from myelinated axons of transgenic mice. The relative proportion of human NF-L protein detected was equivalent to the relative human NF-L mRNA concentrations observed.

25 Homozygous mice of each parental line were crossbred to obtain mice heterozygous for both transgenes. Further crossbreeding ofthe first generation yielded normal mice and mice heterozygous or homozygous for each transgene. The animals used in this study were not pure inbred mice, but were dominantly of C57BL/6 genetic background. The genotypes of transgenic mice were identified by S outhern blotting of tail genomic DNA. Briefly, approximately 1 cm of mouse tail was digested in 10 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.5% SDS, 2 mM EDTA with 0.6 mg/mL of proteinase K (Boehringer Mannheim) at 55 ° C for 4 hours. The digested tissue was then extracted with phenol- chloroform and the aqueous phase precipitated in ethanol. The resulting genomic DNA pellet was resuspended in TE buffer ( 10 mM Tris-HCl pH 8.0, 1 mM EDTA), and 10 μg ofDNA was digested overnight with a selected restriction enzyme. The digestion product was run on an agarose gel, transferred to a charged nylon membrane (GeneScreen Plus, NEN Life Science Products), and hybridized as described in Sambrook etal, (1989) supr . The probe used to detect specifically the hNF-H gene was a PCR product spanning to the fourth exon ofthe hNF-H gene. For detection ofthe hNF-L transgene, the probe corresponded to a Pst I fragment from the first exon ofthe mouse NF-L gene that hybridizes with the gene of both species. Filters were exposed on BioMax MR films (Kodak), using Cronex intensifying screens (Dupont).

Northern Blotting

Total RNA was prepared from freshly isolated or flash-frozen spinal cords from transgenic and normal mice. Homogenization was carried out in 5 mL of Trizol (Gibco-BRL) per gram of tissue and total RNA isolation performed according to manufacturer' s guidelines. Five or 10 μg of total RNA was loaded onto a 1% agarose-formaldehyde gel and processed for northern blotting as described by Sambrooketα/., (\9 9)supra. The radiolabeled probes used for the detection ofhNF-L and hNF-H transgenes were the same as those used for genomic screening. The loading was standardized using a mouse actin cDNA as a probe. Filters were exposed to BioMax MR films (Kodak), using Cronex intensifying screens (Dupont).

Protein analysis Mice were sacrificed and the relevant tissues were dissected to be processed immediately or flash- frozen in liquid nitrogen. To obtain Triton-insoluble cytoskeletal fractions, tissues were homogenized in 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM PMSF, 10 mg/mL aprotinin, 2 mg/mL leupetin, 2 mg mL pepstatin and 1 % Triton X- 100. Homogenates were centrifuged for 20 min at4°C at 13,000xginamicrofuge. The Triton-insoluble pellet was re-homogenized in SUB (0.5% SDS, 8 M urea, 2% β-mercaptoethanol). The resuspended material was centrifuged at room temperature for 15 min in a microcentrifuge. Protein concentrations ofthe resulting supematants were measured using Bio-Rad Protein Assay (Bio-Rad), a Bradford-based protein assay. For immunoblotting,proteinsamplesin62.5 mMTris-HClpH6.8,2% SDS, 10% glycerol and 0.7 Mβ- mercaptoethanol were loaded on 7.5% SDS-PAGE and subsequently transferred onto nitrocellulose. Filters were blocked for 3 hours in gelatin-buffer (20 mM Tris-HCl pH 7.3, 150 mMNaCl, l% fish skin gelatin (Sigma) and 0.1 % Tween 20) and then incubated with primary antibodies for 4 hours at room temperature or overnight at 4 ° C. The primary antibodies were diluted in the gelatin-buffer at the following titers: monoclonal mouse anti-human NF-L DP5- 1121 :2000 (N.T.L. France); monoclonal mouse anti-NF-L RPN.1105 1 : 1000 (Amersham); monoclonal rat anti-human NF-H (OC95) 1 :200 (kindly provided by V. M.- Y. Lee); polyclonal rabbit anti-NF-H 1 : 1000 (Sigma); monoclonal mouse anti-NF-MNN18 1: 1000 (Roche Diagnostics); monoclonal mouse anti-β-tubulin KMX-1 1:200 (Roche Diagnostics); and monoclonal mouse anti-actin C4 1 : 1000 (Roche Diagnostics). After several washes in gelatin-buffer, membranes were incubated for one hour with peroxidase-conjugated secondary antibodies (anti-mouse, rat orrabbit, JacksonlmmunoResearchLaboratoriesInc.) diluted 1 : 1000 in gelatin-buffer. The membranes were washed once in gelatin-buffer and 3 times in 20 mM Tris-HCl pH 7.3, 150 mM NaCl. Detection of the immune complex was performed with the chemoluminescent ECL detection kit (Amersham).

Morphometric Analysis

In order to measure axonal calibers, thin sections ( 1 μm) ofEpon-embedded (Marivac) L5 ventral roots from transgenic and normal mice were stained with 2% Toluidine blue (JB S) . The images of sections were then digitalized using a Hamamatsu CCD camera mounted on a Leitz Diaplan microscope with a 100X objective. The digitalized images were subsequently analyzed using a morphometric software (Image 1, Universal Imaging Corp., USA).

Immunohistochemistry

Anaesthetized mice were perfused with 50 mL of PBS pH 7.4 followed by 50 mL of phosphate buffered 4% paraformaldehyde pH 7.4. Tissues were dissected and further fixed for 2 hours to overnight at 4 ° C in fresh fixative. Samples were sectioned using a vibratome and 25 μm sections were mounted on gelatin-coated slides and permeabilized with 0.3% Triton X- 100 inPB S for 5 minutes at room temperature. Sections were then blocked for 2 hours at room temperature in IF buffer (20 mM Hepes pH 7.9, 250 mMKCl, 1% BSA, 0.2% fish skin gelatin (Sigma), 0.1% Triton X- 100). Sections were then incubated with primary and secondary antibodies as described in the immunohistochemistry section in Example II.

Electron Microscopy

Anaesthetized mice were perfused with 50 mL ofPBS pH 7.4 followed by 50 mL of Jone's fixative pH7.4 (65 mMNaCl, 2.68 mMKCl, 3.26 mMNaH₂P0₄, and 14.42 mMNa₂HP0₄). The tissues were dissected and further fixed for 2 hours to overnight in fresh fixative at 4 ° C . The samples were postfixed in 2% osmium tetraoxide for 2 hours and dehydrated in a graded series of ethanol solutions and Epon (Marivac) embedded according to standard protocols. Ultrathin sections were stained with uranyl acetate and lead citrate prior to observation on a Philips 10 electron microscope.

Axonal Transport Study Two month old hNF-H+/+, hNF-L+/+;hNF-H+/+, and normal mice were anaesthetized using sodium pentobarbital, following which 2μL ofPBS containing 3 OOμCi of ³⁵S-methionine (Amersham) was injected into the ventral horn ofthe spinal cord at the level ofthe first lumbar segments. Twenty-eight days after injection, the injected region ofthe spinal cord, the L5 ventral roots, L5 DRGs, and both sciatic nerves were removed. The nerves were then cut into 8 segments of 3mm each, and corresponding segments ofthe two nerves were pooled. Each fraction was homogenized in 10 mM

Tris-HCl pH7.5, 150mMNaCl, 1 mMEDTA, 2 MPMSF, 10 mg/mL aprotinin, 2 mg/mL leupetin, 2 mg/mL pepstatin, and 1 % Triton X- 100. Triton-insoluble preparations were obtained as described previously in the protein isolation section. Cytoskeleton-enriched preparations and supematants were separated on 7.5% SDS-P GE and stained with Coomassie Brilliant Blue. After destaining in 30% methanol, 10% acetic acid, the gels were incubated 30 min at room temperature in Amplify

(Amersham). Dried gels were exposed to BioMax MR films (Eastman-Kodak, Rochester, NY).

Results It was previously reported that an approximate 3 -fold increase in levels of mRNAs existed for the parental hNF-L and hNF-Hlines (Beaudet etal, (\ 993)Brain Research. Molecular Brain Research 18:23-31; Coteetα/., (1993) Cell 73:35-46). As shown in Fig.3a, the levels ofmRNAinthe spinal cord of doubly hNF-L;hNF-H transgenic mice (3 month old) correspond to those found in singly transgenic mice bearing the hNF-L or hNF-H transgenes alone. The levels of hNF-L or hNF-H transcripts were doubled in mice homozygous for the transgenes, as compared to heterozygous mice.

The increases in hNF-L and hNF-H mRNA levels did not result in comparable increases in protein levels, as shown inFigure 3b and in Table 2. Densitometric analyzes were performed on the Coomassie stained SDS-PAGE of cytoskeletal protein-enriched preparations from the spinal cord. A 3-fold increase of exogenous mRNAs in mice homozygous for hNF-H (hNF-H+/+) or hNF-L (hNF-L+/+) resulted in increased protein levels of 208 ±12% for NF-H and 115 ±3% for NF-L, respectively. It was reported previously that in hNF-L transgenic mice the human NF-L protein species constitutes nearly 80% of the total NF-L protein content in the spinal cord (Beaudet et al, (1993) Brain Research. Molecular Brain Research 18:23-31). It is noteworthy that in doubly transgenic hNF- L+/+;hNF-H+/+ mice, the levels of foreign proteins were further enhanced with a content in NF-L and

NF-H proteins corresponding to 130 ±5% and 251 ±9%, respectively, the levels found in normal mice (see Table 2). This additional increase is likely due to a reciprocal stabilization of additional NF-L and NF-H proteins that are able to form heterodimers (Giasson and Mushynski, ( 1998) J. Neurochem. 70: 1869-1875).

Table 2. Levels of NF-subunits in the Spinal Cord of 6 Month Old Transgenic Mice normal hNF-H+/+ hNF-L+/+; hNF-L+/+; hNF-H+/+ total NF-H 100 % 208 % (±12) 251 % (±9) 97 % (±9) total NF-M 100 % 61 % (±5) 64 % (±4) 110 % (±10) total NF-L 100 % 87 % (±9) 130 % (±5) 115 % (±3)

The expression ofhNF-L and hNF-H species was further confirmed by western blotting, using specific antibodies directed against the human NF-H protein and the human NF-L proteins (Fig.3 b). Whereas the levels ofNF-M were down-regulated in transgenic mice expressing the hNF-H proteins, the levels of tubulin and actin remained similar to those of normal mice.

Reduction ofperikaryal NF accumulations in doubly transgenic mice

The spinal cord from 6 month old transgenic mice was examined by light microscopy (Fig.4). Mice homozygous or heterozygous for the hNF-H transgene developed abnormal accumulations ofNFs in the perikarya and proximal axons of spinal motor neurons (Fig.4c and e) (previously reported in Cote et al , ( 1993 ) Cell 73 : 35-46). In contrast, the 3 -fold increase ofNF-L mRNAs in the hNF-L+/+ mice did not lead to abnormal neurofilamentous accumulations in motor neurons (Fig.4b). Remarkably, the co-expression ofhNF-L proteins in the doubly hNF-L;hNF-H transgenic mice reduced dramatically the number and size ofperikaryal swellings (Fig.4d, f, h). These beneficial effects of extra hNF-L proteins are particularly striking when hNF-H+/- mice (Fig.4c) are compared to hNF-L+/-;hNF-H+/- mice (Fig.4f) whose spinal cord is virtually devoid ofperikaryal swellings, documenting a gene dosage effect. Mice heterozygous for hNF-L and homozygous for hNF-H (hNF-L+/-;hNF-H+/+) developed large perikaryal swellings (Fig.4g), reminiscent of mice expressing hNF-H alone (Fig.4c and e). In contrast, no NF inclusions were detected in perikarya of motor neurons from mice homozygous for hNF-L and heterozygous for hNF-H (hNF-L+/+;hNF-H+/-) (Fig.4h, small arrows); however, hNF- L+/+;hNF-H+/- mice exhibited some giant proximal axons (large arrows) . Similar results were obtained with one year old ice ofthe various genotypes (data not shown).

Axonal atrophy in hNF-H mice is alleviated by extra hNF-L The L5 ventral roots from 6 month old mice were examined by light microscopy. A dramatic atrophy of motor axons in hNF-H+/+ and hNF-H+/- transgenic mice could be observed (Fig. 5c and e). The axonal atrophy was more pronounced in the hNF-H+/+ animals (Fig.5c) than in the hNF-H+/- animals (Fig. 5e), emphasizing again the gene dosage effect of transgenes. In doubly hNF-L;hNF-H transgenic mice, co-expression of hNF-L restored the radial growth of axons (Fig. 5d, f and h). Remarkably, in the doubly heterozygous hNF-L+/-;hNF-H+/- mice (Fig. 5f) and in the hNF-L+/+;hNF-H+/- mice

(Fig.5h), some ventral root axons oflarger caliber than normal were observed. To quantify the changes of axonal calibers, cross-sectional areas ofL5 ventral root axons were analyzed using a morphometric software (Image 1 , Universal Imaging Corp., USA). Normal mice and hNF-L mice showed abimodal distribution of axonal calibers with peaks at 2-3 μm and 7-8 μm (Fig. 6a). In contrast, no bimodal distribution and a significant increase in the percentage of small axons were observed in mice expressing hNF-H alone (Fig.6b) . The co-expression of hNF-L together with hNF-H restored the radial growth of axons. Thus, the bimodal distribution of axonal calibers was completely reestablished in the doubly heterozygous hNF-L+/-;hNF-H+/- mice. In addition, a rescue of axonal atrophy in the hNF-

L+/+;hNF-L+/+ mice (Fig. 6b) and the hNF-L+/-;hNF-H+/+ mice (Fig. 6d) could be detected, although the bimodal distribution was not fully recovered (Fig. 6b). Note the presence of axons with diameters exceeding 13 μm in doubly transgenic mice.

Integrity of axonal cytoskeleton recovered in doubly transgenic mice The L5 ventral roots of 6 months old animals were analyzed by electron microscopy (EM). Transverse sections of large motor axons from normal mice revealed an abundance ofNF profiles (Fig. 7a). In motor axons of hNF-L+/+ mice, an increased density ofNFs as compared to normal could be observed (Fig.7b). In contrast, in homozygous or heterozygous hNF-H transgenic mice, (Fig. 7c and e), the cytoskeleton was markedly perturbed and the number of intact NF structures was reduced dramatically. In hNF-H+/+ mice at 12 and 24 months of age, EM revealed in these shrunken axons a general disruption ofthe NF network and fewer microtubules, as compared to younger animals (Collard et al, (1995) Nature 375:61-4). Consistent with the above morphometric data, the co- expression of hNF-L with hNF-H led to the reestablishment of a normal cytoskeleton in axons from young and old mice. The expression of hNF-L in hNF-L+/-;hNF-H+/- mice (Fig. 7f) and in hNF- L+/+;hNF-H+/- mice (Fig.7h) restored a normal NF density and distribution across the axoplasm (Fig.

7d and f). Analysis of 12 month old animals yielded similar results: fewer degenerative axonal profiles could be detected in doubly transgenic mice as compared to mice expressing the hNF-H transgene alone (data not shown). It is remarkable that a relatively low protein ratio ofNF-L to NF-H in hNF- L+/-;hNF-H+/+ transgenic mice was sufficient to dramatically improve the cytoskeletal integrity in motor axons (Fig. 7g).

Improved axonal transport in doubly transgenic mice

Defects in axonal transport has been proposed to underlie the pathogenic mechanism in hNF-H transgenic mice (Collard et al, (1995) Nature 375:61-4); therefore, the rate of transport of cytoskeletal proteins into axons of 2 month old doubly transgenic mice was studied by monitoring radiolabeled, slowly transported polypeptides, 28 days after the injection of ³⁵S-methionine into the spinal cord. In each segment along the length ofthe sciatic nerve, the transported radiolabeled polypeptides present in the Triton-insoluble fraction were loaded onto SDS-PAGE and analyzed by fluorography. Whereas normal mice showed a leading peak corresponding to a transport rate of ~0.75 mm/day for the three NF subunits (Fig. 8), axonal transport was impaired in h_NF-H+/+ mice with a leading peak for NF-L and NF-M corresponding to an axonal transport rate of ~0.64 mm/day (see arrows). The transport of tubulin was also altered in hNF-H+/+ mice with leading edge at -0.96 mm/day instead of ~1.18 mm day in normal mice (Fig. 8, asterisk). The co-expression of hNF-L enhanced the anterograde axonal transport rate, not only for NF proteins, but also for tubulin, with transport rate of -0.86 mm/day and -1.18 mm/day, respectively (Fig. 8, bottom panel).

No overt phenotypes in mice co-expressing human NF-L and NF-H

Whereas hNF-H+/+ and hNF-H+/- mice acquired progressive motor dysfunction and weaknesses, mice co-expressing hNF-L and hNF-H did not develop overt clinical symptoms. Moreover, they rarely exhibited the hind limb contraction reflex, characteristic of motoneuronal disorders, observed in mice expressing the hNF-H transgene alone (Fig. 9). We also noted that the hNF-H+/+ mice lost body weight during aging. At one year of age, the hNF-H+/+ mice have a weight of 31.5 ±3.6 g (n=12) instead of 48.5 ±5.2 g (n= 10) for normal mice. At the same age, the hNF-L+/+;hNF-H+/+ mice had an average body weight of 39.0 ±4.6 g (n=9), which is closer to normal.

The above results demonstrate that NF-L proteins can suppress motor neuron disease. A gene delivery approach based on the use ofthe recombinant viral vectors encoding NF-L proteins ofthe present invention offer a means of up-regulating NF-L levels in a sustained manner. These vectors can be used for gene therapy to treat neurodegenerative diseases, neural injuries, and neural degeneration due to aging.

EXAMPLE V

PROTECTIVE EFFECT OF ELEVATING LEVELS OF NF-H PROTEINS IN ALS MICE To investigate the effect of elevating the levels of neurofilament protein in neurodegenerative diseases, excess levels of human NF-H protein were expressed in a transgenic mouse model of ALS . To this end, transgenic mice expressing a human SOD 1 mutation (G37R) were bred with transgenic mice from two lines overexpressing normal forms ofthe human gene encoding NF-H protein.

Materials:

The transgenic mouse lines overexpressing human NF-H proteins (lines 200 and 398) were obtained by the microinjection into one-cell mouse embryos of large genomic fragments for the two normal NF- H alleles flanked by 5 ' promoter region and 3 ' sequences as described previously (Cote et al. , ( 1993) Cell 73 :35-46). Line 200 (hNF-H⁴³) carries a N 3 F-H allele having 43 Lys-Ser-Pro (KSP) phosphorylation sites whereas line 398 (hNF-H⁴⁴) bears a NF-H allele with 44 KSP repeats

(Figlewicz et al. (1994)).

The heterozygous NF-H transgenic mice from these two lines develop abnormal neurofilament accumulations in spinal motor neurons and exhibit relatively mild neurological abnormalities with fine tremors and abnormal limb contraction reflexes after 8 months of age. The NF-H-induced pathology progresses with atrophy and slow degeneration of motor axons in very old transgenic mice (≥ 18 months) but with no significant loss of spinal motor neurons and with no noticeable effect on life expectancy. The total amount of human NF-H protein detected was similar in heterozygous mice of both transgenic lines and corresponds to approximately 1.5 fold the level of endogenous mouse NF-H expression.

The transgenic mice heterozygous for an S OD 1 mutation (G37R) referred to as line 29 are described in Wong etal,(\995)Neuron 14: 1105-1116. TheG37R mice were mated with both the line 200 and line 398 hNF-H mice to produce transgenic mice expressing both the hNF-H and the S OD 1 mutation (S0D1^G37R;N. F-H transgenic mice).

Western Blot Analysis: Total protein extracts were obtained by homogenization of spinal cord in SUB buffer (0.5% SDS, 8M

Urea and 2% β-mercaptoethanol). Samples were electrophoresed on 15% polyacrylamide SDS- PAGE and transferred to nitrocellulose filter. Antibodies against human NF-H were obtained from V.M.-Y Lee, Univ. ofPennsylvania. Antibodies against SODl (Biodesignlnc.) recognize bothmouse and human SODl proteins with apparent molecular weights of 18 kDa and 20 kDa respectively. Peroxidase-coupled secondary antibody was detected using ECL (Amersham) chemoluminescence kit (Figure 10).

The levels of human NF-H and SOD 1^G37R proteins in spinal cord from doubly S0D1^G37R;NF-H transgenic mice (3 month old) corresponded to those found in singly transgenic mice bearing either the human NF-H or the S0D1^G37R transgenes alone (Figure 10).

Physiological Characteristics and Life Expectancy: Figure 11 shows the survival curves of S0D1^G37R transgenic mice. Transgenic mice expressing

S0D1^G37R alone in a normal neurofilament background had a mean life expectancy of 9.5 ± 2.8 months (n=20) and most of them (75%) died before 1 year old. In contrast, 100% of SOD 1^G37R;NF- H⁴³ transgenic mice were still alive after 1 year for an average life span of 15.8 ± 1.5 months (n=9) . The expression ofthe human NF-H⁴³ transgene thus dramatically extended the mean longevity of S0D1^G37R expressing mice (approximately 6 months).

The protective effect ofhuman NF-H protein was also important in SOD 1 ^^NF-H⁴⁴ transgenic mice, derived from the other NF-H transgenic line. In this case, the median of life probability of doubly transgenic mice was extended by approximately 2 months (n=23) (Figure 11). After the onset of paralysis in doubly SOD 1 ^G37R;NF-H transgenics, the SOD 1 -induced pathology evolved rapidly to death in a manner similar to S0D1^G37R mice.

Microscopy:

Light microscopy examination of spinal cord and lumbar (L5) spinal root axons from 1 year old transgenic mice, carrying the SOD 1 ^G37R transgene alone or together with a NF-H transgene further corroborated the protective effect of NF-H against SOD 1^G37R toxicity (Fig.12). Whereas massive axonal loss and cell death had occurred in one-year old SOD 1 ^G37R mice (Fig.12 D, E, F), the motor and sensory axons were generally spared in SOD 1 ^G37R;NF-H⁴³ transgenic mice (Fig.12 J, K). Note that, like mice ofthe parental NF-H⁴³ line 200, the doubly SOD 1^G37R;NF-H⁴³ transgenic mice exhibited axonal atrophy (Fig.12 J,K) and prominent perikaryal neurofilamentous accumulations (Fig.12 L). To a lesser degree, the rescue of motor and sensory axons was also evident in doubly transgenic mice expressing the NF-H⁴⁴ (Fig.12 G, H, I). Remarkably, the different degrees of protection conferred by the two human NF-H transgenes in the SODl -induced disease correlated with the extent of neurofilament accumulations occurring in motor neurons of each NF-H transgenic line.

This overexpression ofhuman NF-H protein represents the first successful approach to substantially delay the onset of disease in transgenic mice bearing a SODl mutant found in ALS: the NF-H overexpression was able to extend the life-span ofthe SODl transgenics by up to 65%.

EXAMPLE VI

PROTECTIVE EFFECT OF ELEVATING LEVELS OF NF-L PROTEINS IN ALS MICE

To investigate the effect of elevating the levels of neurofilament protein in neurodegenerative diseases, excess levels ofhuman NF-L protein were expressed in a transgenic mouse model of ALS . To this end, transgenic mice expressing a human SODl mutation (G37R) were bred with transgenic mice overexpressing normal forms ofthe human gene encoding NF-L protein.

Materials and Methods:

The transgenic mice overexpressing hNF-L were generated as described in Julien etal, (1987) Genes & Development 1.1085-1097. The transgenic mice heterozygous for an SODl mutation (G37R) referred to as line 29 are described in Wong etal, (\995)Neuron 14: 1105-1116. The G37Rmice were mated with mice heterozygous for the human NF-L transgene to produce the following offspring:

S0D1^G37R transgenics, NF-L transgenics, doubly S0D1^G37R;NF-L transgenics, and normal mice.

Physiological Characteristics and Life Expectancy:

The NF-L mRNA was overexpressed by approximately 1.5 fold in the SOD 1 ^G37R;NF-L transgenic mice. Figure 10 shows the survival curves of SOD 1^G37R;NF-L transgenic mice as compared to the SOD 1 ^G37R transgenic mice. Remarkably, the increased levels ofthe human NF-L protein extended the life spans ofthe S0D1^G37R transgenic mice by approximately 8 weeks.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications to the invention to adapt it to various usages and conditions. Such changes and modifications are properly, equitably, and intended to be within the full range of equivalence ofthe following claims.

Claims

WE CLAIM:

1. A use ofincreased levels of one or more neurofilament proteins or fragments thereof to provide protection against neurodegeneration associated with oxidative stress in a subject.

2. The use of claim 1 , wherein the neurofilament proteins are selected from the group consisting of NF-L, NF-M, NF-H, and any combination thereof.

3. The use of claim 1, wherein the subject is an animal.

4. The use of claim 3, wherein the animal is a mammal.

5. The use of claim 4, wherein the mammal is a human.

6. The use of claim 1 , wherein the neurodegeneration associated with oxidative stress is due to a neurodegenerative disorder.

7. The use of claim 6, wherein the neurodegenerative disorder is selected from the group consisting of Amyotrophic lateral sclerosis (ALS), Alzheimer' s disease, Parkinson' s disease, Giant Axonal Neuropathy, neuropathies associated with diabetes, toxic neuropathies such as those induced by ╬▓,╬▓'-iminodipropionitrile (IDPN), 2,5-hexanedione, acrylamide, and aluminum, Lewy body Dementia, or Guam-Parkinsonism.

8. Theuseofclaim 1, wherein the neurodegeneration associated with oxidative stress is due to aging.

9. The use of claim 1 , wherein the neurodegeneration associated with oxidative stress is due to injury.

10. Theuseofclaim 1, wherein the increased levels of neurofilament proteins are obtained by the administration of neurofilament proteins or fragments thereof.

11. The use of claim 10, wherein the neurofilament proteins are purified neurofilament proteins or fragments thereof.

12. The use of claim 11 , wherein the neurofilament proteins are synthetic neurofilament proteins or fragments thereof.

13. The use of claim 1 , wherein the increased levels of neurofilament proteins are obtained by gene therapy.

14. The use of claim 13, wherein the gene therapy involves the administration of one or more viral vectors encoding one or more neurofilament proteins.

15. The use of claim 1, wherein the increased levels of neurofilament proteins are obtained by the administration of one or more chemical compounds.

16. The use of claim 15, wherein the chemical compound increases the expression of an NF protein.

17. A use ofincreased levels of one or more neurofilament proteins or fragments thereof to provide protection against reactive oxygen species in a subject.

18. The use of claim 17, wherein the neurofilament proteins are selected from the group consisting of NF-L, NF-M, NF-H, and any combination thereof.

19. The use of claim 17, wherein the subject is an animal.

20. The use of claim 19, wherein the animal is a mammal.

21. The use of claim 20, wherein the mammal is a human.

22. The use of claim 17, wherein the reactive oxygen species is selected from the group consisting of oxygen-derived free radicals, hydroxy radicals, superoxide anions, peroxynitrite, and hydrogen peroxide.

23. The use of claim 17, wherein the increased levels of neurofilament proteins are obtained by the administration of neurofilament proteins or fragments thereof.

24. The use of claim 23 , wherein the neurofilament proteins are purified neurofilament proteins or fragments thereof.

25. The use of claim 23 , wherein the neurofilament proteins are synthetic neurofilament proteins or fragments thereof.

26. The use of claim 17, wherein the increased levels of neurofilament proteins are obtained by gene therapy.

27. The use of claim 26, wherein the gene therapy involves the administration of one or more viral vectors encoding one or more neurofilament proteins.

28. Theuseofclaim 17, wherein the increased levels ofneurofilament proteins are obtained by the administration of one or more chemical compounds.

29. The use of claim 28, wherein the chemical compound increases the expression of an NF protein.