Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, 3, 329-340 329 The Molecular Pathology of Huntington’s Disease (HD)

David C. Rubinsztein*

Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2XY, UK

Abstract: Huntington’s disease (HD) is one of nine known neurodegenerative conditions caused by CAG trinucleotide repeat expansions that are translated into abnormally long polyglutamine tracts in the mutant protein. This review describes the clinical and pathological features of HD and considers the genetic and transgenic/knockout mouse data supporting gain-of-function vs. loss-of-function mechanisms whereby the mutation may cause disease. Intraneuronal aggregates (also known as inclusions) are one of the pathological hallmarks of all of the polyglutamine expansion diseases. A major focus of the review is a detailed consideration of the debate as to whether aggregates/aggregation are pathogenic, deleterious or epiphenomena, drawing on data from cell-based and animal models of different polyglutamine diseases and also from the polyalanine codon reiteration disease, oculopharyngeal muscular dystrophy, which manifests intramuscular nuclear inclusions. I will describe how cells deal with aggregate- prone and misfolded proteins using chaperones and various degradation pathways. Using data from animal and cell-based models, the review considers some of the different but non-mutually exclusive mechanisms whereby the HD mutation may cause disease, including early changes in gene transcription, production of reactive oxygen species, aberrant protein- protein interactions and abnormal cellular susceptibility to glutamate. Understanding possible pathogenic mechanisms for HD has provided a rational basis for intervention strategies, ranging from antibodies/peptides that prevent mutant protein aggregation to drugs that enhance mitochondrial function and protect against excitotoxicity.

SYMPTOMATOLOGY OF HD psychiatric syndrome [5,6]. The rate of suicide has been estimated at 5-10% [7,8]. HD is an autosomal dominant neurodegenerative disease associated with a triad of symptoms, including movement NEUROPATHOLOGY disorders, cognitive deterioration and psychiatric distur- bances. Symptoms of the disease begin insidiously, most HD is characterised by a striking specificity of neuronal commonly between the ages of 35 to 50, but the age of onset loss. The regions most affected are the striatum, where there can vary from early childhood until old age. The disease is is typically 50-60% loss of cross-sectional area from the relentlessly progressive and fatal 15–20 years after the onset caudate nucleus and the putamen in advanced disease. In the of symptoms. Disturbances of motor function are classical striatum, the most sensitive cell population are the medium features of the disease. They include choreiform involuntary spiny neurons, which show dentritic changes including movements of the proximal and distal muscles, and progres- recurving of dentrites and altered density, shape and size of sive impairment of co-ordination of voluntary movements spines. Among spiny neurons, enkephalin-containing [1-3]. In patients with juvenile-onset HD, the signs and neurons projecting to the external globus pallidus externa are symptoms are somewhat different; they include brady- more susceptible compared to substance-P-containing kinesia, rigidity and dystonia, and chorea may be completely neurons projecting to the internal globus pallidus [9,10]. absent. Involuntary movements may take the form of tremor, Although the pathology of HD is most obvious in the and affected children often develop epileptic seizures [4]. striatum, it is likely that much of the degeneration occurs elsewhere - the average brain of someone who has died of The cognitive difficulties usually start with a slowing of HD weighs about 300 g less than normal and the striatal intellectual processes. In contrast to Alzheimer’s disease, atrophy can only account for about 50g of this loss [11]. patients with HD have problems with retrieval of established memory rather than formation of new memories. Cognitive There is loss of cortical volume, which affects predomi- losses accumulate progressively and late-stage patients with nantly the large pyramidal neurons in layers III, V and VI. HD have profound dementia. Emotional disturbances in the While this loss is overt in advanced cases, it may be one of form of depression and manic-depressive behaviour are the earlier changes in the disease [12]. In advanced cases, common features. Personality changes, including irritability, there is also loss of neurons in the thalamus, substantia nigra apathy and sexual disturbances often accompany the pars reticulata and in the subthalamic nucleus. There is also loss of volume in the globus pallidus, which is primarily due to loss of striatal fibre connections, rather than to loss of *Address correspondence to this author at the Department of Medical neurons. Cerebellar atrophy is most frequently reported in Genetics, Cambridge Institute for Medical Research, Wellcome Trust/MRC cases with juvenile onset disease. Fibrillary astrogliosis Building, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2XY, UK; mirrors the loss of neurons as the disease progresses Tel: 01223 762608; Fax: 01223 331206; E-mail: [email protected] (reviewed in [9,10]).

1568-0134/03 $41.00+.00 © 2003 Bentham Science Publishers Ltd. 330 Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 David C. Rubinsztein

Vonsattel and colleagues described a grading system for hemizygous loss of one of the two huntingtin genes has been standardisation of HD pathology based on gross and observed as a result of either a terminal deletion of one microscopical examinations [9]. It is interesting that the chromosome 4 (which includes the HD gene) in patients with mildest category, grade 0, is grossly indistinguishable from Wolf-Hirschhorn syndrome [22], or a balanced translocation normal brains but cell counting can show up to 40% loss of with a breakpoint between exons 40 and 41, physically neurons in the head of the caudate nucleus in such brains. disrupting the HD gene in one female patient [23]. However, this hemizygous inactivation of huntingtin does not cause an GENETICS OF HD HD phenotype. In addition, mice that have only one functioning HD gene do not show features of the disease The HD gene and mutation were identified in 1993 [13]. [24-27]. The gene is located on the short arm of chromosome 4 (4p16.3) and encodes a large protein called huntingtin that A gain-of-function is also supported by observations in contains more than 3000 residues. Exon 1 of the wild-type the related polyQ disease spinobulbar muscular atrophy gene contains a stretch of uninterrupted CAG trinucleotide (SBMA), which is caused by a CAG repeat expansion in the repeats, which is translated into a series of consecutive androgen receptor. Males with SBMA develop progressive glutamine residues, a polyglutamine (polyQ) tract. weakness and muscle atrophy due to loss of their motor Asymptomatic individuals have 35 or fewer CAG repeats nerve supply and mild androgen insensitivity [28]. This and HD is caused by expansions of 36 or more repeats [14]. contrasts with the effects of point mutations elsewhere in the There is an inverse relationship between CAG repeat number gene, which can result in severe androgen insensitivity but and the age of onset of symptoms; the greater the number of never cause a neuromuscular disease. Indeed, the CAG repeats, the earlier the age of onset [15]. Most adult neuromuscular features of SBMA are not even seen in a onset cases have 40-50 CAG repeats, whereas expansions of patient with a complete androgen receptor gene deletion. >55 repeats frequently cause juvenile-onset disease. This gain-of-function model was elegantly demonstrated Incomplete penetrance has been observed in individuals with th th in a mouse model, where 146 CAG repeat sequence was 36-39 repeats - some individuals in their 9 and 10 decades inserted into the hypoxanthine phosphoribosyltransferase with alleles in this size range have no signs, symptoms or (HPRT) gene, which is not involved in any CAG-repeat gross neuropathological features of HD [14,16]. About 70% disorders. While previous work had shown that inactivation of the variance in the age at onset of HD can be accounted of the HPRT gene in mice does not cause deleterious effects, for by CAG repeat number. Family studies suggest that a these mutant mice produced a polyglutamine-expanded form component of the residual variance not associated with the of the HPRT protein and developed a late-onset neurological CAG repeat number, may be accounted for by additional phenotype that progressed to premature death [29]. genetic factors [17]. One possible candidate modifying gene is the GluR6 kainate receptor [18] – the effect that we Wild-type huntingtin is widely expressed and is found reported of genotypes at this locus on age-at-onset of HD, mainly in the cytoplasm, where, in neurons, it is associated after accounting for the CAG repeat length, has been with vesicle membranes and microtubules. Huntingtin replicated by MacDonald et al. [19]. appears to be associated with clathrin via the huntingtin interacting protein Hip-1 [30-34]. Wild-type huntingtin is HD has been considered to be a true dominant disease necessary for development, as homozygous knockout mice where the severity is similar in patients with one or two show embryonic lethality [24-26]. This may be related to mutant alleles. However, this interpretation is based on data observations that overexpression of wild-type huntingtin from only a few homozygote cases (reviewed in [20]). A protects cells against a variety of apoptotic stimuli [35], recent study on the largest cohort of homozygotes assessed probably by inhibiting pro-caspase-9 processing [36]. Wild- to date suggests that while the age at onset is similar between type huntingtin can protect against apoptosis in the testis of homozygotes and heterozygotes matched for the larger mice expressing full-length huntingtin transgenes with repeat size, the progression of disease may be more rapid in expanded CAG repeats [37] and overexpression of wild-type homozygotes [20]. One interpretation of these data is that huntingtin significantly reduced the cellular toxicity of CAG repeat length is a more important factor than gene mutant HD exon 1 fragments in both neuronal and non- dosage. However, since cleavage of the full-length protein to neuronal cell lines [38]. This suggests that overexpression of an N-terminal toxic fragment may be a key step in the wild-type huntingtin can be protective in different cell types pathogenic pathway (see later), another possibility is that the and that it can act against the toxicity caused by mutant processing events leading to the formation of this toxic huntingtin. fragment or fragments is rate-limiting. In addition to protecting against polyglutamine expansion HD is a member of a growing group of diseases caused toxicity, wild-type huntingtin may also play a role in HD by by CAG repeat/polyglutamine expansions that include upregulating the transcription of brain-derived neurotrophic spinocerebellar ataxias types 1,2,3,6, 7 and 17, spinobulbar factor (BDNF) [39]. This activity appears to be reduced in muscular atrophy (SBMA) and dentatorubral-pallidoluysian the disease state and Zuccato and colleagues [39] have atrophy (DRPLA) (reviewed in [21]). proposed that this may contribute to HD pathology by HD is an autosomal dominant condition – one mutated reducing neurotrophic support from cortical neurons to gene is sufficient to cause the disease, in spite of the striatal neurons. presence of a normal gene inherited from the other parent. Gervais et al. have proposed that the polyglutamine Genetic and transgenic data suggest that the mutation confers expansion in huntingtin may cause disease as it reduces a novel deleterious function on the protein. In humans, The Molecular Pathology of Huntington’s Disease Curr. Med. Chem. – Imumn., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 331 binding of the mutant protein to Hip-1 [40]. Free Hip-1 binds many parallel and randomly oriented fibrils. Intraneuronal to Hippi (Hip-1 protein interactor) forming pro-apoptotic aggregates are a pathological common denominator for Hippi-Hip-1 heterodimers. This heterodimer can recruit polyglutamine diseases, since similar structures are seen in procaspase-8 into a complex of Hippi, Hip-1 and procaspase- brains from cases with all of these diseases. Inclusions in 8, and launch apoptosis through components of the juvenile onset cases occur predominantly in the nucleus, ‘extrinsic’ cell-death pathway. Gervais et al. argued that the while a greater proportion of aggregates in adult-onset cases polyglutamine expansion in mutant huntingtin liberates Hip- are cytosolic [46]. This may be a factor accounting for some 1 so that it can form a caspase-8 recruitment complex with of the differences in presentation between adult-onset and Hippi [40]. This process is unlikely to be the main or only juvenile HD. mechanism for disease - HD-like pathology is seen in A causal role for these inclusions in polyglutamine transgenic mouse models expressing both full-length and N- disease pathology has been suggested, as they appear before terminal mutant huntingtin constructs in mice with two the signs of disease in a transgenic mouse expressing exon 1 endogenous huntingtin genes and while mutant huntingtin of the HD gene with expanded repeats [43]. In addition, the does not bind Hip-1 as well as the wild-type protein it does numbers of inclusions in the cortex of HD patients correlates still bind Hip-1 [41]. Therefore, these transgenic mice are with CAG repeat number [47]. Inclusion formation in vitro expected to have more Hip-1 bound to huntingtin, compared in cultured cells also correlates with susceptibility to cell to wild-type mice. death [48-52]. In striatal cultures, neuritic aggregates block protein transport in neurites, and cause neuritic degeneration POSSIBLE DISEASE MECHANISMS before nuclear DNA fragmentation occurs [53]. These A wide range of different mechanisms have been authors suggested that the early neuropathology of HD may proposed to explain how the HD mutation causes cell originate from axonal dysfunction and degeneration associa- dysfunction and death. I will discuss selected processes that I ted with huntingtin aggregates. Reduction of polyglutamine believe to be important. There are possible connections inclusion formation, by overexpression of heat shock between many of the processes discussed, based on proteins and bacterial and yeast chaperones (that are unlikely knowledge from other systems (e.g. reactive oxygen species to directly affect cell death pathways), is also associated with and apoptosis, energy production and ubiquitin-proteasome decreased cell death in vitro [54-56] (Fig. 1A). function etc.). However, since the roles of many of the proposed mechanisms as primary factors in pathogenesis in HD patient brains have not been conclusively demonstrated, Protein monomer Monomer Monomer I believe that it would be unwise and possibly misleading to speculate on too many connections between putative pathogenic pathways. It is possible and indeed likely that a number of processes are acting in concert in HD as the mutation results in a chronic insult acting over decades. Furthermore, there may some pathways that are shared between the different polyglutamine diseases and even Aggregation Aggregation Death Death between different diseases associated with abnormal intracellular protein aggregation.

HUNTINGTIN AGGREGATES In 1996, Bates and co-workers created a transgenic mouse which expressed exon 1 of the human HD gene containing different numbers of CAG repeats, under the Death/dysfunction Aggregation control of the human huntingtin promoter [42]. Mice expressing 18 CAG repeats developed normally and remained healthy. By contrast, mice that expressed 113-156 Fig. (1A). Formal possibilities of the relationship of aggregation CAG repeats developed progressive neurological symptoms to cell dysfunction/death in HD. together with some other clinical features similar to HD. These mice developed intraneuronal aggregates (inclusions) On the left, aggregation leads to death; in the middle, aggregation is in nuclei and in neuronal processes [43]. Similar aggregates an epiphenomenon unconnected to dysfunction/death; on the right, were identified in post mortem human HD brains in cortical death/dysfunction causes aggregation. Experiments in cell culture and striatal neurons [44] and in dystrophic neurites [45], but and in vivo support the model on the left – chaperones and not in the globus pallidus or cerebellum [46]. In HD brains antibodies that reduce aggregation but do not affect cell death these inclusions comprise truncated derivatives of the mutant pathways reduce cell death [54-58,80]. On the other hand, reduction proteins, which only appear to be recognised by antibodies to of cell death (e.g. with caspase inhibitors) does not reduce epitopes close to the expanded polyglutamines. The aggregation of HD exon 1 constructs in cell culture [166]. The inclusions are spherical, ovoid or elliptical in shape and are central model is unlikely, since it predicts that chaperones would concentrated in neurons in areas of the brain which reduce aggregation but increase cell death. degenerate in HD. Electron microscopy revealed that the inclusions were heterogeneous in composition and contained A number of recent studies strengthen the claim for a mixture of granules, straight and tortuous filaments and aggregates playing a role in disease. In the first, Koshnan et 332 Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 David C. Rubinsztein al. studied a panel of monoclonal antibodies that they Klement et al. suggested that aggregate formation may developed to various epitopes on huntingtin [57]. In cell not be a prerequisite for pathology, since similar Purkinje culture models they showed that the antibody that cell pathology and ataxic phenotypes were observed in mice significantly inhibited aggregation also inhibited cell death. expressing the SCA1 gene with 77 repeats, with or without a In contrast, the two antibodies that stimulated huntingtin deletion of the ataxin-1 self-association domain [64]. The aggregation increased cell death. Kazantsev and coworkers mice expressing transgenes without the self-association have developed a related approach by designing suppressor domain had no intranuclear inclusions in their Purkinje cells, polypeptides that bind mutant Htt and interfere with the in contrast to the mice expressing the entire mutant gene. process of aggregation in cell culture [58]. In a Drosophila This conclusion may be simplistic, since later reports suggest model, the most potent suppressor inhibits both adult that the phenotype in the mice with the deleted self- lethality and photoreceptor neuron degeneration. Further- association domain progressed significantly more slowly more, this work showed a strong correlation between the than in mice with the full-length mutant gene [65]. Thus, development of aggregates and cell death in the Drosophila inclusion formation may affect disease progression. These model [58]. data need to be interpreted carefully, since no data were Wetzel and colleagues provided further support for a role presented for mice with normal repeat lengths containing of aggregates in experiments where they synthesized polyQ deletions of the self-association domain in SCA1 and Perutz suggested that removal of the self-association domain from peptides in vitro and introduced these into cells in culture the normal SCA1 gene may itself cause abnormal protein [59]. The addition of a nuclear localisation signal to the peptides resulted in nuclear aggregate formation and cell folding and a SCA-like phenotype [66]. death [59]. A pathological role for aggregation is also Lansbury and colleagues have suggested that the consistent with the observation that the predicted lag times aggregation process itself may be toxic, possibly with the for polyQ aggregation based on in vitro data correlate with most toxic species being oligomers/microaggregates, rather the age-at-onset/CAG repeat curves seen in HD [60,61]. than aggregates that are easily visible using light microscopy The failure to always see a correlation between inclusion [67]. Microaggregates have been identified by electron formation and cell death/pathology in HD models has led microscopy in the YAC transgenic mouse model made by Hayden’s lab [68]. They can be around 50nm in diameter some to question the role of aggregates. Saudou et al. observed a dissociation between inclusion formation and cell [68]. Such microaggregates may be very difficult to detect or death in primary striatal neurons after inhibition of exclude in humans or animal models. A typical cortical neuron is about 25 m diameter and EM sections are usually ubiquitination [62]. They tested the effect of inhibiting u 50 nm thick – 500 sections per neuron. Thus, if there were ubiquitination on inclusion formation and cell viability. The one 50nm microaggregate per cell (or five microaggregates inclusions seen in patients’ brains and in in vivo and in vitro in 20% of cells), one would only see such an aggregate in models of polyglutamine diseases are ubiquitinated. This about 1/500 EM cell/sections. Thus, the failure to observe process is used by cells to tag misfolded proteins and target such microaggregates using EM does not exclude their them for degradation. Saudou et al. showed that expression existence even in the majority of neurons. Recent bio- of a dominant-negative ubiquitin-conjugating enzyme mutant physical data from Ross and colleagues have confirmed that reduced the proportion of cells with aggregates but increased mutant huntingtin fragments form protofibril and globular cell death caused by huntingtin constructs containing intermediates that eventually become aggregates [69]. expanded repeats, in the cells remaining on the dishes after 6 days [62]. However, inhibition of ubiquitination also resulted Further work needs to be done in order to clarify the in increased cell death in cells expressing “wild-type” controversy regarding the pathogenic role of aggregates. huntingtin constructs with 17 repeats. It is not clear if the This may have a much wider relevance, since the results were simply due to an additive effect of the polyglu- phenomenon of ubiquitinated inclusion bodies is not tamine insult and the defective ubiquitination, resulting in confined to polyglutamine diseases. Indeed, it seems to be an earlier death of cells expressing mutant huntingtin with emerging theme in many other late-onset neurodegenerative smaller inclusion loads. This scenario would result in fewer diseases like Alzheimer’s disease, Parkinson’s disease, adherent cells with visible inclusions after the 6 day amyotrophic lateral sclerosis and prion diseases [70]. experiment – dead cells do not remain attached to culture Recently, another class of codon reiteration disease dishes for long. associated with b-sheet aggregates has been discovered, exemplified by autosomal dominant oculopharyngeal Cummings and colleagues showed that a mutation in the muscular dystrophy (OPMD) [71]. OPMD usually presents E6-AP ubiquitin ligase reduced nuclear inclusion frequency in the sixth decade with progressive swallowing difficulties, but accelerated polyglutamine-induced pathology in SCA1 ptosis and proximal limb weakness. Its pathological hallmark mice [63]. While these data suggest that large visible is the presence of nuclear filamentous inclusions in skeletal inclusions may not be required for cell death, the authors muscles. OPMD is caused by the abnormal expansion of a consider other possibilities which are consistent with a (GCG) trinucleotide repeat in the coding region of the pathological role for inclusions. For instance, ubiquitination n poly(A) binding protein 2 gene ( ): a (GCG) repeat is of ataxin-1 may not be E6-AP-dependent. The deletion of PABP2 6 this enzyme may affect the turnover of other proteins, which expanded to (GCG)8-13 in patients [71]. In PABP2, (GCG)6 at abnormally high steady-state levels may enhance the codes for the first 6 alanines in a homopolymeric stretch of ten alanines. Thus, disease is associated with expansions of cellular sensitivity to the SCA1 mutation (or aggregates) [63]. 12 or more uninterrupted alanines in this exclusively nuclear protein. A number of other autosomal dominant diseases, The Molecular Pathology of Huntington’s Disease Curr. Med. Chem. – Imumn., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 333 including synpolydactyly and a form of cleidocranial dysplasia, are also caused by polyalanine expansions in Monomer nuclear proteins (the transcription factors HOXD13 and CBFA1) [72,73]. A gain-of-function mechanism for polyalanine expansion mutations in OPMD would be consistent with experiments from our lab [74] and from Rouleau’s group [75]: cells overexpressing long alanine repeats, either in the context of Aggregation-prone monomer Death PABP2 (17 alanines - A17) or fused to green fluorescent protein (GFP) (A19-37), show aggregate formation and increased cell death, compared to wild-type constructs which did not aggregate (PABP2 - A10; GFP – A7). PABP2 regulates the elongation of poly(A) tails and polyadenylation of mRNAs and this function is not impaired in OPMD myoblasts [76]. Further evidence against a loss of function mechanism in OPMD comes from studies of the Drosophila Aggregation [non-toxic] PABP2 orthologue [77], which lacks the first 54 residues of the mammalian proteins [71,78]. Despite lacking the domain that is mutated in OPMD (starting at residue 2 of human Fig. (1B). A further model for the relationship of aggregation to PABP2), the Drosophila protein stimulates poly(A) cell dysfunction/death in HD. polymerase and is able to control poly(A) length in Here the mutant protein can exist in two monomer conformations. reconstituted mammalian polyadenation systems [77]. The one conformation is benign, the other is toxic and would be A causal role for aggregation in the cell death in tissue aggregate-prone. In this model, chaperones may prevent the culture models of OPMD is supported by complementary conversion of the benign to the toxic monomeric conformation. data from our lab and Rouleau’s group. Rouleau and However, the data on Congo Red suggest that it prevents the colleagues found that oligomerization of PABP2 is mediated conversion of protofibrils to mature fibrils and therefore acts in the by two potential oligomerization domains (ODs) – deletions aggregation process [69]. Since Congo Red is protective in cell in either of these domains inactivated oligomerization of models and in vivo, this argues for a deleterious. role for mutant PABP2 and also reduced the cell death caused by this aggregation in disease pathogenesis [81]. protein [79]. In our experiments, we showed that a number of different chaperones, including bacterial and yeast derived Recent data suggest that interruption of aggregation may constructs reduced the aggregate formation and cell death indeed be beneficial. Congo red blocks the conversion of caused by mutant PABP2. Since two of the chaperones that mutant huntingtin protofibrils into mature fibrils [69] and were protective could not protect against the pro-apoptotic Sanchez et al. showed that Congo red reduced aggregation and cell death in HD cell models [81]. The latter study also effects of H2O2 or staurosporine, it is likely that the protection against cell death was related to their effects on reported that infusion of Congo red into an HD mouse model reducing PABP2 aggregation or misfolding [80]. While the by the intraperitoneal and intracerebroventricular routes mechanisms, structures and contents of polyA aggregates improved survival, weight loss and motor function, may differ from polyQ aggregates to some extent, it is compared to untreated mutant mice [81]. These data provide tempting to speculate that the process of intracellular protein important insights into the role of aggregation of aggregation may perturb some similar processes. I believe polyglutamine disease pathology and suggested that the that this is an important hypothesis to consider and test, beneficial effects of Congo red were due to its anti-aggregate given the large number of different diseases associated with properties. Unfortunately, the therapeutic potential for this phenotype. Congo red in HD may be minimal due to its very poor blood- brain barrier permeability [82]. While the effects of chaperones on aggregation and cell death strongly support a correlation between the appearance THE ROLE OF UBIQUITIN, PROTEASOMES AND of aggregates and cell death/dysfunction, they may be CHAPERONES compatible with another theoretical model where aggregation would not necessarily be causally related to cell Cells have a complex array of chaperone proteins that death. The mutant monomeric proteins may exist in two sets assist in the folding of normal proteins, and recognition and of conformations: a properly-folded non-toxic species and an handling of abnormally-folded or aggregate-prone proteins. aggregate-prone toxic form(s) (Fig. 1B). If the Several of the heat shock proteins (HSPs) are involved in chaperones/antibodies promote the conversion of the toxic modulation of protein folding pathways, promoting proper aggregate-prone monomers to the non-toxic form, then one protein assembly in an ATP-dependent manner and would see a reduction of both aggregation and cell death in preventing misfolding and aggregation under both normal the presence of chaperones. This scenario may be difficult to and stress conditions [83]. HSP70 and HSP40 family distinguish from the model where aggregation is toxic, members are associated with huntingtin exon 1 aggregates in unless one can identify a means of distinguishing and cell models [52,84,85]. Overexpression of the human HSP40 quantifying different monomeric forms of these aggregation- homologues HDJ-1 and HDJ-2 and/or HSP70 family prone proteins from cell models or in vivo experiments. members in vitro reduced aggregation of ataxin-1, ataxin-3 and the androgen receptor with expanded polyglutamine 334 Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 David C. Rubinsztein proteins in cell models [54,55,86]. However, overexpression enzymes normally involved in crosslinking of glutamine of HDJ-2 did not alter the aggregation of exon 1 of residues in different proteins. Huntingtin is a substrate of huntingtin containing 72 repeats in two neuronal-precursor transglutaminase in vitro and the rate of the reaction cell lines and actually increased aggregation in COS-7 cells increases with length of the polyglutamine tract [95]. An [52]. Thus, overexpression of this HSP may increase or expanded polyglutamine stretch could result in increased decrease aggregates in different cell types or with different cross-linking between mutant huntingtin and itself or other polyglutamine containing proteins. proteins and precipitation with slow intraneuronal An important intracellular pathway for reducing the accumulation of huntingtin aggregates. The activity of levels of misfolded proteins is the ubiquitin-proteasome transglutaminases has been seen to be increased in HD patients compared to normal individuals [96], and it has been pathway. Intranuclear inclusions seen in patients’ brains and proposed that the level of transglutaminase activity affects in in vitro models of polyglutamine diseases are ubiquitina- ted and sequester components of the proteasome [52,54,87]. the age of onset in individuals with equal numbers of CAG Proteasome inhibitors increase steady-state-levels of such repeats [97]. However, tissue transglutaminase is not required and does not facilitate huntingtin aggregate proteins [88] consistent with the hypothesis that the protea- some degrades proteins containing expanded polyglutamine formation [98], although this enzyme does appear to modify tracts. Another major route for degradation of proteins with proteins that associate with truncated mutant huntingtin [99]. expanded polyglutamine tracts is the autophagy-lysosome The data of Chun et al.[99] suggest that this modification is more likely to involve polyamination than crosslinking. It is pathway [88]. We suggested that autophagy may be particularly relevant to aggregate-prone species [88]. Indeed, tempting to speculate that this phenomenon may play a role autophagy may be the natural default pathway for clearance in HD pathology, but further work is required. A role for of aggregated proteins, since recent data suggest that soluble, transglutaminase is aggregate formation is however but not aggregated polyQ expansion-containing proteins, can supported by the observation of reduced aggregate formation and disease progression in an HD exon 1 transgenic mouse be degraded by the proteasome [89]. This may arise because a protein must be unfolded before entering the central 20S treated with the transglutaminase inhibitor cystamine [100]. proteolytic subunit of the proteasome complex. If redistribu- These data do not allow one to draw conclusions about the tion of proteasomes into inclusions depletes the neuron of pathogenic role of aggregates, as cystamine has been shown functional proteolytic activity, the results would be to block caspase activation [101]. deleterious. This possibility is supported by the work of Kopito and colleagues, who showed that cells containing 3) Linear Lattice Model polyglutamine (or other) inclusions have impaired protea- A third model for aggregation may be simply that some activities, compared to cells without aggregates [90]. A polyglutamine tracts are sticky and that longer polyQ major role of the proteasome is to degrade short-lived stretches have a greater multiplicity of binding sites, proteins, like some transcription factors. The concentrations allowing more intermolecular interactions [102]. of some of these proteins needs to be carefully controlled, as they are critical regulators of cellular metabolism, hence the THE TOXIC FRAGMENT HYPOTHESIS short half-lives. If the proteasome is impaired, the levels of such short-lived proteins rise abnormally and apoptosis often It appears that full-length huntingtin may be cleaved to ensues. form an N-terminal fragment containing the glutamine repeats that is more toxic and which is also more prone to PROPOSED MECHANISMS OF PROTEIN AGGRE- aggregation, compared to the full-length protein [103]. In GATION HD knock-in mutant mice, there appears to be nuclear localization of full-length mutant huntingtin, followed by the Three non-mutually exclusive mechanisms have been formation of an N-terminal fragment and insoluble aggregate proposed to explain the aggregation of mutant huntingtin. formation [104]. These changes show a repeat length- dependency and precede any clinical features of disease. Li 1) The Polar Zipper Model et al. compared the patterns of inclusion formation in HD transgenic mice expressing only exon 1 with expanded One possibility is that the normal protein conformation is repeats to knock-in mice expressing full-length mutant destabilized by the presence of the expanded polyglutamine mouse huntingtin homologues [105]. The former develop tract, which, in turn, leads to abnormal protein-protein inclusions in most brain regions, while the knock-in mice interactions and the formation of insoluble b-pleated sheets develop inclusions that are confined to the striatum [105]. by linking b-strands together into barrels or sheets via While this study was not perfectly controlled, since the hydrogen bonding, forming so-called polar zipper structures transgenes are expressed by different promoters (the mouse [91,92]. This model is supported by in vitro studies, which and human HD promoters differ [106]) and inclusion showed that purified recombinant proteins with glutamine formation is not a direct measure of cell dysfunction/death, it expansions formed stable aggregates exhibiting polarisation supports the hypothesis that tissue-specificity of huntingtin with Congo Red, characteristic of proteins that have b- proteolysis may be an important determinant of which cell pleated sheets [93]. populations are affected. The cleavage/toxic fragment model described above has recently been challenged by data 2) The Role of Transglutaminases suggesting that wild-type huntingtin is more susceptible to Aggregation of mutant huntingtin products has been cleavage compared to mutant huntingtin [107]. While further postulated to be mediated by transglutaminases [94], work will be required to resolve this important controversy, The Molecular Pathology of Huntington’s Disease Curr. Med. 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Hayden and colleagues have shown that huntingtin cleavage caspases, which produce more toxic fragments in a positive precedes neurodegeneration in a YAC transgenic mouse feedback loop, ultimately resulting in cell death [117]. model expressing full-length mutant human huntingtin and In a transgenic HD mouse model, expression of a that cleavage is apparent in early-stage human disease [108]. dominant negative caspase-1 mutant that inhibits the activity If cleavage is a rate-limiting step in HD pathogenesis, then of caspase-1, delays onset of symptoms and extends survival. cell-type differences in the complement of the relevant active Direct intraventricular administration of a caspase-1 inhibitor proteases coupled with differences in gene expression may also delayed disease progression and mortality in the mouse account for the differential cell-type vulnerability to the HD model of HD [118]. mutation, compared to the other polyQ diseases (some others which are also associated with cleavage of the mutant Caspase 8 appears to be a necessary mediator of death in protein). primary rat neurons transiently transfected with a construct expressing an expanded polyglutamine repeat [119]. This CELL DEATH IN HD study showed that caspase 8 is recruited into inclusions and suggested that it is activated as part of the HD disease Apoptosis is programmed cell death – a conserved process. cellular mechanism initiated by diverse stimuli that leads to activation of aspartate-specific proteases (caspases), Another pathway which may be relevant to HD culminating in DNA fragmentation and cell death. A subset pathogenesis is the activation of c-Jun amino-terminal of neurons and glia in the neostriatum of post-mortem HD kinases. This pathway is induced by a variety of oxidative brains show DNA strand breaks (as assayed with terminal stress stimuli and can induce apoptosis. Expression of mutant transferase-mediated deoxyuridine triphosphate–biotin nick- huntingtin with expanded polyglutamine repeats in rat end labelling (TUNEL) [109-111]. Isolated medium spiny hippocampal neuronal cells in vitro has been shown to neurons were stained, most intensely in the putamen, stimulate JNKs activity and induce apoptotic cell death, followed by the globus pallidus and caudate. Labelling was whereas expression of normal huntingtin had no toxic effect. also increased in more advanced cases of the disease. There JNK activation preceded apoptosis and co-expression of a appears to be a positive correlation between the number of dominant negative mutant form of the stress signalling CAG repeats in HD and the degree of nuclear fragmentation kinase (SEK1) almost completely blocked JNK activation in the HD striatum [112]. However, TUNEL recognises and apoptosis [120]. some forms of necrosis and is thus not selective for apoptosis [113]. EARLY CHANGES IN GENE EXPRESSION Apoptosis may not be the major mode of cell death in A number of studies have used cDNA expression and HD. Davies and colleagues found that the R6/2 HD Affymetrix arrays to interrogate early changes in gene transgenic mouse lines do develop late onset expression in HD transgenic mice [121-125] and in stable neurodegeneration within the anterior cingulated cortex, inducible cell models [85]. The expression of many classes dorsal striatum, and in the Purkinje neurons of the of genes that could participate in neurodegeneration are cerebellum [114]. Dying neurons characteristically exhibited perturbed, including neurotransmitter receptors and proteins neuronal intranuclear inclusions, condensation of both the involved in different signalling pathways. One group of cytoplasm and nucleus, and ruffling of the plasma membrane genes that are downregulated in mutant vs. wild-type while maintaining ultrastructural preservation of cellular mice/cells are those that are controlled by cAMP response organelles. These cells do not develop blebbing of the elements (CRE) [85, 122]. A reduction of CRE-mediated nucleus or cytoplasm, apoptotic bodies, or fragmentation of transcription is also likely in human HD, since reduced DNA. Neuronal death occurs over a period of weeks not levels of the CRE-responsive genes, somatostatin, hours. Degenerating cells of similar appearance were corticotrophin releasing hormone, proenkephalin and observed within these same regions in brains of patients who substance P, are seen in HD vs. control brains, even in early had died with HD. Thus, these workers suggested that the stages of the disease [126-129]. Such genes may be relevant mechanism of neuronal cell death in both HD and a to the increased susceptibility to cell death and decreased transgenic mouse model of HD is neither by apoptosis nor by neurite outgrowth seen in HD and related cell models, as necrosis [114]. However, a failure to observe apoptotic cells these phenotypes can be partly attenuated by stimulating may be a function of their rapid clearance. Therefore, it is CRE-mediated transcription by overexpressing not clear which cell death pathway(s) are operating in HD. transcriptional co-activators like CRE-binding protein (CREB) or TAF 130, or by treating cells with cAMP or Caspases are a family of cysteine proteases which are II forskolin (which stimulates adenylyl cyclase) [85, 130-132]. activated in apoptosis. A number of studies have suggested a role for these proteases in polyglutamine diseases. During Decreased CRE-mediated transcription may also be apoptosis, caspase-3 cleaves structural and nuclear proteins, important in relation to neurite outgrowth, which may impact as well as other caspases [115]. Caspase-3 also specifically on cell dysfunction that occurs in the early stages of polyQ cleaves huntingtin and long polyglutamine sequences make diseases prior to cell death [85,133]. This may be relevant in huntingtin more susceptible to this cleavage [116]. In vitro vivo, since CREB-mediated signalling is crucial for long- studies suggest that N-terminal cleavage products of mutant term potentiation (LTP), the synaptic analogue for memory huntingtin are more toxic and more prone to aggregate [134]. LTP is impaired in a number of HD mouse models formation than the full-length protein. Toxic fragments from and memory difficulties are a feature of HD [135, 136]. proteins with polyglutamine expansions may further activate 336 Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 David C. Rubinsztein

There are a number of potential non-mutually exclusive further supported by the finding that genotypes at the GluR6 mechanisms that may act upstream of CREB in polyQ kainate receptor locus may modify the age-at-onset of diseases. Decreased adenylyl cyclase activity has been symptoms independently of the effect of the CAG repeat observed in HD transgenic mice [122]. It is possible that number [18,19]. A contribution of excitotoxicity to HD some of these changes may be mediated by the increased pathology is supported by Zeron et al., who have shown that levels of protein phosphatase 2A suggested by our mutant full-length huntingtin enhances the excitotoxic cell expression assays [85]. This enzyme can deactivate death in HEK293 cells expressing NR1A2B N-methyl- D- phosphorylated CRE-binding protein (CREB) [137], one of aspartate (NMDA) glutamate receptors after treatment with the main transcription factors that bind to CRE elements. 1mM glutamate [150]. This group have gone on to show that Another appealing model invokes the sequestration of medium spiny neurons in a full-length mutant huntingtin coactivators like CREB-binding protein (CBP) and TAF II130 YAC transgenic mouse are particularly prone to NMDA- by mutant polyQ stretches into inclusions, as these co- mediated cell death, compared to wild-type animals. This activators are important positive regulators of CRE-binding effect could be abolished with an NR2B NMDA-receptor protein (CREB)-mediated transcription. CBP has been subtype specific antagonist [151]. observed in aggregates in spinobulbar muscular atrophy Excitotoxicity in HD may be the result of a reduced [130], HD and DRPLA [132, 138] and TAFII130 in SCA3 threshold for glutamate toxicity that would occur in neurons and DRPLA [131]. It may be difficult to unravel the relative with compromised energy metabolism, causing otherwise importance of these different mechanisms. normal levels of this excitatory neurotransmitter become Transcription in HD may also be more widely affected, toxic [152,153]. A role for mitochondrial dysfunction in HD as the mutant protein binds to the acetyltransferase domains is further supported by recent work from Greenamyre and of histone acetylases like CBP and p300 [139]. This is colleagues who reported that lymphoblast mitochondria from associated with impaired histone acetylation and this defect patients with HD have a lower membrane potential and can be partially reversed in cell culture models by treating depolarize at lower calcium loads than do mitochondria from with histone deacetylase inhibitors. Histone deacetylase controls [154]. Similar defects were observed in brain inhibitors slowed the progression of the disease in a mitochondria from transgenic mice expressing full-length Drosophila HD model [139]and have modest beneficial mutant huntingtin, and this defect preceded the onset of effects in the R6/2 mouse model [140], suggesting that such pathological or behavioral abnormalities by months. drugs may be beneficial in the human disease. Interestingly, Impaired energy metabolism has been observed in the brains histone acetylation appeared normal in the R6/2 mouse of HD patients using nuclear magnetic resonance brains [140]. Thus, the histone deacetylase inhibitors may be spectroscopy [155], positron emission tomography [156] and protecting indirectly [141], as opposed to rescuing a pathway using biochemical methods [157,158]. The relevance of that is significantly compromised in HD. impaired energy metabolism to HD pathology is also suggested by the effects of the toxin 3-nitroproprionic acid Another class of genes that may be downregulated in HD (3-NP), which irreversibly inhibits succinate dehydrogenase, are those controlled by Sp1, a transcription factor that binds an enzyme involved in the tricarboxylic acid cycle and the to GC-rich elements in certain promoters and activates electron transport chain during ATP synthesis. When 3-NP is transcription of the corresponding genes. Li and colleagues administered chronically in low doses to animals it repro- [142] and Dunah et al. [143] showed that polyglutamine duces the slow progressive nature of human HD and the expansion enhances the interaction of N-terminal huntingtin neuropathological and neurological outcomes closely with Sp1. In HD transgenic mice (R6/2) that express N- mimick human HD [159,160]. Humans surviving 3-NP terminal-mutant huntingtin, Sp1 binds to the soluble form of toxicity develop choreiform movements and dystonia mutant huntingtin but not to aggregated huntingtin. Mutant [161,162]. huntingtin inhibited the binding of nuclear Sp1 to relevant target promoters and suppressed their transcriptional Oxidative stress has been implicated in late onset activities in cultured cells. Dunah et al. suggested that neurodegeneration and there is in vivo support for oxidative mutant huntingtin decreased the interactions between Sp1 damage to mitochondrial DNA in HD parietal cortex [163], and TAF II130 and that the combination of these two factors evidence for free radical production in the brains of HD reduced the cellular toxicity of mutant huntingtin [143]. patients and transgenic mice [158, 164] and SOD-2 upregulation in an HD cell model [165]. We have recently EXCITOTOXICITY AND IMPAIRED ENERGY observed that mutant huntingtin causes increased levels of PRODUCTION ROS in neuronal and non-neuronal cells. Our data suggested that ROS contributed to cell death and was not simply a Exitotoxicity refers to death of neurons as a result of consequence of apoptosis because both N-acetyl-L-cysteine exposure to excitatory amino acids, like glutamate. and glutathione in its reduced form suppressed polyQ- Excitotoxic pathways could be important modifiers of HD mediated cell death [166]. Interestingly, the polyQ neuropathology, since animals injected intracranially with expansion-dependent increase in ROS production and excitatory amino acids, such as kainate [144, 145] and toxicity can be suppressed by HSP27. This hsp mediates its quinolinic acid have similar striatal pathology to that seen in protection without reducing aggregate formation [166]. HD [146-148]. HD brains also show a loss of binding sites for excitatory amino acids [149], suggesting that cells It is not clear if mitochondrial dysfunction and/or expressing these receptors are made vulnerable by the oxidative stress are primary changes or a consequence of the mutant HD protein. The role of excitotoxicity in HD is early neuropathological changes in HD. Guidetti et al. have The Molecular Pathology of Huntington’s Disease Curr. Med. Chem. – Imumn., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 337 argued that mitochondrial changes may be secondary events, The availability of a number of different mouse models since measurements of mitochondrial electron transport of HD has provided powerful tools for preclinical testing of Complexes I-IV did not reveal changes in the striatum and therapeutic strategies, since mice have uniform mutations cerebral cortex in symptomatic HD transgenic mice without (similar CAG lengths) and genetic backgrounds. overt neuronal death [133]. The neostriatum and cerebral Furthermore, compounds can be tested in the animals prior cortex in human presymptomatic and pathological Grade 1 to onset of disease and data can be accumulated fairly HD cases also showed no change in the activity of rapidly, given the short lifespan of many of the HD mouse mitochondrial Complexes I-IV. Nevertheless, mitochondrial models. Promising results have been reported with dysfunction, even if it is a secondary event, may impact on minocycline, a tetracycline derivative that inhibits the pathological processes. transcription of caspases and the inducible form of nitric oxide synthase, among other pathways, and creatine, which THERAPEUTIC STRATEGIES can buffer energy levels [178,179]. At present, there is no treatment that can arrest the course Other approaches that may be successful are those which of HD. Recent trials in humans have used remacemide, a inhibit polyglutamine aggregation. Huntingtin aggregation non-competitive NMDA receptor antagonist, and and cell death have been reduced in cell models with human lamotrigine, which blocks voltage-gated sodium channels single chain Fv intracellular antibodies [180], a inhibiting glutamate release – these compounds were polyglutamine antibody IC12 [181], and peptides that selected on the assumption that excitotoxicity plays an interfere with polyglutamine aggregation [182]. important role in HD [167,168]. Coenzyme Q10, an antioxidant and cofactor involved in mitochondrial electron ACKNOWLEDGEMENTS transfer has also been tested on its own and in combination I am grateful to Mrs Denise Schofield for secretarial with remacemide [169-171]. Although these and previous assistance and Dr Janet Davies for critical comments. The trials using baclofen [172] idebenone [173] and vitamin E work in my laboratory on HD is funded by The Wellcome [174] showed no clear effects, it is possible small beneficial Trust, Medical Research Council, The Hereditary Disease effects may have been missed. For instance, the recent Foundation, The Violet Richards Charity and The Isaac “large” trial of remacemide and coenzyme Q10 was designed Newton Trust. with power to detect a 40% slowing of functional decline in a 30 months period on early HD patients [168]. It is possible REFERENCES that such drugs may show clear beneficial effects if used for longer, or in larger studies, and may delay onset of disease if [1] Brandt, J.; Strauss, M.E.; Larus, J.; Jensen, B.; Folstein, S.E. and used in presymptomatic patients. Clearly, the logistical and Folstein, M.F. J. Clin. Neurophysiol., 1984 6, 401. 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