Annu. Rev. Neurosci. 2000. 23:217–247Copyright q 2000 by Annual Reviews. All rights reserved

0147–006X/00/0301–0217$12.00 217

GLUTAMINE REPEATS AND

NEURODEGENERATION

Huda Y. Zoghbi1 and Harry T. Orr2

1Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030;e-mail: hzoghbi@bcm.tmc.edu; 2Institute of Human Genetics, University of Minnesota,Minneapolis, Minnesota 55455; e-mail: harry@lenti.med.umn.edu

Key Words polyglutamine diseases, triplet repeat, protein aggregates, nuclearinclusions, ubiquitin, Huntington disease, SBMA, SCA, DRPLA

Abstract A growing number of neurodegenerative diseases have been found toresult from the expansion of an unstable trinucleotide repeat. Over the past 6 years,researchers have focused on identifying the mechanism by which the expanded poly-glutamine tract renders a protein toxic to a subset of vulnerable neurons. In this review,we summarize the clinicopathologic features of these disorders (spinobulbar muscularatrophy, Huntington disease, and the spinocerebellar ataxias, including dentatorubro-pallidoluysian atrophy), describe the genes involved and what is known about theirproducts, and discuss the model systems that have lent insight into pathogenesis. Thereview concludes with a model for pathogenesis that illuminates the unifying featuresof these polyglutamine disorders. This model may prove relevant to other neurode-generative disorders as well.

INTRODUCTION

In 1991, a new mutational mechanism for disease was discovered: the expansionof unstable trinucleotide repeats (Fu et al 1991, La Spada et al 1991). “Tripletrepeat” expansions have since been found to cause 15 neurological disorders,eight of which are neurodegenerative diseases that result from expansion of CAGrepeats coding for polyglutamine tracts in the respective proteins: spinobulbarmuscular atrophy (SBMA), Huntington disease (HD), and the spinocerebellarataxias [including dentatorubropallidoluysian atrophy (DRPLA)]. With the excep-tion of SBMA, these neurodegenerative disorders are dominantly inherited. Alleight disorders are progressive, typically striking in midlife and causing increas-ing neuronal dysfunction and eventual neuronal loss 10–20 years after onset ofsymptoms. Several other features characterize this group of diseases: the greaterthe number of CAG repeats on expanded alleles, the earlier the age of onset andthe more severe the disease. The repeats show both somatic and germline insta-bility. Successive generations of affected families experience anticipation, or ear-

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lier age of onset and more rapid disease progression, due to intergenerationalrepeat instability that is particularly marked in paternal transmissions. Lastly, andmost puzzling, only a certain subset of neurons is vulnerable to dysfunction ineach of these diseases, despite the widespread expression of the relevant proteinthroughout the brain and other tissues. Table 1 summarizes what is known aboutthe disease-causing genes, their protein products, and the brain regions charac-teristically affected in these disorders; this information is presented in greaterdetail in the next section.

CLINICAL AND MOLECULAR DATA

Spinobulbar Muscular Atrophy

Clinicopathology SBMA, or Kennedy disease, is unique among the polyglu-tamine diseases in showing an X-linked recessive pattern of inheritance. Affectedmales may suffer muscle cramps for years before presenting with proximal muscleweakness in the fourth or fifth decade. Tendon reflexes are usually absent andsensation is normal. Weakness, wasting, and fasciculations spread to involve theface and distal musculature; gynecomastia is common, and late hypogonadism,progressive loss of the libido, difficulty in maintaining an erection, and late ste-rility have been reported (Arbizu et al 1983).

Pathologically, SBMA causes anterior horn cell, bulbar neuron, and dorsalroot ganglion cell degeneration (Sobue et al 1989). Electromyography studies onSBMA patients indicate a neurogenic basis of atrophy with the presence of fib-rillations and fasciculations. Muscle biopsies show evidence of chronic dener-vation, often with collateral reinervation.

Mutation and Gene Product The expanded polymorphic CAG repeat tract inSBMA patients lies in the coding region of the androgen receptor (AR) gene.Wild-type alleles of the AR gene have from 9 to 36 CAG repeats whereas affectedalleles have from 38 to 62 CAGs.

The AR is a member of the steroid hormone receptor family. Like other mem-bers of this protein family, it contains domains for hormone and DNA binding.On binding of androgen in the cytoplasm, the receptor/ligand complex is trans-ported to the nucleus where it activates the transcription of hormone responsivegenes (Zhou et al 1994). The AR polyglutamine tract is located at the NH2 ter-minus outside of both the hormone and the DNA binding regions; its function isunknown. An AR lacking the polyglutamine tract is still able to transactivatehormone responsive genes (Jenster et al 1991). Expansion of the glutamine repeathas no effect on hormone binding and only slightly reduces its ability to transac-tivate responsive genes. The expansion may cause partial loss of receptor functionthat could be responsible for some signs of androgen insensitivity in affectedmales. Complete absence of AR leads to testicular feminization rather than a

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TABLE 1 Molecular characteristics of polyglutamine neurodegenerative diseases

Disease Gene locusGeneproduct

NormalCAG(n)

ExpandedCAG(n)

Proteinlocalization Special features Brain regions most affected

SBMA Xq11-12 Androgenreceptor

9–36 38–62 Nuclear andcytoplasmic

Anterior horn and bulbarneurons, dorsal root ganglia

HD 4p16.3 Huntingtin 6–34 36–121 Cytoplasmic Intermediate alleles:29–35

Striatum, cerebral cortex

SCA1 6p22-23 Ataxin-1 6–44 39–82 Nuclear inneurons

Normal alleles .21repeats interruptedwith 1–4 CAT units

Cerebellar Purkinje cells,dentate nucleus; brainstem

SCA2 12q23-24 Ataxin-2 15–31 36–63 Cytoplasmic Normal allelesinterrupted with 1–2CAA units

Cerebellar Purkinje cells, brainstem, fronto-temporal lobes

SCA3 14q24.3-31 Ataxin-3 12–41 62–84 Cytoplasmic Cerebellar dentate neurons,basal ganglia, brain stem, spinalcord

SCA6 19p13 CACNA1A 4–18 21–33 Cell membrane Cerebellar Purkinje cells,dentate nucleus, inferior olive

SCA7 3p12-p21.1 Ataxin-7 4–35 37–306 Nuclear Intermediate alleles:28–35

Cerebellum, brain stem, macula,visual cortex

DRPLA 12q Atrophin-1 6–36 49–84 Cytoplasmic Cerebellum, cerebral cortex,basal ganglia, Luys body

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motor neuron disease; heterozygous SBMA females may be protected by X-inactivation patterns, if the toxic effect of CAG expansion in the AR is cellautonomous, or by low androgen levels, if the toxic effect is ligand dependent.

Huntington Disease

Clinicopathology Chorea, the most common motor symptom in adult patients,usually presents along with memory deficits, affective disturbance, and changesin personality; other forms of motor dysfunction such as parkinsonism, dystonia,and involuntary motor impairments may all be present. Juvenile-onset patientsshow bradykinesia, rigidity, epilepsy, severe dementia, and a more rapidly pro-gressing disease. Caudate atrophy is visible in computer tomography or magneticresonance imaging scans.

Diffuse, severe atrophy of the neostriatum (perhaps worse in the caudate thanthe putamen) is the pathologic hallmark of HD (Vonsattel et al 1985). Medium-sized spiny striatal neurons containing c-amino butyric acid (GABA) areextremely vulnerable, whereas larger neurons positive for nicotinamide adeninedinucleotide phosphate diaphorase (NADPH-d) are relatively preserved. In partbecause of the loss of striatopallidal projection fibers, the globus pallidus canshow a volume loss comparable to that seen in the striatum. Early onset cases ofHD most often show loss of cerebellar Purkinje cells (Vonsattel & DiFiglia 1998),along with generalized atrophy of the brain.

Mutation and Gene Product Wild-type chromosomes with a stable CAG repeathave 6–34 repeat units; more than 36 repeats results in an unstable, expanded,disease-causing allele. Juvenile forms of HD are associated with alleles containingmore than 70 repeats; the longest allele described to date contains 121 CAGrepeats. Intergenerational instability shows a dramatic transmitting parent effect:80% of juvenile patients inherit the mutant HD gene from their father (Harper etal 1991, Zuhlke et al 1993). The most likely explanation for this augmentedpaternal repeat instability is the greater number of cellular divisions that takeplace during spermatogenesis.

Genes homologous to HD are present in the genome of other vertebrates. Forexample, the murine homologue, Hdh, shares 90% homology with human HD(Lin et al 1994). Despite this overall high level of sequence identity, the mouseprotein has only seven consecutive glutamines.

HD appears to be a true dominant disorder due to a gain of toxic function bya mutant allele, because HD homozygote patients have a disease similar in sever-ity to the disease present in their heterozygote siblings (Wexler et al 1987).Genetic evidence in both humans (Ambrose et al 1994) and mice (Duyao et al1995, Nasir et al 1995, Zeitlin et al 1995) demonstrates that HD deletion doesnot result in HD. Nullizygous mouse embryos are developmentally retarded anddie in gestation between days 8.5 and 10.5, which suggests that huntingtin maybe developmentally important at a time of organogenesis. It is important that the

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GLUTAMINE REPEATS 221

embryonic function of huntingtin is retained with expansion of the polyglutaminetract: Replacing wild-type Hdh with an Hdh allele containing 50 glutaminesresults in proper embryonic and brain development (White et al 1997).

HD is expressed widely throughout the brain, primarily as a soluble cytoplas-mic protein (DiFiglia et al 1995, Persichetti et al 1995, Trottier et al 1995a).Subcellular localization studies reveal huntingtin to be a cytoplasmic protein insomatodendritic regions and in axons; it is associated with microtubules in den-drites and with synaptic vesicles in axon terminals, perhaps serving some functionin synaptic transmission. Huntingtin is not associated with mitochondria, so adirect role in oxidative metabolism is less likely. One report suggests that in celllines huntingtin is located in both the cytoplasmic and the nuclear compartments(De Rooij et al 1996). Relatively high levels of huntingtin show a patchy distri-bution in both large striatal interneurons and medium spiny neurons; huntingtinis also abundant in cortical pyramidal cells and cerebellar Purkinje cells (Vonsattel& DiFiglia 1998).

Huntingtin-associated protein 1 (HAP1) was the first protein found to interactwith huntingtin (Li et al 1995); it is interesting that the interaction strengthenswith increasing polyglutamine length. Although HAP1 expression is highest inthe brain, its role in either the natural or pathogenic function of huntingtin isunknown. Kalchman et al found that huntingtin can interact with a ubiquitin-conjugating enzyme (Kalchman et al 1996). This interaction is not dependent onthe polyglutamine length and might indicate a catabolic route for huntingtin. Anintriguing interaction between huntingtin and the enzyme glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) was found using immobilized protein (Burke etal 1996). If relevant to pathogenesis, an interaction between huntingtin andGAPDH would support the hypothesis that development of HD involves a dis-ruption of energy production in the brain. The human homologue of the yeastprotein Sla2p, designated huntingtin-interacting protein 1 (HIP1) (Kalchman etal 1997), colocalizes with huntingtin, and its interaction with huntingtin isinversely correlated to the polyglutamine chain length. Sla2p is essential forproper function of the cytoskeleton in Saccharomyces cerevisiae, so impairedinteraction between huntingtin and HIP1 may alter membrane-cytoskeletal func-tion in the HD brain. Numerous additional proteins interact only with the NH2

terminus of huntingtin (Faber et al 1998). Several huntingtin interactors are mem-bers of the WW domain family of proteins, which are present throughout evo-lution and seem to play a critical role in a number of cellular processes, such asnonreceptor signaling, channel function, protein processing, and pre-mRNA splic-ing. Thus, based on the variety of huntingtin interacting proteins identified, hun-tingtin may play a role in a number of cellular events. This could make thedisruption of cellular function by mutant huntingtin complex.

Studies of patient tissue at the electron microscopy level reveal both cytoplas-mic and nuclear abnormalities that include the presence of large nuclear inclusions(NI) or aggregates (Roizin et al 1979). Protein expression studies using antibodiesto huntingtin clearly demonstrate that expanded protein is expressed (Jou et al

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1995). Mutant huntingtin is often seen at a lower level in HD heterozygote lym-phoblast or brain protein extracts than in its wild-type counterpart (DiFiglia et al1995, Persichetti et al 1995, Trottier et al 1995a,b). However, pulse-chase exper-iments fail to reveal an alteration in mutant huntingtin turnover or processing tosmaller fragments (Persichetti et al 1996), which suggests that the decreased sig-nal from the protein is due to either a difference in gene expression or decreasedbinding to the antisera. In HD patient brain tissue, huntingtin localizes with ubiq-uitin in neuronal nuclear inclusions and dystrophic neurites in the cortex andneostriatum but not in globus pallidus or cerebellum. The NI stain only withantibodies directed to the NH2-terminal portion of huntingtin and not with anti-bodies to the COOH terminus. Moreover, an NH2-terminal fragment of huntingtinof about 40 kDa is detectable in nuclear extracts from a patient brain and not inan extract prepared from control cortex (DiFiglia et al 1997). These data suggestthat an NH2-terminal fragment of mutant huntingtin might need to translocate tothe nucleus to contribute to pathogenesis.

The Spinocerebellar Ataxias

The dominantly inherited spinocerebellar ataxias (SCAs) are a heterogeneousgroup of neurologic disorders characterized by variable degrees of degenerationof the cerebellum, spinal tracts, and brain stem (Greenfield, 1954, Koeppen &Barron 1984). Numerous attempts at classification were foiled by the vexing inter-and intrafamilial variability in clinicopathological findings that are as character-istic of these diseases as incoordination—indeed, patients from the same kindredwere occasionally ascribed two or more distinct types of SCA. It was not untilthe genetic bases of these diseases were discovered that it became possible toreliably distinguish among them. Only those SCAs for which the gene has beencloned are discussed here.

Some clinical features are common to all the SCAs. The eponymous cerebellardegeneration dictates that ataxia and dysarthria, for example, will be hallmarksof each of these diseases. But there are variable features, seen either in somesubtypes or in some families with a specific subtype, that help distinguish certainataxias. Hypo- or areflexia and extremely slow saccades suggest SCA2; it isinteresting that dementia, which occurs in a fraction of patients, was the earlysign in all members of one kindred (Durr et al 1995, Geschwind et al 1997a, Pulstet al 1993). Bulging eyes, early faciolingual fasciculations, and Parkinsonismsuggest SCA3, but the curious history of this ataxia—its early segregation fromwhat was thought to be a separate disease entity, Machado-Joseph disease—evinces the extreme variability of clinical and pathological signs that dog thediagnostician (Durr et al 1996, Giunti et al 1995). Slowly progressive or episodicataxia can presage SCA6, but the most notable difference between this diseaseand the other SCAs is the complete absence of extrapyramidal signs, spasticity,cognitive impairment, and visual deficits (Geschwind et al 1997b, Jodice et al1997, Schols et al 1998, Zhuchenko et al 1997). SCA7 patients may first present

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GLUTAMINE REPEATS 223

with cerebellar ataxia or visual deficits: Half present with both, and the majorityof the remaining half present with ataxia and may maintain normal vision fordecades. The minority who first present with visual deficits, which are secondaryto pigmentary macular degeneration, usually develop ataxia within a few years.Behavioral abnormalities such as auditory hallucinations and progressive psy-choses occur in some SCA7 patients, whereas the infantile phenotype is uniquein involving nonneuronal tissue (the heart) (Benton et al 1998, David et al 1998,Johansson et al 1998). DRPLA is distinct from most SCAs in its characteristicchoreoathetosis, myoclonic epilepsy, and dementia; juvenile-onset individualsmay also suffer mental retardation (Naito & Oyanagi 1982, Warner et al 1994).With these variabilities in mind, we can present what is known about the neu-ropathology and mutant protein relevant to each SCA.

Spinocerebellar Ataxia Type 1 The predominant neuropathologic findings inSCA1 are cerebellar atrophy with severe loss of Purkinje cells, dentate nucleusneurons, and neurons in the inferior olive and cranial nerve nuclei III, IV, IX, X,and XII. Eosinophilic spheres, also known as torpedoes, are present in the internalgranule cell layer and some are related to Purkinje cell bodies. The dorsal andventral spinocerebellar tracts and dorsal columns are demyelinated; gliosis of themolecular layer of the cerebellum is marked, whereas gliosis of the anterior hornof the spinal cord is milder (Zoghbi & Ballabio 1995).

A highly polymorphic CAG repeat (heterozygosity rate of 84%) lies in thecoding region of SCA1 and encodes a glutamine tract. Normal alleles contain 6–44 repeats, and those that contain over 20 repeats, are interrupted with 1–4 CATrepeat units encoding histidine, which most likely maintains CAG repeat tractstability (Chung et al 1993). Disease alleles, in contrast, not only contain a largernumber of repeats (39–82), they are also uninterrupted by CAT sequences (Chonget al 1995, Goldfarb et al 1996, Jodice et al 1994, Quan et al 1995, Ranum et al1994). Mosaicism is observed in SCA1, particularly in sperm cells. Paternal trans-missions tend to produce expansions, whereas maternal transmissions tend toshow contractions (Chung et al 1993, Jodice et al 1994).

The SCA1 gene product, ataxin-1, is a novel protein that shares no homologywith other proteins. It seems to play a role in synaptic plasticity and neuronalfunctions underlying some learning tasks function: Sca1 null mice displayimpaired spatial and motor learning and decreased paired-pulse facilitation in theCA1 area of the hippocampus (Matilla et al 1998). The fact that mice eitherheterozygous or homozygous for the null mutation do not develop ataxia confirmsthat SCA1 is not caused by loss of normal ataxin-1 function. Furthermore, inhumans, large deletions in 6p22–23 spanning the SCA1 gene do not result inSCA1 but do cause mental retardation and seizures (Davies et al 1999). Althoughthis phenotype could result from the loss of genes in addition to SCA1, it confirmsthat haploinsufficiency does not lead to ataxia.

Wild-type ataxin-1 is predicted to encode 792–830 amino acids, depending onthe size of the repeat tract (Banfi et al 1994). The murine Sca1 gene product

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shares 89% identity with the human protein; a notable difference in mice is thepresence of two glutamines and three prolines in place of the long glutaminerepeat (Banfi et al 1996). Wild-type ataxin-1 is predicted to be approximately 87kDa but has an altered electrophoretic mobility, likely due to the glutamine tract,because the mobility of the mutant protein varies according to the number ofCAG repeats (Servadio et al 1995). Ataxin-1 is expressed in the central nervoussystem (CNS) at two to four times the levels found in peripheral tissues. In lym-phoblasts, heart, skeletal muscle, and liver, the protein is localized to the cyto-plasm; in neurons, however, it is predominantly nuclear, with some cytoplasmicstaining in Purkinje cells and brain stem nuclei (Cummings et al 1998, Koshy etal 1998, Servadio et al 1995, Skinner et al 1997). In SCA1 patients, mutant ataxin-1 localizes to a single large nuclear inclusion in brain stem neurons (Cummingset al 1998, Skinner et al 1997). These inclusions stain positively for ubiquitin,the porteasome, and the molecular chaperone HDJ-2/HSDJ (Figure 1). The pro-tein may be more abundant in these cells, or it may undergo specific interactionsor modifications unique to these neurons.

The 247–amino acid, leucine-rich acidic nuclear protein (LANP) associateswith ataxin-1 in a glutamine-repeat, length-dependent manner (Matilla et al 1997).The highest level of Lanp expression occurs in Purkinje cells at approximatelypostnatal day 14 (Matsuoka et al 1994), contemporaneous with a transient burstof Sca1 expression in mice (Banfi et al 1996). Furthermore, like ataxin-1, thisprotein localizes primarily to the nucleus, and in transfected cells it is redistributedinto nuclear inclusions formed by mutant ataxin-1. LANP belongs to the familyof leucine-rich repeat proteins that mediate protein-protein interactions pivotal toprocesses as wide-ranging as morphogenesis, cell adhesion, and signaling. It maywell be that LANP and possibly other nuclear proteins bind to mutant ataxin-1in a manner that either hinders its normal activity or causes deleterious effects.

Spinocerebellar Ataxia Type 2 Necropsies of SCA2 patients reveal that thecerebellum and brain stem are atrophied, with severe degeneration of Purkinjeand granule cells along with conspicuous neuronal loss and gliosis in the inferiorolive and pons. Atrophy of the fronto-temporal lobes and degeneration of thesubstantia nigra have been reported (Durr et al 1995, Orozco et al 1989).

The SCA2 cDNA encodes a novel protein predicted to be 140 kDa. Ataxin-2shares no homology with proteins of known function, but it does share sequencehomology with mouse Sca2 gene product and another novel protein, ataxin-2–related protein (Pulst et al 1996). The SCA2 transcript is widely expressed inbrain, heart, placenta, liver, skeletal muscle, and pancreas and is estimated to be4.5 kbp (Imbert et al 1996, Pulst et al 1996, Sanpei et al 1996). Unlike ataxin-1,ataxin-2 localizes to the cytoplasm. It is interesting that although there are nonuclear inclusions in SCA2 brain samples to parallel those seen in many otherSCAs, there is a clear increase in ataxin-2 immunostaining in the cytoplasms ofPurkinje cells and dentate neurons, which suggests that the mutant protein may

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Figure 1 (A, B) The subcellular distribution of ataxin-1 in nucleus pontis centralisneurons from a spinocerebellar ataxia 1 patient. Nuclear inclusions (NI) magnified(bottom right) and containing ubiquitin (B). (C) Redistribution of the 19S proteasometo aggregates in patient tissue; (D) control shown for comparison. (E) Molecular chap-erone HDJ-2/HSDJ localizes mainly to cytoplasm except for the NI; (F) control shownfor comparison.

be accumulating and thereby contributing to cellular dysfunction (Huynh et al1999).

The CAG repeat is not as polymorphic as those that undergo expansion inother neurodegenerative diseases—95% of alleles are 22 or 23 repeats (Imbert et

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al 1996, Pulst et al 1996, Riess et al 1997, Sanpei et al 1996)—and is normallyinterrupted by two CAA sequences. As in SCA1, expanded alleles contain aperfectly uninterrupted CAG repeat tract. Normal alleles may contain as few as15 or as many as 31 repeats, though the latter are rare; 36–63 repeats have beenfound in disease alleles, with the majority being between 36 and 43 repeats.Intermediate alleles, ranging between 32 and 34 repeats, have been found inmembers of SCA2 families who carry the disease haplotype but whose clinicalstatus is normal at the time of evaluation. It is not clear whether these alleles arein the pathogenic range.

Machado-Joseph Disease/Spinocerebellar Ataxia Type 3 Neuropathologicalstudies show that SCA3 degeneration is most prominent in the basal ganglia,brain stem, and spinal cord. Neuronal loss is mild in the cerebellum and not typicalin the inferior olive (Durr et al 1996, Takiyama et al 1994, Woods & Schaumburg1972).

The size of the CAG tract ranges from 12 to 41 repeats on normal chromo-somes and from 62 to 84 repeats on disease chromosomes (Durr et al 1996, Giuntiet al 1995, Maciel et al 1995, Maruyama et al 1995)—making MJD/SCA3 oneof the few trinucleotide repeat diseases in which there is a distinct gap betweennormal and affected repeat sizes. Intergenerational instability is common and ismore pronounced when paternally transmitted.

The MJD1 gene encodes an intracellular protein of unknown function pre-dicted to be 42 kDa (Kawaguchi et al 1994). The MJD1 gene product ataxin-3 ispredominantly a cytoplasmic protein, although it has been found within thenucleus in patient tissues (Paulson et al 1997). Ataxin-3 shares no homology withother proteins outside of the glutamine tract and is predicted to contain a nuclearlocalization signal (Tait et al 1998). Both the transcript and the protein are widelyexpressed, without any evidence supporting higher levels in tissues that areaffected in MJD/SCA3. Ataxin-3 localizes to ubiquitin-positive nuclear aggre-gates in affected neurons (Paulson et al 1997); various components of the pro-teasome and cellular chaperones also colocalize to ataxin-3 aggregates (Chai etal 1999).

Spinocerebellar Ataxia Type 6 Neuropathologic findings in SCA6 includemarked cerebellar atrophy and mild atrophy of the brain stem. There is severeloss of cerebellar Purkinje cells with moderate loss of cerebellar granule cells,dentate nucleus neurons, and neurons of the inferior olive (Ikeuchi et al 1997,Zhuchenko et al 1997).

The SCA6 gene codes for the a1A-voltage–dependent calcium channel(CACNA1A). The CAG repeat lies within the coding region and is predicted toencode a glutamine tract (Zhuchenko et al 1997). Voltage-sensitive calcium chan-nels are multimeric complexes made of an a1A subunit, which is sufficient to formthe channel structure, and a2 and b subunits, which are regulatory (Catterall1995). The a1A subunit encodes P- and Q-type calcium channels that were iden-

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GLUTAMINE REPEATS 227

tified in cerebellar Purkinje cells and granule neurons, respectively (Llinas et al1989, Zhang et al 1993). The a1A subunit gene is expressed abundantly in theCNS, with highest levels in the cerebellum (Fletcher et al 1996).

The pathogenic range for the repeat is 21–33, whereas normal alleles range insize from 4 to 18 repeats (Moseley et al 1998, Schols et al 1998, Zoghbi 1997).The SCA6 CAG repeat differs from other disease-causing CAG repeats in thatthe expansion is small and the repeat is relatively stable. Intergenerational insta-bility is apparently rare: In over 50 parent-child transmissions evaluated, only asingle expansion event has been observed (a 24 repeat expanded to 26 in a father-son pair) (Matsuyama et al 1997).

Missense and splicing mutations in the a1A-voltage–dependent calcium chan-nel have been identified in other neurological disorders, such as hereditary par-oxysmal cerebellar ataxia (or episodic ataxia type 2) and familial hemiplegicmigraine. The tottering and tottering-leaner mice, which develop ataxia and sei-zures, also bear mutations in this subunit (Fletcher et al 1996, Ophoff et al 1996).The exact mechanism by which the various mutations in this channel cause dis-ease is not determined. For familial hemiplegic migraine and episodic ataxia, itis possible that the mutations cause haplo insufficiency of the gene product orlead the mutant product to act in a dominant negative manner. Even modestexpansions may alter the normal physiology of the channel, disrupting calciumhomeostasis. Alternatively, the CAG repeat expansion may confer a novel toxicfunction onto the protein, as it does in other polyglutamine neurodegenerativedisorders.

Spinocerebellar Ataxia Type 7 The predominant neuronal loss in SCA7 is inthe cerebellum, inferior olive, and some cranial nerve nuclei. There is hypomye-lination of the optic tract and some gliosis of the lateral geniculate body andvisual cortex (David et al 1998, Martin et al 1994).

The polyglutamine tract is in the NH2-terminal region (codons 30 to 39) ofSCA7. It is polymorphic, ranging in size from 4 to 35 repeats on normal alleles;the majority (;75%) contain 10 repeats. In contrast to the CAG repeats in SCA1and -2, all normal alleles contain pure CAG tracts without any evidence of inter-ruption (Benton et al 1998; David et al 1997, 1998; Del-Favero et al 1998; Gouwet al 1998; Johansson et al 1998; Moseley et al 1998).

The SCA7 repeat is one of the most unstable CAG repeats known. Patientscarry expansions ranging in size from 37 to 306 repeats, and intermediate allelesare known to expand into pathogenic range on intergenerational transmission. Forexample, an allele containing 35 CAG repeats expanded to 57 during paternaltransmission and resulted in the SCA7 phenotype in successive generations (Ste-vanin et al 1998). The largest known intergenerational repeat expansion enlargedthe CAG tract by 263 repeats in a father-to-son transmission (Benton et al 1998).Although paternal transmission causes much greater expansions, the disease ismore often observed on maternal transmission. This suggests that paternally trans-mitted alleles may be embryonic lethal or are at a disadvantage for fertilization

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(Benton et al 1998, Gouw et al 1998). Somatic mosaicism of this repeat has alsobeen demonstrated in leukocyte DNA.

Ataxin-7, a nuclear protein of 892 amino acids, shares no significant homologywith known proteins. It ranges in size from 130 to 180 kDa in SCA7 patients(Stevanin et al 1996, Trottier et al 1995b). The 7.5-kbp transcript is ubiquitous,with the highest expression levels in the heart, placenta, skeletal muscle, andpancreas and lower levels in brain, liver, and kidney. Within the CNS, the SCA7transcript is expressed at higher levels in the cerebellum than in other tissues. Inaffected neurons, ataxin-7 aggregates in single large nuclear inclusions that stainpositively for ubiquitin (Holmberg et al 1998).

Dentatorubropallidoluysian Atrophy The neuropathology of dentatorubropal-lidoluysian atrophy (DRPLA) is extensive: pronounced neuronal loss in the cere-bral cortex, globus pallidus, striatum, and cerebellar cortex and in the subthalamic(Luys body), red, and dentate nuclei. There is intense fibrillary gliosis at the sitesof neuronal degeneration; severe demyelination and axonal degeneration in thesuperior cerebellar peduncle is common and in some families it is also observedin the subcortical white matter along with calcifications of the basal ganglia(Burke et al 1994, Naito & Oyanagi 1982, Smith 1975, Takahashi et al 1988).DRPLA patients with late-onset disease often have symmetrical high-signallesions on magnetic resonance imaging in the cerebral white matter and brainstem suggestive of leukodystrophic changes (Potter et al 1995, Uyama et al 1995,Warner et al 1994).

The normal range for the DRPLA repeat is 6–36; expanded alleles have 49–84 repeats. Intergenerational instability is more pronounced in paternal transmis-sions (Ikeuchi et al 1995, Koide et al 1994). The DRPLA cDNA is predicted tocode for 1185 amino acids; the gene product, atrophin-1, is widely expressed andshares no homology with other known proteins. Atrophin-1 is distributed through-out the cytoplasm in neurons and peripheral tissues of both unaffected andaffected individuals (Knight et al 1997, Yazawa et al 1995). Nuclear inclusionscontaining mutant atrophin-1 are found only in neurons in regions of the brainaffected by DRPLA (Igarashi et al 1998).

Salient Features Shared by the Polyglutamine Diseases

Before discussing pathogenesis, we draw a few interesting observations from thepolyglutamine diseases as a whole.

Inverse Relationship of Repeat Size and Clinical Presentation Analysis ofrepeat size and symptomatology in polyglutamine disease patients has clearlydemonstrated an inverse relationship between the size of the repeat and the ageof onset, though the contribution of the repeat size to age of onset varies accordingto protein context (Figure 2). The variability in the clinical phenotype of SCA7,for example, is particularly well correlated with the size of the CAG repeat.

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Figure 2 Longer CAG lengths cause earlierdisease onset in polyglutamine disorders.SCA, spinocerebellar ataxia; HD, Huntingtondisease; DRPLA, dentatorubropallidoluysianatrophy. (Courtesy of James F. Gusella andCell Press.)

Typically, 30–40 SCA7 repeats cause disease after 20 years of age; 50–80 repeatswill cause disease between 5 and 20 years of age, and usually with visual deficitsin addition to the cerebellar symptoms. Patients with more than 100 repeats havean infantile onset occurring shortly after birth or in the first few months of life.There are three infants known to have SCA7 who, in addition to the visual andneuronal deficits, had somatic features: severe hypotonia, patent ductus arteriosus,congestive heart failure, and death in the first year of life (Benton et al 1998,

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Johansson et al 1998, Neetens et al 1990). Although in HD, SCA1, SCA6, andSCA7 over 70% of the age-of-onset variability is accounted for by the numberof CAG repeats, in MJD/SCA3 the contribution of the repeat size is estimated tobe approximately 45–48%. Furthermore, it is clear from careful study of thevarious kindreds that other factors, probably both environmental and genetic,contribute to the onset of disease.

Phenotypic Overlap and Loss of Cell Specificity It is interesting that when theexpansions are large, leading to severe, juvenile-onset disease, there is significantoverlap in the phenotypes of these disorders. Juveniles with SCA1 manifest notonly the characteristic ataxia and brain stem dysfunction but also some cognitiveimpairment and dystonic features. Juvenile-onset Huntington patients developdystonia and seizures in addition to the classical phenotype of chorea and demen-tia seen in adult patients. This loss of cell specificity with large expansions isintriguing and hints that toxicity is probably much more widespread throughneuronal subtypes that are normally spared when the repeat sizes are in the mod-erate range. Even more interesting, the very massive expansions observed inSCA7 cause not only loss of neuronal specificity but also involvement of non-neuronal tissue (e.g. the heart).

Protein Aggregation One aspect of the subcellular distribution of polyglutam-ine proteins in SCA1, -3, -7, HD, SBMA, and DRPLA is the localization ofmutant proteins to ubiquitinated microscopic nuclear inclusions or aggregateswithin neuronal nuclei. Nuclear inclusions containing the polyglutamine proteinhave been detected in brains of HD, SCA1, MJD/SCA3, SCA7, and DRPLApatients and in motor neurons of SBMA patients (Li et al 1998). In some diseases(HD, SCA1, MJD/SCA3, DRPLA, SBMA), nuclear inclusions are found selec-tively in neurons affected by the disease. In one juvenile-onset patient, ataxin-7–containing nuclear inclusions were not restricted to sites of severe neuronal lossusually associated with disease (Holmberg et al 1998). The significance of thisfinding must be tempered by two facts: It derived from only one patient, a severejuvenile-onset patient. In all the polyglutamine diseases, juvenile onset is asso-ciated with loss of tissue specificity. Although nuclear inclusions are not seen inSCA2 brain samples, there is a clear increase in ataxin-2 immunostaining inPurkinje cells and dentate neurons, which suggests that there might be cytoplas-mic accumulation of the mutant protein (Huynh et al 1999). The dramatic pres-ence of nuclear inclusions in patient neurons has led to the suggestion that proteinaggregation is a critical molecular component of polyglutamine diseases (Davieset al 1998, Ross 1997).A

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PATHOGENESIS STUDIES OFPOLYGLUTAMINE DISEASES

Mouse Models

Spinocerebellar Ataxia Type 1 The first transgenic mouse model of a polyglu-tamine disease utilized a strong Purkinje cell-specific promoter from the Pcp2/L7gene to direct expression of the human SCA1 cDNA encoding full-length ataxin-1 (Burright et al 1995). These lines expressed high levels of either a wild-typeSCA1 allele with 30 repeats (30Q) or an expanded allele with 82 repeats (82Q).The 82Q transgenic mice developed severe ataxia and progressive Purkinje cellpathology, whereas mice expressing wild-type ataxin-1 displayed no neurologicabnormalities and were indistinguishable from nontransgenic littermates (Clarket al 1997). These studies demonstrated that pathological changes are induced bythe expression of ataxin-1 with an expanded polyglutamine tract.

In transgenic mice from a 30Q line, ataxin-1 localized to several ;0.5-lmnuclear inclusions. In contrast, in 82Q mice, ataxin-1 localized to a single ;2-lm ubiquitinated nuclear aggregate, as it does in patient tissue (Skinner et al1997). The appearance of these aggregates, which stained positive for the 20Sproteasome and the HDJ-2/HSDJ (Hsp40) chaperone protein, preceded the onsetof ataxia by approximately 6 weeks (Cummings et al 1998). The only notabledifference between the pathology observed in SCA1 transgenic mice and that ofSCA1 patients is that the mice lack axonal dilatations (torpedoes). SCA1 trans-genic animals from the 82Q line had mild cerebellar impairment at 5 weeks ofage; there was no evidence of gait abnormalities or balance problems at that age(Clark et al 1997). By 12 weeks, this slight motor skill impairment progressed toovert ataxia, which worsened over time.

The first histologic change was the development of cytoplasmic vacuoleswithin Purkinje cell bodies at postnatal day 25; by 5 weeks, loss of proximaldendritic branches and a decrease in the number of dendritic spines became appar-ent, indicating that mutant ataxin-1 may impair the maintenance of dendritic arbo-rization. By 12–15 weeks, the complexity of the dendritic arborization of Purkinjecells was markedly reduced, the molecular layer atrophied, and there were severalheterotopic Purkinje cells within the molecular layer. The heterotopia, notdetected in young animals, is not a developmental abnormality but most likelyan attempt to preserve synapses in the face of severely reduced dendritic arbori-zation. Cell loss was minimal at the time of progressive gait abnormality.Although it had long been assumed that the neurological phenotype in SCApatients results from neuronal death, these mice demonstrated that the neurolog-ical impairment is due instead to neuronal dysfunction.

To ascertain whether ataxin-1 must be in the nucleus to cause disease, Klementet al (1998) generated and characterized transgenic mice that express expandedataxin-1 (82 glutamines) with a mutated nuclear localization sequence, ataxin-1K772T. Although these mice expressed high levels of ataxin-1 in Purkinje cells,

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similar to those observed in the original SCA1 (82Q) transgenic mice, they neverdeveloped Purkinje cell pathology or motor dysfunction. Ataxin-1 was diffuselydistributed throughout the cytoplasm and formed no aggregates, even when themice were a year old. Nuclear localization is clearly critical for pathogenesis andataxin-1 aggregation. Furthermore, these studies provided direct evidence thatexpanded CAG repeats are toxic at the protein and not the RNA level (Klementet al 1998).

To assess the role of nuclear aggregates in causing disease, Klement et al(1998) generated transgenic mice using ataxin-1 (77Q) with amino acids deletedfrom the self-association region found to be essential for ataxin-1 dimerization.These mice developed ataxia and Purkinje cell pathology similar to the originalSCA1(82Q) mice, but without apparent nuclear ataxin-1 aggregation. Thus,although nuclear localization of ataxin-1 is necessary, nuclear aggregation ofataxin-1 is evidently not required to initiate pathogenesis in transgenic mice.Deletion of 122 amino acids might compromise the protein in various ways (fold-ing, turnover rate, interactions), but this truncated ataxin-1 retained its ability tointeract with its known partner, LANP, and produced all the neurobehavioral andunique pathologic features observed in the (82Q) mice. It seems safe to say, then,that this protein with an expanded polyglutamine tract exerts similar pathogenicityto full-length expanded ataxin-1 in spite of the fact that it does not accumulatein visible aggregates.

The best available model of SCA1 pathogenesis thus holds that sequenceswithin ataxin-1 in addition to the polyglutamine tract are critical in specifying thesite and course of disease. The neuronal dysfunction that causes SCA1 symptomsbegins with the localization of ataxin-1 to the nucleus. Once there, mutant ataxin-1 misfolds and is somehow altered in its distribution. It is likely that changes innuclear architecture and the interaction of mutant ataxin-1 with other nuclearproteins alter gene expression, all of which may well contribute to neuronal dys-function, symptomatology, and eventual cell loss. The extent to which this modelof SCA1 pathogenesis may be applicable to other polyglutamine disordersremains to be seen.

Machado-Joseph Disease/Spinocerebellar Ataxia Type 3 Ikeda and colleagues(1996) generated transgenic mice expressing full-length and truncated versionsof the MJD/SCA3 protein using the Pcp-2/L7 promoter region. Mice expressingfull-length ataxin-3 with 79 glutamines (an expanded allele) were designatedMJD79; mice expressing a truncated form comprising the glutamine tract and 42amino acids C terminal to the repeat were designated Q79C or Q35C, dependingon whether they harbored an expanded or a normal repeat size. Mice expressingonly a 79 glutamine residue tract (Q79) were also generated. Both the Q79C andQ79 transgenic mice developed ataxia by 4 weeks of age. None of the miceexpressing full-length expanded ataxin-3 (MJD79) or a truncated ataxin-3 with35 repeats (Q35C) developed ataxia.

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Q79C transgenic mice 8 weeks old showed massive degeneration of the cer-ebellum, which occupied approximately one eighth its normal volume. Thedegeneration involved all three layers of the cerebellum, with thinning of themolecular layer, altered morphology of Purkinje cells with attenuation of theirdendrites and reduced calbindin immunoreactivity, and loss of granule neurons.The main conclusion that can be drawn from these data is that the expandedpolyglutamine tract can induce neuronal cell death. This is in contrast to thetoxicity induced by full-length ataxin-1, which causes slow and progressive neu-ronal dysfunction rather than massive Purkinje and granule cell loss. Unfortu-nately, we cannot be certain that full-length ataxin-3 is not toxic, because data onexpression levels from the various transgenes were not provided. Based on thefact that the truncated protein is toxic, Ikeda and colleagues (1996) proposed thatcell-specific proteolytic cleavage of the mutant protein liberates an elongatedpolyglutamine tract that then induces cell death. Data demonstrating that ataxin-3 can be cleaved by a caspase (Wellington et al 1998), a member of a family ofcysteine proteases involved in apoptosis, are consistent with the hypothesis thatataxin-3 exerts its toxicity after cleavage and release of a polyglutamine tract.However, there is no direct evidence that cleavage of ataxin-3 occurs in affectedbrain regions of MJD/SCA3 patients.

Huntington Disease Mangiarini and colleagues (1996) generated transgenicmice expressing the human huntingtin promoter and the first exon with a CAGrepeat size of approximately 140 repeats. These mice express a truncated peptidethat includes the first 69 amino acids of huntingtin and an expanded glutaminetract. Northern and Western blot analyses demonstrated that the message andpeptide are widely expressed in all 18 tissues and brain regions tested. Three linesof transgenic animals (R6/1, R6/2, and R6/5) were developed from a malefounder, and each was shown to have a progressive neurological phenotype. Micefrom the R6/2 line, which have been characterized most extensively, are indistin-guishable from nontransgenic littermates at weaning, but at about 2 months ofage they develop an irregular gait, resting tremor, stereotypic grooming activity,sudden shuddering movements, and occasional seizures. This neurological phe-notype is progressive, and by 12 weeks it is accompanied by decline in bodyweight, an increase in frequency of urination, and sterility. Control transgenicmice expressing the same peptide with 18 CAG repeats do not display any ofthese abnormalities. Neuropathological analysis found no evidence of neuronaldegeneration, but brain weight diminished by about 20% 4–6 weeks prior to theloss in body weight.

Immunocytochemical and electron microscopy studies using antibodies raisedto the N terminus of huntingtin detected altered subcellular distribution of hun-tingtin in the R6/2 mice. In control mice, huntingtin was diffusely distributed inthe cytoplasm, dendrites, axon terminals, and vesicle membranes; in R6/2 mice,huntingtin staining was nuclear and localized to dense neuronal intranuclear inclu-sions (Davies et al 1997). Antibodies raised to C-terminal huntingtin peptides

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(deleted in the transgene) revealed no huntingtin in the NI, which suggests thatthe inclusions contain only truncated mutant huntingtin and not the endogenousfull-length protein. The NI were seen throughout the CNS in cortical neurons,Purkinje cells, striatum, spinal cord, and, to a lesser degree, in other brain regions.They were first detected at 3.5–4.5 weeks of age prior to any neurobehavioralabnormalities; by 5–6 weeks they stained positively for ubiquitin. Ultrastructuralanalysis of NI from R6/2 brains revealed that they are amembranous with finegranular and filamentous morphology. NI typically occupy about 1% of thenucleus and are invariably accompanied by invaginations of the nuclear mem-brane. These ultrastructural changes are reminiscent of the changes observed byelectron microscopy in HD brain tissue (for review, see Vonsattel & DiFiglia1998).

Recently, Hurlbert and colleagues (1999) reported that the R6/2 mice developinsulin-responsive diabetes at 8.5 weeks. Immunocytochemical studies foundlowered levels of glucagon and insulin and, more interesting, some intranuclearinclusions in the pancreatic islets. The hypoglycemia in these mice might accountfor some of the phenotypic features in these mice, such as polyuria and weightloss. It is unlikely that the diabetes gives rise to the neurological phenotype, giventhe prominence of the neurological findings with only modest elevations of bloodglucose (two times normal levels).

Ordway and colleagues (1997) examined polyglutamine toxicity in anothermouse model in which an expanded polyglutamine tract (146 CAG units) wasintroduced into the mouse hypoxanthine phosphoribosyl transferase (Hprt) gene.Mice with this mutation developed seizures, tremors, and motor dysfunction anddied prematurely. The mice did not show graded neuropathological abnormalitiesor reduced brain weight as in other polyglutamine disorders, but they did developNI in several brain regions. The authors concluded that CAG repeats are neuro-toxic, irrespective of the protein context. Protein context must influence the selec-tive neuronal loss characteristic of polyQ diseases, however, as this selectivity islost in the HPRT mouse.

Reddy et al generated several transgenic mouse lines expressing the full-lengthHD cDNA with either 16, 48, or 89 CAG repeats under the control of the cyto-megalovirus promoter. These mice had wide expression of huntingtin throughoutthe CNS and in peripheral tissues (Reddy et al 1998). Mice with 48 or 89 repeatsdeveloped progressive behavioral abnormalities characterized initially by abnor-mal limb clasping and generalized hyperactivity, unidirectional rotations, back-flips, and excessive grooming. As the mice aged (to approximately 24 weeks),they developed urinary incontinence and decreased locomotor activity, whichprogressed to akinesia. Once volitional movements ceased, the mice did notrespond to sensory stimuli and typically died within a week. The hypokinesiawas accompanied by neuronal loss and gliosis in the striatum in a pattern remi-niscent of that seen in HD patients. Neuronal loss also occurred in the hippocam-pus, thalamus, and cerebral cortex. Despite expression of the transgene in otherbrain regions (e.g. the cerebellum), neuronal loss was limited to areas typically

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affected in HD. Intranuclear inclusions were detected in several regions, includingthose typically unaffected by the disease (e.g. Purkinje cells); paradoxically, thefrequency of the inclusions was less than 1% in the striatum, where the neuronalloss was most prominent (Reddy et al 1998). These findings provide a counter-poise to the mouse models that express a truncated HD peptide or an unrelatedprotein (HPRT) with an expanded long glutamine tract: Selective neuronal lossoccurs only when the full-length protein with an expanded glutamine tract isexpressed, which again suggests that protein context helps mediate this selectivity.The paucity of nuclear aggregates in the striatum when neuronal loss and a clinicalphenotype are evident supports the notion that the aggregates do not initiatepathogenesis.

Invertebrate Models

To date, two Drosophila models expressing peptides with expanded polyglutam-ine tracts have been generated. Warrick et al (1998) expressed a C-terminal frag-ment of ataxin-3 with either 27 or 78 repeats (MJD-Q27 and MJD-Q78,respectively) in photoreceptors using a bipartite system in which the Glass Mul-timer Reporter promoter drives the expression of the yeast transcription factorGAL4, which in turn activates GAL4-activating sequences upstream of ataxin-3.This results in high expression levels of truncated ataxin-3 in eyes. MJD-Q78flies showed retinal degeneration during midpupal development and had nuclearinclusions in the photoreceptor cells of developing eye discs. Jackson et al (1998)expressed an amino terminal fragment of HD (the first 142 amino acids excludingthe repeat) containing either 2, 75, or 120 glutamines (Q2, Q75, and Q120, respec-tively) in Drosophila by directing fusion of the Glass Multimer Reporter promoterto the huntingtin peptide. Photoreceptor neurons expressing Q120 degeneratedwithin 10 days posteclosion, whereas those expressing Q75 had mild degenerationat 30 days posteclosion. The subcellular localization of the peptides was pre-dominantly nuclear for Q120, with numerous nuclear aggregates; Q75 wasnuclear in a few photoreceptor neurons. The age of onset and severity of thephenotype correlated well with the number of glutamine repeats in these twolines. It is interesting that partial ataxin-3 produced an earlier phenotype than thehuntingtin peptide with comparable repeat numbers (78 vs 75); this could be dueto different expression levels of the transgenes and/or to the flanking peptidesequences. Both groups evaluated the effect of overexpression of the viral antia-poptotic protein P35 on photoreceptor degeneration. A weak depigmentation phe-notype in MJD-Q78 flies—possibly reflecting cell death or abnormal developmentof pigment cells—was partially rescued by coexpression of P35, but no protectiveeffect on neuronal degeneration was detected in either model.

Polyglutamine toxicity was evaluated in Caenorhabditis elegans by expressingan amino-terminal fragment of huntingtin containing various numbers of gluta-mines (Htn-Q2, Htn-Q23, Htn-Q95, and Htn-Q150) in ASH sensory neuronsusing the osm-10 gene promoter (Faber et al 1999). Animals expressing Htn-

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Q150 had filling defects in ASH neurons at 8 days of age, based on decreaseduptake of the vital dye DiD. There was no cell death associated with this fillingdefect. When Htn-Q150 was coexpressed with a second toxic protein, OSM-10::GFP, which at subthreshold levels causes filling defects in less than 2% ofASH neurons at 8 days, the percentage of cells exhibiting filling defects increasedto ;27% and cell death became apparent. Furthermore, a phenotype of fillingdefects was detected in worms expressing Htn-Q95 under these conditions. ASHneuronal cell death required ced-3 caspase function, which suggests that polyglu-tamine-mediated cell death is apoptotic. Yet the phenotype of neuronal dysfunc-tion preceded the onset of neuronal death and Htn-Q150 protein aggregation bythree days.

In Vitro and Cell Culture Models

Overexpression of full-length ataxin-1 in COS cells caused the protein to behavemuch as it does in vivo: It localized to the nucleus and formed multiple aggregateswhose size correlated with the number of glutamine repeats (Skinner et al 1997).Deletion of the self-association domain prevented aggregation; mutation of thenuclear localization sequence resulted in both cytoplasmic and nuclear localiza-tion. Within the nucleus, ataxin-1 associated with the nuclear matrix, and themutant form caused redistribution of the promyelocytic oncogenic domain (Skin-ner et al 1997). Overexpression of full-length huntingtin, ataxin-3, atrophin-1,and AR revealed that these proteins have predominantly cytoplasmic and/or peri-nuclear localization with some propensity to aggregate (Butler et al 1998, Igarashiet al 1998, Ikeda et al 1996, Lunkes et al 1998, Martindale et al 1998, Merry etal 1998, Paulson et al 1997). Full-length ataxin-3 was found to be nuclear and toassociate with the nuclear matrix (Tait et al 1998). For most of these proteins,nuclear localization and aggregation became apparent when truncated proteinpeptides harboring expanded polyglutamine tracts were overexpressed. Aggre-gation in each case was enhanced with expansion of the glutamine tract or use ofshorter truncated peptides harboring long CAG repeats. The only exception is thefull-length expanded AR, which did induce aggregate formation in the nucleusin a repeat-dependent manner when androgen hormone was added (Stenoien etal 1999). These data suggest that for some polyglutamine proteins, processing ofthe full-length protein may be necessary to liberate a peptide that translocates tothe nucleus and then exerts its toxic effect. Such proteolytic fragments have beendetected when full-length expanded huntingtin and AR were expressed (Goldberget al 1996, Lunkes et al 1998, Merry et al 1998). Studies of both transfected cellsand recombinant proteins show that huntingtin, AR, ataxin-3, and atrophin-1 aresubstrates for one or more caspases, although the CAG length does not modulatecleavage for any of the proteins tested (Wellington et al 1998). It is interestingthat in patient material, an amino-terminal huntingtin fragment of 40 kDa isdetected in nuclear extracts, which suggests that huntingtin processing occurs invivo (DiFiglia et al 1997). This fragment is smaller, however, than those typically

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generated during caspase cleavage. How cleavage of huntingtin or other polyglu-tamine proteins might be involved in pathogenesis remains to be determined, butit is possible that cleavage could release a truncated peptide that contains a toxic,expanded polyglutamine tract, allowing it to wreak havoc in the cell.

Two important aspects of pathogenesis have been elucidated by cell culturestudies. The first, that protein misfolding and proteolysis may play a role in patho-genesis, was discovered by Cummings et al (1998), who demonstrated that ataxin-1 aggregates induce Hsp70 expression and redistribute it to the site of aggregation,along with HDJ-2/HSDJ and components of the proteasome. Overexpression ofthe HDJ-2/HSDJ chaperone decreased both the size and frequency of mutantataxin-1 nuclear aggregates. Similar results were obtained with ataxin-3 and AR(Paulson et al 1997, Stenoien et al 1999), which suggests that polyglutamine tractexpansion casues protein misfolding and that the cellular levels of a chaperonemay directly regulate the propensity of an expanded polyglutamine protein toaggregate. The observation that proteasome inhibitors promote aggregation ofmutant ataxin-3 suggested that intact proteasomal function is important for han-dling expanded polyglutamine proteins (Chai et al 1999). The second importantfinding came from studies that evaluated the effects of overexpression of anamino-terminal fragment of huntingtin on striatal neurons and showed that nuclearaggregates may be dissociated from pathogenicity (Saudou et al 1998). Expandedtruncated huntingtin aggregated in the nuclei and induced apoptosis, unlessblocked by specific apoptosis inhibitors and brain-derived and ciliary neuro-trophic factors. Adding a nuclear export signal forced mutant huntingtin to local-ize to the cytoplasm and significantly suppressed aggregation and cell death.Mutant huntingtin must therefore be nuclear to induce apoptosis. It need notaggregate, however: coexpression of a dominant negative mutant form of a ubiq-uitin-conjugating enzyme dramatically suppressed huntingtin’s aggregation whileaccelerating huntingtin-induced cell death in a repeat-dependent manner. Thissuggests a role for the ubiquitination pathway in aggregation. As shown by theSCA1 77Q mice (see above), nuclear inclusions do not induce pathogenesis butmay represent a cellular attempt to contain or degrade a misfolded protein.

IS THERE A UNIFYING PATHOGENIC MODEL?

All pathogenesis models posit that the expansion of CAG repeats confers a toxic“gain of function,” i.e. disease develops because the mutant form of the proteingains a new function, not because the protein loses its normal function. Data insupport of this model come from both patient studies and mice with targeteddeletions of huntingtin and ataxin-1 (see above). For some proteins, the expandedpolyglutamine tract may cause a partial loss of function, as is the case with AR:SBMA patients have some androgen insensitivity, manifesting as gynecomastiaand infertility.

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Given that the neuronal degeneration of most of the polyQ diseases is causedby a gain-of-function mechanism, what features of long polyglutamine tracts ren-der them and/or the proteins harboring them toxic to neurons? Two hypotheseshave been put forth. Green (1993) proposed that the polyglutamine tract is atransglutaminase substrate that, in the presence of an active enzyme, can becomecross-linked via an isopeptide to polypeptides containing lysyl groups. Theexpected products will contain multimers of the polyglutamine proteins as wellas other copolymers, leading to the formation of aggregates. Synthetic peptidescontaining consecutive glutamine residues and the polyglutamine tract withinhuntingtin are excellent substrates of transglutaminase and preferentially incor-porate into polymers in vitro (Kahlem et al 1998). It is not known whether trans-glutamination of polyglutamines occurs in vivo. However, it is interesting to notethat transglutaminase inhibition in cell culture did affect atrophin-1 polymeriza-tion: COS-7 cells expressing truncated forms of atrophin-1 with expanded poly-glutamine stretches developed filamentous peri- and intranuclear aggregates andeventually underwent apoptosis (Igarashi et al 1998). Apoptosis was partiallysuppressed by the addition of transglutaminase inhibitors, indicating that trans-glutamination may be involved in atrophin-1 aggregation and cell death. Perutzproposed an alternate mechanism by which two antiparallel b-strands of polyglu-tamine repeats can be linked together by hydrogen bonds and undergo multimer-ization and subsequent aggregation by polar zipper formation (Perutz et al 1994).Multimerization of chymotrypsin inhibitor 2 protein containing 10 glutamines hasbeen suggested based on circular dichroism data (Stott et al 1995), but resolvingthe crystal structure of four glutamines failed to support a model in which hydro-gen bonds between neighboring b-strands could lead to multimerization (Chen etal 1999). An amino terminal fragment of huntingtin containing 51 glutaminesand approximately 120 amino acids has been shown to aggregate in vitro, formingamyloid-like fibrils (Scherzinger et al 1997). However, the addition of 243 aminoacids from the glutathione S-transferase protein prevented its aggregation. Thesedata, together with the finding that full-length huntingtin with an expanded glu-tamine tract does not aggregate spontaneously in vitro, suggest that if multimer-ization of the polyglutamine tract is to occur, it has to first be cleaved fromhuntingtin.

The findings that mutant polyglutamine proteins aggregate in vulnerable neu-rons and that these aggregates stain positively for ubiquitin and the proteasomeprovided the first evidence that there are features common to the pathogeneses ofpolyQ disorders. Data from cell culture studies suggest that one such feature mightbe protein misfolding and/or conformational alterations due to polyglutamineexpansion. Such alterations may lead to aberrant protein-protein interactions andtargeting of the misfolded protein (and possibly its copolymers) for degradation.The altered distribution of the proteasome may eventually compromise the effi-ciency of proteoloysis in the affected neurons and contribute to neuronal dys-function. The cell specificity of the various phenotypes could be due to a numberof factors, such as relative levels of the polyglutamine protein, protein interactors,

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and cell specificity of certain modifying proteins. The latter group may includecell-specific transglutaminases and cysteine proteases.

Is the polyglutamine tract sufficient to induce disease? Data from both cellculture and animal studies clearly demonstrate that a long polyglutamine tract istoxic to neurons and peripheral cells alike. However, the typical gradual CNSpathology is seen only when full-length proteins are expressed. Additional peptidesequences must be contributing to the late onset of these diseases and selectiveneuronal vulnerability. As noted above, this neuronal selectivity disappears in theearliest juvenile-onset cases, when the polyglutamine tract becomes dispropor-tionately large relative to the rest of the protein; there may be a threshold forglutamine size beyond which it becomes the predominant toxic moiety.

And what of the aggregates? Do they induce neuronal degeneration? The factthat nuclear inclusions are present in vulnerable neurons supports the notion thattheir presence is relevant to pathogenesis. However, both animal and cell culturestudies indicate strongly that aggregation is neither necessary nor sufficient forneuronal dysfunction. It may be that mutant proteins are most toxic when roamingfreely, and that the aggregates themselves represent the cell’s effort to corral thetoxic strands into a more innocuous clump. Three lines of evidence support thismodel: (a) A variant of expanded ataxin-1 that does not aggregate still inducesPurkinje cell pathology and the ataxic phenotype in mice; (b) inhibition of aubiquitin-conjugating enzyme in cell culture abolishes huntingtin aggregation butenhances its toxicity; and (c) overexpression of full-length huntingtin can lead toselective neuronal loss, but aggregates are rare in degenerating neurons and moreprevalent in cells that have not yet manifested pathology. It is possible that overdecades the cell, no longer able to cope with accumulating mutant polyglutamineproteins (because of impairment of the proteolytic machinery), eventually suc-cumbs to the large inclusions, which then disrupt cellular functions at the latterstages of pathogenesis. It should also be noted that cells under stress show alteredtranslocation of proteins to the nucleus, i.e. proteins that are normally cytoplasmicbecome nuclear (Wilkinson & Millar 1998). This raises a more fundamental ques-tion: Is the nuclear localization of expanded polyQ proteins pathogenic or indic-ative of a stressed cell?

Notwithstanding evidence that aggregates are likely not the first step in patho-genesis, it is striking that all polyglutamine diseases and possibly other neuro-degenerative diseases such as Alzheimer, amyotrophic lateral sclerosis, andParkinson disease involve the gradual accumulation of mutant proteins in thenuclei, cytoplasm, or extracellular space. Misfolded or otherwise altered in con-formation because of the mutation, these proteins may all interact with otherproteins in an aberrant fashion, undergo altered processing (huntingtin, amyloidprecursor protein), and possibly decreased ubiquitin-dependent degradation.Future research investigating the effects of these mutations on protein interac-tions and turnover together with studies aimed at enhancing their proper foldingand degradation could mitigate the disease process and possibly delay onset byyears.

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ACKNOWLEDGMENTS

We wish to thank V Brandt and C Cummings for critical input on the manuscript,and we gratefully acknowledge the support of the NIH/NINDS and the HowardHughes Medical Institute. Because of space constraints, we were unable to citeall the papers that merit note; we apologize for these sometimes arbitraryomissions.

Visit the Annual Reviews home page at www.AnnualReviews.org.

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Annual Review of Neuroscience Volume 23, 2000

CONTENTSCortical and Subcortical Contributions to Activity-Dependent Plasticity in Primate Somatosensory Cortex, Edward G. Jones 1Microtubule-Based Transport Systems in Neurons: The Roles of Kinesins and Dyneins, Lawrence S. B. Goldstein, Zhaohuai Yang 39Apoptosis in Neural Development and Disease, Deepak Nijhawan, Narimon Honarpour, Xiaodong Wang 73Gain of Function Mutants: Ion Channels and G Protein-Coupled Receptors, Henry A. Lester, Andreas Karschin 89The Koniocellular Pathway in Primate Vision, Stewart H. C. Hendry, R. Clay Reid 127Emotion Circuits in the Brain, Joseph E. LeDoux 155Dopaminergic Modulation of Neuronal Excitability in the Striatum and Nucleus Accumbens, Saleem M. Nicola, D. James Surmeier, Robert C. Malenka 185Glutamine Repeats and Neurodegeneration, Huda Y. Zoghbi, Harry T. Orr 217Confronting Complexity: Strategies for Understanding the Microcircuitry of the Retina, Richard H. Masland , Elio Raviola 249Adaptation in Hair Cells, Ruth Anne Eatock 285Mechanisms of Visual Attention in the Human Cortex, Sabine Kastner and Leslie G. Ungerleider 315The Emergence of Modern Neuroscience: Some Implications for Neurology and Psychiatry, W. Maxwell Cowan, Donald H. Harter, Eric R. Kandel 343Plasticity and Primary Motor Cortex, Jerome N. Sanes, John P. Donoghue 393Guanylyl Cyclases as a Family of Putative Odorant Receptors, Angelia D. Gibson, David L. Garbers 417Neural Mechanisms of Orientation Selectivity in the Visual Cortex, David Ferster, Kenneth D. Miller 441Neuronal Coding of Prediction Errors, Wolfram Schultz, Anthony Dickinson 473Modular Organization of Frequency Integration in Primary Auditory Cortex, Christoph E. Schreiner, Heather L. Read, Mitchell L. Sutter 501Control of Cell Divisions in the Nervous System: Symmetry and Asymmetry, Bingwei Lu, Lily Jan, Yuh-Nung Jan 531Consciousness, John R. Searle 557The Relationship between Neuronal Survival and Regeneration, Jeffrey L. Goldberg, Ben A. Barres 579Neural Representation and the Cortical Code, R. Christopher deCharms, Anthony Zador 613Synaptic Plasticity and Memory: An Evaluation of the Hypothesis, S. J. Martin, P. D. Grimwood, R. G. M. Morris 649Molecular Genetics of Circadian Rhythms in Mammals, David P. King, Joseph S. Takahashi 713Parallel Pathways for Spectral Coding in Primate Retina, Dennis M. Dacey 743Pain Genes?: Natural Variation and Transgenic Mutants, Jeffrey S. Mogil, Lei Yu, Allan I. Basbaum 777

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