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Does loss of the normal protein function contribute to the pathogenesis of Huntington's disease?

Does loss of the normal protein function contribute to the pathogenesis of Huntington's disease? BioscienceHorizons Volume 8 2015 10.1093/biohorizons/hzv005 Review article Does loss of the normal protein function contribute to the pathogenesis of Huntington’s disease? Heidi Paine Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, England *Corresponding author: St Mary’s Hospital, Praed Street, London W2 1NY, England. Email: heidipaine@doctors.org.uk Supervisor: Nigel Hooper, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, Manchester, M13 9PT, England. Neurodegenerative disorders such as Huntington’s, Alzheimer’s, Parkinson’s and prion diseases are progressive and without a cure. A common finding is one of misfolded protein aggregates, conventionally believed to underlie pathogenesis via a toxic gain of function. Recently, a potential contribution of loss of normal protein function has come under the spotlight. With a focus on huntingtin, the protein involved in Huntington’s disease, this review examines the evidence for the conventional ‘gain of function’ model, before considering the hypothesis that a loss of function contributes to pathogenesis. In support of a primarily toxic gain of function are findings that huntingtin aggregates are neurotoxic in vitro. Additionally, aggregates of mutant huntingtin pro- teins have been detected prior to neuropathological changes, supporting a causal role. However, a dissociation between the neurons containing mutant huntingtin aggregates and those that are most vulnerable in Huntington’s disease indicates the pos- sibility of a contribution from a loss of protein function. Evidence suggests a neuroprotective role for huntingtin; loss of its func- tions could feasibly lead to neurodegeneration. An exclusive role of loss of function is contradicted by the finding that genetic ablation of huntingtin protein does not cause Huntington’s disease, but a contribution from loss of function is supported by simi- larities between neuropathological and behavioural phenotypes in animal models of Huntington’s Disease and those produced by loss of the normal functions of huntingtin. Perhaps, therefore, both loss and gain of function are necessary processes in Huntington’s pathogenesis, with neither one sufficient to cause the disease alone. Review of the current evidence fails to eluci- date an exact role for loss of function in Huntington’s disease pathogenesis. More information is required on the extent to which depletion of the normal protein causes, rather than accompanies, disease. In the meantime, attempts at drug discovery should be mindful of the possibility of a contribution from loss of function when designing treatments and interpreting trial results. Key words: Huntington’s disease, huntingtin, neurodegenerative, striatal neurons, gain of function, loss of function Submitted on 4 January 2015; accepted on 22 July 2015 Introduction death of a cell population; the location or function of this population impacts upon phenotype. Because neurons are not Neurodegenerative disorders, which affect the central nervous a dividing cell population, those lost in disease cannot be system, include Alzheimer’s, Parkinson’s and Huntington’s renewed or replaced. These disorders are therefore progressive diseases (HDs), amyotrophic lateral sclerosis and the prion and currently have no cure. diseases. They can be inherited or sporadic, with infectious forms additionally seen in prion disease. Though aetiology A finding common to many of these diseases is that of mis- and pathogenesis of each disease differs, a common feature is folded protein aggregates (Taylor, Hardy and Fischbeck, © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Review article Bioscience Horizons • Volume 8 2015 2002) such as amyloid-β and α-synuclein in Alzheimer’s and and Clark, 2009, p. 1149). Most cases show classical autoso- Parkinson’s diseases, respectively. A large body of evidence mal dominant inheritance, with ∼3% due to de novo muta- support the hypothesis that these aggregates confer a toxic tions. HD is caused by a mutation in the Huntingtin gene on gain of function that underlies disease (mechanisms of toxicity chromosome 4, which codes for huntingtin protein (htt). The reviewed in Ross and Poirier (2004)), and the common fea- mutation is a dynamic CAG repeat expansion in the coding tures shared by neurodegenerative disorders imply successful region of the gene causing expansion of the N-terminus of the treatment of different diseases may involve parallel mecha- huntingtin protein, with 41 or greater CAG repeats seen in nisms. As such, drug discovery has targeted these large protein affected individuals. HD shows anticipation, whereby age of aggregates. Disappointing results from trials of some drugs, onset decreases between generations. This is due to meiotic which theoretically seem mechanistically sound, raise two key instability, which causes an increase in the number of CAG questions. Firstly, are the large protein aggregates toxic or is repeats. toxicity conferred by an intermediate? Support for the latter is The clinical features typically manifest around the fourth emerging, particularly in the case of Alzheimer’s disease, decade, beginning with memory loss, personality changes and where evidence now suggests that oligomers, rather than amy- chorea. Bradykinesia and rigidity occur later, and cognitive loid fibrils, are the toxic species, though definitive character - impairment progresses to dementia. Death usually occurs ization of specific oligomers involved in Alzheimer’s disease is within 15–20 years of symptom onset, often as a consequence lacking (reviewed in Benilova, Karran and De Strooper of heart failure or aspiration pneumonia (Gil and Rego, (2012)). Despite this, the principle is supported by the finding 2008). Gross atrophy of the striatum is commonly cited as the in HD that neurons containing large huntingtin aggregates pathological hallmark of HD. The most severely afflicted neu- show improved survival compared with surrounding neurons rons are the GABAergic medium-sized spiny striatal neurons, devoid of inclusions (Arrasate et al., 2004). The need for fur- which constitute around 95% of the striatal neuronal popula- ther characterization of the toxic species in these diseases is tion. Specific subpopulations of striatal neurons are affected therefore indicated. at different stages of disease, which is reflected by the tempo- Secondly, is the disease exclusively caused by a gain of ral cascade of symptoms. Current therapies provide only function or does a loss of the normal protein function contrib- symptomatic relief, and despite improving quality of life, they ute? The latter would certainly help to explain the failure of fall short of halting or reversing the disease. some drugs to show efficacy in trials. Research has therefore seen a shift towards the identification of the normal protein Huntingtin protein (htt) functions and whether their loss contributes to, or is purely a secondary finding of, neurodegenerative diseases. With par - Huntingtin protein (Fig.  1) is ubiquitously expressed, but ticular focus on HD, this work will discuss the normal protein its concentration is highest in the central nervous system and functions before examining the hypothesis that a loss of the the testes. normal protein function contributes to the disease process. Huntingtin in development Also considered in this work are the subsequent implications of this upon current and future therapeutic avenues. Huntingtin is critical for embryonic development in mice, and generation of nullizygous huntingtin mice (Hdh−/−) causes embryonic death between Day 8.5 and 10.5 (Zeitlin et  al., Huntington’s disease 1995). However, the same study found that mice heterozygous HD is the most common polyglutamine neurodegenerative for the null mutation did not die during embryogenesis and disorder, with a worldwide prevalence of 5 in 100 000 (Kumar were histopathologically and phenotypically indistinguishable Figure 1. Schematic diagram of huntingtin protein. The polyglutamine repeat region begins at amino acid 18, spaning up to 34 glutamine residues in unaffected individuals. Red squares indicate the three main clusters of HEAT repeats that are involved in protein–protein interactions. These are conserved in vertebrates, indicating that huntingtin interacts with similar proteins across vertebrates. Adapted by permission from Macmillan Publishers Ltd: [NATURE REVIEWS NEUROSCIENCE] (Cattaneo et al., 2005), copyright (2015). Accessed at: http://www.nature.com/nrn/index.html (3 January 2015). 2 Bioscience Horizons • Volume 8 2015 Review article from control mice, indicating a single htt allele is sufficient to physical interaction between caspase-3 and wild-type htt in carry out the developmental function of huntingtin in mice. mouse N2a neuroblastoma cells and found an inverse correla- Furthermore, evidence suggests that mutant huntingtin can tion between wild-type htt expression and caspase-3 activa- carry out this developmental role. Expression of human hun- tion. A role for htt in anti-apoptosis has also been demonstrated tingtin with a pathological polyglutamine expansion (72 CAG in vivo. Overexpression of full-length wild-type htt in yeast repeats) rescued Hdh−/− mice from embryonic lethality artificial chromosome (YAC18) transgenic mice conferred (Leavitt et al., 2001); moreover, patients homozygous for the protection against apoptosis triggered by NMDA receptor- mutation in HD express no wild-type htt but are born with no induced excitotoxicity in a gene dose-dependent manner apparent developmental defects (Wexler et  al., 1987). This (Leavitt et al., 2006). suggests that mutant htt can perform the developmental func- tion of htt, and thus, loss of this function is not implicated in BDNF transcription and vesicle trafficking Huntington’s disease. Brain-derived neurotrophic factor (BDNF) is important for Anti-apoptosis and neuroprotection survival of striatal neurons and the activity of cortico-striatal synapses, and is found co-localized with htt in cortical neu- Rigamonti et al. (2000) exposed in vitro ST14A striatal cells rons that project to the striatum (Cattaneo, Zuccato and transfected with wild-type htt to pro-apoptotic conditions and Tartari, 2005). Wild-type huntingtin promotes cortico-striatal found that any decrease in cell viability correlated with a BDNF expression by enhancing transcription of exon II of the reduction in the level of the exogenous protein; conversely, BDNF gene (Fig. 2A and B). In vivo mouse studies show that overexpression of wild-type htt increased cell viability. this function is exclusive to wild-type huntingtin; mice over- Importantly, cells transfected with mutant htt showed a faster expressing wild-type huntingtin showed a significant increase decline in cell viability and mitochondrial activity compared in cortical production and striatal levels of BDNF compared with those transfected with normal htt under pro-apoptotic with control littermates, whereas those overexpressing mutant conditions, indicating the reduced ability of mutant htt to htt demonstrated a decrease compared with control (Zuccato carry out htt’s neuroprotective role. This study also revealed et al., 2001). that wild-type htt directly influences cell survival and acts as an anti-apoptotic protein in neural cells, partially by preven- A further finding reported by Cattaneo, Zuccato and tion of caspase-3 activation. Zhang et  al. (2006) showed a Tartari (2005) was one of the role of htt in BDNF vesicle Figure 2. Huntingtin activity: BDNF transcription and trafficking. (A) Wild-type huntingtin interacts with and sequesters Repressor element 1-silencing transcription factor (REST ) in the cytoplasm. This prevents binding of REST to the neuron-restrictive silencer element (NRSE) site and the BDNF gene is transcribed. (B) In Huntington’s disease, mutant huntingtin is less able to retain REST in the cytoplasm, allowing it to enter the nucleus where it binds to NRSE and represses transcription of BDNF. (C) Wild-type huntingtin binds to huntingtin-associated protein 1 (HAP1) and indirectly regulates the assembly of p150 (grey) with the dyenin (blue) and dynactin (pink) complexes that ultimately control the microtubule transport of BDNF vesicles (yellow). Arrows indicate the movement of BDNF vesicles. (D) Mutant huntingtin binds more tightly to HAP1, reducing the transport of BDNF vesicles along microtubules. Adapted by permission from Macmillan Publishers Ltd: [NATURE REVIEWS NEUROSCIENCE] (Cattaneo et al., 2005), copyright (2015). Accessed at: http://www.nature.com/nrn/index.html (3 January 2015). 3 Review article Bioscience Horizons • Volume 8 2015 trafficking. BDNF vesicle transport along microtubules is observations strongly support a largely toxic gain of function, enhanced in the presence of wild-type htt and reduced either they do not rule out an increased dominant-negative effect on in the presence of mutant htt (Fig. 2C and D) or when levels the remaining wild-type allele (if the patient is heterozygous), of wild-type htt are reduced using RNA interference (Gauthier whereby increased mutant htt toxicity would cause increased et al., 2004). loss of wild-type protein function. HD mouse models suggest a predominant Gain of function in Huntington’s GOF disease Initial experiments involving depletion of wild-type htt, in the presence of mutant htt, have produced results indicative of a The conventional model of neurodegenerative diseases such as predominantly toxic gain of function in HD pathogenesis. Huntington’s has been one of the predominantly gain of toxic Van Raamsdonk et al. (2005b) generated YAC128−/− mice, function, and efforts have focused on identifying and character- which express mutant htt without wild-type htt. The behav- izing the toxic species in each disease. Evidence underpinning ioural and histopathological phenotypes of these mice were such a concept in Huntington’s disease is considered below. compared with that of YAC128+/+ mice, which express the same level of mutant htt with a background of normal levels Expanded CAG is toxic of wild-type htt. Since wild-type htt is neuroprotective over An expanded CAG sequence is itself toxic: expression of an striatal neurons, a reasonable prediction states that its loss in expanded CAG sequence alone, or in the context of a small YAC128−/− mice should predispose to more severe striatal part of the huntingtin protein, causes neurological symptoms pathology, specifically a more pronounced loss of striatal neu- in animals (Cattaneo et al., 2001). In addition, Ordway et al. rons. However, although both YAC128−/− and +/+ mice (1997) introduced a 146-unit CAG repeat into the mouse showed a decrease in striatal volume and striatal neuron num- hypoxanthine phosphoribosyltransferase gene (HPRT). The ber compared with control (as expected), there was no differ- HPRT gene is not involved in any known CAG repeat disor- ence between the former two groups. This finding suggests ders, and inactivation of this gene alone does not have any that loss of striatal neurons, a pathological hallmark of HD, is deleterious effects in mice. The mice developed a late-onset primarily due to a toxic gain of function of mutant huntingtin. neurological phenotype and premature death. Interestingly, this model showed the same inverse relationship between Different gene sets are affected by CAG CAG repeat number and age of onset of symptoms as HD. The expansion and wild-type htt knockout fact that a neurological phenotype can be produced by inser- tion of an expanded CAG fragment into a gene unrelated to Jacobsen et al. (2011) examined genome-wide gene expres- HD, a phenotype that therefore does not require inactivation sion changes correlated with CAG size across an allelic series or depletion of wild-type htt, lends great support to a toxic of heterozygous CAG knock-in mouse embryonic stem cells gain of function in HD. The fact that this model does not pro- and compared them with genes differentially expressed duce the phenotype specific to HD, however, does suggest that between huntingtin-null and wild-type parental embryonic reduced function of wild-type huntingtin might guide the pat- stem cells. Any model in which the expanded CAG tract con- tern of neuronal dysfunction and death seen in HD. In this tributes to a pathogenic loss of htt function (a dominant-neg- sense, similarities between the nine known human CAG ative effect, for example) would predict an extensive overlap expansion disorders could in part be explained by an empiri- in the molecular responses observed; something a simple gain cal gain of toxic function attributable to CAG expansion, of function hypothesis does not require. They found that the while the differences in neuronal populations affected and CAG-correlated gene set was distinct from the huntingtin-null subsequent clinical manifestations may be governed by the gene set and that the pathways that were specifically corre- wild-type proteins upon which each disease impacts. lated with CAG size were not predictive of the pathways altered by knocking out wild-type htt; observations that fail to CAG repeat length correlates to age of support a model of HD involving loss of normal function. onset and severity of neuropathology Cleavage of mutant htt produces fragments The fact that CAG repeat number is inversely proportional to that mediate toxicity in HD animal models age of onset of HD symptoms in humans (Imarisio et  al., 2008) provides further support to a toxic gain of function, Huntingtin is susceptible to cleavage by caspases 2, 3 and 6. whereby larger CAG repeats cause enhanced toxicity. Though N-terminal fragments of huntingtin have been found Accordingly, greatly expanded CAG sequences lead to juvenile in both control and HD brains, Wellington et al. (2002) report onset forms of HD, and length of CAG repeat is positively that cleavage of mutant huntingtin leads to the release of a correlated with neuropathological severity and disease pro- toxic N-terminal fragment. To ascertain the role of these frag- gression (Furtado et  al., 1996). Accordingly, patients with ments, the brains of YAC72 mice (express mutant htt with 72 juvenile onset HD demonstrate a greater degree of pathologi- CAG repeats) were compared with those of controls cal severity than those with adult-onset HD. Though these (Wellington et al., 2002). Detection of fragments derived from 4 Bioscience Horizons • Volume 8 2015 Review article mutant htt occurred several months prior to onset of behav- Similarities between wild-type htt knockout ioural or neuropathological changes, suggesting a possible and HD mouse models causal role. Adding significance to these results was the find- ing that the staining pattern of the fragments closely paralleled In further support of an involvement of loss of function is the that found in post-mortem human HD samples. Similar tem- finding that conditional inactivation of wild-type htt in mice poral proximity between detection of mutant htt fragments has revealed a striking similarity between the loss of htt func- and onset of behavioural abnormalities was reported by tion and the HD mouse model phenotypes (Dragatsis, Levine Van Raamsdonk et al. (2005a) in YAC128 HD mice; the addi- and Zeitlin, 2000). The wild-type htt knockout mice showed tional observation that the nuclear localization of the htt frag- progressive behavioural and motor deficits, as well as decreased ments occurred earliest and to the greatest extent in the striatum, survival, that were similar to findings in HD mice. Histological the area most affected in HD, further implicates a causal role for examination revealed tissue degeneration in the striatum and these fragments in disease. Indeed, further work has implicated cortex, again paralleling that seen in HD mouse models. these fragments as a critical step in HD pathogenesis, whereby Loss of wild-type huntingtin worsens prevention of cleavage of by specific caspases rescues the HD phenotype in mice. Graham et al. (2006) showed that expres- clinical disease progression sion of caspase-6-resistant mutant htt in HD mice was found to Discussed earlier were the findings from Van Raamsdonk et al. prevent striatal volume loss, and the mice showed the same level (2005b) that in mouse models of HD, the presence of wild-type of motor performance as controls, unlike HD mice with htt htt has no effect on striatal neuron number and striatal volume, amenable to caspase-6 cleavage. Nuclear translocation of htt arguing against a role for loss of function in histopathological fragments did occur in these mice, albeit at a later time point, changes in HD. However, the same study found that HD mice suggesting other htt cleavage mechanisms do exist. However, lacking wild-type htt were hypoactive at 2 months compared the absence of any neuropathological or behavioural deficits in with both those with wild-type htt and control. Accordingly, the these mice implicates caspase-6-mediated huntingtin cleavage as YAC128−/− mice performed less well on the rotarod test of a critical rate-limiting step in the pathogenesis of HD, lending motor coordination from 2 to 12 months of age. These findings further support to a gain of function hypothesis. were echoed by a later study in a Drosophila model, which showed that removal of endogenous huntingtin accelerated the neurodegenerative phenotype associated with polyglutamine Loss of function in Huntington’s toxicity (Zhang et al., 2009). Therefore, though loss of normal disease protein function does not seem to worsen the histopathology in the HD models, perhaps it is a factor in the timing and severity Though a loss of some of the aforementioned functions of hun- of the clinical manifestations of HD. tingtin could conceivably lead to reduced neuroprotection, less clear is whether a loss of function in HD is a causative factor, an Depletion of wild-type huntingtin parallels exacerbating factor or an epiphenomenon. Attempts to clarify disease progression in HD mice this have involved genetically manipulating wild-type htt levels and comparing phenotypes to those of HD animal models. R6/2 mice express exon 1 of human huntingtin with an expanded CAG repeat and develop a progressive HD-like phe- Not a simple loss of function, but loss of notype. Zhang et al. (2003) evaluated levels of wild-type htt at different stages of disease in R6/2 mice and compared them function may contribute with the levels of unaffected mice. Unaffected mice showed an It is logical to firstly consider a hypothesis of an exclusive loss increase in wild-type htt at 5, 7 and 12 weeks in comparison of function in HD. Compelling evidence against this however to at 1 week. The same was seen in R6/2 mice at 5 weeks, but comes from Wolf–Hirschhorn Syndrome: a disease character- at 7 and 12 weeks these mice showed a progressive reduction ized by a deletion of the short arm of chromosome 4, which in wild-type htt. This reduction was not due to reduced pro- contains the Huntingtin gene. These patients do not develop duction, suggesting increased protein metabolism or seques- HD, despite the loss of 50% of the normal levels of huntingtin tration, indicating a dominant-negative effect of the mutant protein (Gottfried, Lavine and Roessmann, 1981). Lending fur- huntingtin. Importantly, disease progression in R6/2 mice, ther support to this is the case of one phenotypically normal evaluated using measures of weight loss and tests of motor individual possessing a balanced translocation bisecting the HD coordination, closely paralleled the depletion of wild-type htt, gene locus (Ambrose et al., 1994), which indicates that inacti- supporting a causal role for loss of function. vation of one huntingtin allele is not sufficient to cause HD. However, this does not rule out a pathogenic contribution from Mice lacking BDNF show similarities to loss of function in conjunction with a toxic gain of function. HD mouse models Accordingly, the finding that mutant htt can recruit wild-type htt into insoluble aggregates both in vitro and in vivo (Cattaneo Wild-type htt promotes transcription of BDNF. If a loss of et al., 2001) suggests that the toxic effect might at least partially function was implicated in HD pathogenesis, one might lie in a dominant-negative effect on wild-type htt. expect to find a reduction in levels of BDNF in the brains of 5 Review article Bioscience Horizons • Volume 8 2015 those with HD. Accordingly, Zhang et al. (2003) found a sig- cause of HD and suggesting a dissociation between mutant nificant reduction in BDNF concentration in the brains of protein aggregates and toxicity, lending support to a contribu- symptomatic HD mice, which paralleled wild-type htt deple- tion from loss of function. However, recent suggestions that tion and disease progression. This is supported by observa- large mutant protein aggregates are not the toxic species in tions by Zuccato et al. (2001) of reduced BDNF protein in the neurodegenerative disorders propose a mechanism whereby cortices of HD patients. Though this evidence is consistent smaller intermediate species are instead pathogenic. Therefore, with a role for reduced BDNF in HD, the fact that the mice further study is required to clarify the implications of this dis- were symptomatic at time of analysis means it is difficult to sociation between NIIs and vulnerable neurons. elucidate whether reduced BDNF is a cause or effect of HD. Complementing the above studies are some comparing Implications upon treatment of BDNF knockout mice with HD mouse models; similarities in Huntington’s disease the histological or clinical pictures between the two would suggest that BDNF depletion contributes to at least those Preventing expression of the mutant htt allele is an attractive aspects of HD pathogenesis, rather than being an incidental or target, and mice with only one working copy of the Huntingtin secondary finding. BDNF cortical knockout mice show gene show no overt abnormalities (Imarisio et  al., 2008). reduced cross-sectional area, dendritic diameter and spine Though direct intraventricular injections of short interfering density of medium striatal neurons (Baquet, Gorski and Jones, RNA against mutant htt in HD mice have shown efficacy in 2004), changes seen in both HD mice and htt knockout mice. delaying onset of motor symptoms and prolonging survival In addition, behavioural phenotypes observed in BDNF (Wang et al., 2005), this technique may not be safe or practical knockout mice parallel those of HD mice, such as progressive in humans. Additionally, it would not be applicable to homozy- hind limb clasping. These similarities suggest a causal role in gous HD patients if huntingtin has critical functions. A more both the histopathological and clinical manifestations of HD. acceptable target may be to up-regulate autophagy, the cell’s Interestingly, however, these mice do not show poor rotarod own mechanism of mutant huntingtin clearance. Ravikumar performance compared with wild-type controls, unlike HD et  al. (2004) found that rapamycin enhanced clearance of mice. On face value, this discrepancy between models ques- mutant huntingtin exon 1 fragments in a cell model and slowed tions the role of BDNF depletion in HD. The explanation for neurodegeneration in fly and mouse models expressing mutant this may however lie in the design of the mouse model. The htt. Although no improvement was seen in the lifespan of mice mice used in this study were cortical BDNF knockout mice, treated with rapamycin, the delayed onset of symptoms was yet ∼15% of medium striatal neurons receive BDNF input found to be signic fi ant. If onset could be delayed beyond the from the substantia nigra (Johnston et al., 1990). In the corti- normal life expectancy, the disease would effectively be ‘cured’. cal BDNF knockout mice, some striatal neurons may still have been receiving BDNF from the substantia nigra, thus An advantage of the above treatment strategies is that as perhaps not truly recapitulating the global effects on BDNF of gain of function almost certainly contributes to HD pathogen- loss of huntingtin function. Comparison of rotarod perfor- esis, indicated by evidence supporting gain of function in con- mance of cortical BDNF knockout mice and htt knockout junction with the conclusion that loss of function alone does mice is therefore indicated, but certainly this apparent dis- not lead to HD, targeting the toxic species should, at the very crepancy cannot currently rule out a causal role for loss of least, slow progression. However, the fact that rapamycin did BDNF in HD pathogenesis. Accordingly, supporting a proac- not halt disease progression provides indirect support of a loss tive rather than reactive role for a reduction in BDNF in HD, of function contribution to disease pathogenesis, suggesting Strand et al. (2007) found that of all HD-related mouse mod- the need to address this contribution when designing thera- els, BDNF knockout mice have striatal mRNA expression peutic options. A logical strategy in parallel with those men- patterns that best recapitulate those seen in human HD, sug- tioned above would therefore be to restore normal functioning gesting that the striatal neurons in Huntington’s disease and of wild-type htt, either by replacing the protein itself or by BDNF-deprived environments suffer from similar insults. modifying its downstream targets. A potential problem with the former may arise in the event that loss of function occurs Huntingtin inclusions do not correlate via a dominant-negative effect. This may largely be circum- with neuronal vulnerability vented by the latter, though this would first require identifica- tion of the function, if any, whose loss contributes to disease. A classical finding in HD is neuronal intracellular inclusions (NIIs) and protein aggregates in dystrophic neurites in striatal Though theoretically promising, further studies are needed and cortical neurons. While the number of cortical inclusions to test efficacy of these strategies in treating HD rather than has been found to correlate well with the length of CAG simply reducing mutant or increasing wild-type protein levels. repeat expansion and age of disease onset (Becher et  al., Alongside this, other treatment options, largely focusing on 1998), neurons bearing NIIs do not correspond to the most more generalized neuroprotective strategies, have been sug- vulnerable ones. Kuemmerle et  al. (1999) found that some gested (Fig. 3). Ultimately, any approach to treating HD will interneurons with high numbers of NIIs were spared in the benefit from a further clarification on the specific pathological processes underlying HD. disease, indicating that NIIs may be a marker rather than 6 Bioscience Horizons • Volume 8 2015 Review article Figure 3. Therapeutic strategies for Huntington’s disease. Drug discovery has focused on removal of mutant htt or restoration of the normal htt functions. Additionally, more generalized neuroprotective strategies have been tested, such as attempts to decrease excitotoxic cell death or enhance neurotrophin release. Adapted from Imarisio et al. (2008). Huntington’s disease: from pathology and genetics to potential therapies. Biochemical Journal, 412(2), 191–209. Portland Press. Accessed at: doi: 10.1042/BJ20071619 (3 January 2015). seems a reasonable conclusion that loss of function can Conclusion worsen phenotype, but does that mean it truly contributes to pathogenesis; if any contribution from loss of function were There are three main scenarios regarding pathogenesis of neu- removed, would the disease follow a similar pattern of neuro- rodegenerative disorders. Firstly, disease is caused by a gain of pathology and clinical features? That question remains unan- toxic function, perhaps conferred by mutant protein aggre- swered, and until more is understood about the normal gates. Though evidence supports a correlation between protein functions, as well as the true impact of their loss under mutant proteins, neuropathology and clinical symptoms, as disease rather than normal conditions, it is likely to remain so, yet none definitively rules out a contribution from loss of leaving the contribution of loss of function to pathogenesis function. Secondly, a loss of normal protein function is the ambiguous. What the evidence reviewed here does suggest, cause, and aggregations of mutant protein are either second- however, is that loss of function has a role in directing toxicity, ary to disease or incidental findings. Conditions such as Wolf– whereby reduced BDNF transcription as a result of loss of Hirschhorn Syndrome largely contradict an exclusive role for huntingtin function may contribute to the selective vulnerabil- loss of function, however. A final option is that disease is ity of striatal neurons. caused by a combination of loss and gain of function. An increasing repertoire of studies supports this, and certainly the It is useful to consider any potential relevance of issues dis- proteins involved in these diseases carry functions that, if lost, cussed here within the wider arena of neurodegenerative dis- could conceivably lead to neurodegeneration. eases; interestingly, several parallels can be drawn. With regard A key question regarding the hypothesis addressed in this to loss of function directing toxicity, a similar state may be true review is what is meant by a loss of function contributing to in prion diseases, whereby loss of the neuroprotective signalling disease? Evidence from animal models of Huntington’s dis- role of prion protein at synapses (Zanata et  al., 2002) ease suggests that a loss of function accelerates disease pro- corresponds to the early loss of synapses in the disease (Jeffrey gression. In this way, and others discussed in this review, it et  al., 2000). Similarly, this review discusses a dissociation 7 Review article Bioscience Horizons • Volume 8 2015 Cattaneo, E., Rigamonti, D., Goffredo, D. et al. (2001) Loss of normal hun- between neuronal intracellular inclusions of mutant huntingtin tingtin function: new developments in Huntington’s disease and vulnerable neurons; a well-characterized observation in research, Trends in Neurosciences, 24 (3), 182–188. Alzheimer’s disease is that distribution of the toxic species, amyloid-β plaques, correlates poorly with the pattern and Cattaneo, E., Zuccato, C. and Tartari, M. (2005) Normal huntingtin func- severity of dementia (Terry et al., 1991). tion: an alternative approach to Huntington’s disease, Nature Reviews Neuroscience, 6 (12), 919–930. Armed with the knowledge that further characterization of the role of normal huntingtin protein in health and disease Dragatsis, I., Levine, M. S. and Zeitlin, S. (2000) Inactivation of Hdh in the is required, one must be mindful when developing treat- brain and testis results in progressive neurodegeneration and steril- ments of the possible detrimental effects of compromising ity in mice, Nature Genetics, 26 (3), 300–306. normal functions, as well as the potential beneficial effects of restoring these. In turn, failures and successes in trials involv- Furtado, S., Suchowersky, O., Rewcastle, N. B. et al. (1996) Relationship ing replacement of particular protein functions may feed between trinucliotide repeats and neuropathological changes in back into the understanding of the involvement of loss of Huntington’s diease, Annals of Neurology, 39 (1), 132–136. function in the disease. Encouragingly, similarities can be Gauthier, L. R., Charrin, B. C., Borrell-Pagès, M. et al. (2004) Huntingtin consistently drawn between several neurodegenerative disor- controls neurotrophic support and survival of neurons by enhancing ders, and thus, the principles discussed may impact upon the BDNF vesicular transport along microtubules, Cell, 118 (1), 127–138. direction of future research and drug discovery programmes in several neurodegenerative disorders in an attempt to suc- Gil, J. M. and Rego, A. C. (2008) Mechanisms of neurodegeneration in cessfully treat these increasingly prevalent and devastating Huntington’s disease, European Journal of Neuroscience, 27 (11), diseases. 2803–2820. Gottfried, M., Lavine, L. and Roessmann, U. (1981) Neuropathological Author’s biography findings in Wolf-Hirschhorn (4p-) syndrome, Acta Neuropathologica, 55 (2), 163–165. This review was adapted from Heidi’s BSc dissertation, which Graham, R. K., Deng, Y., Slow, E. J. et al. (2006) Cleavage at the caspase-6 studied loss of protein function in the context of both site is required for neuronal dysfunction and degeneration due to Huntington’s and Prion disease. She obtained a First Class mutant huntingtin, Cell, 125 (6), 1179–1191. Honours BSc in Neuroscience as an intercalated degree and was awarded the Neuroscience Scientifica University prize for Imarisio, S., Carmichael, J., Korolchuk, V. et al. (2008) Huntington’s dis- the highest overall degree mark. She subsequently completed ease: from pathology and genetics to potential therapies, her MBChB at the University of Leeds, graduating with Biochemical Journal, 412 (2), 191–209. Honours. 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(2005b) Loss of wild- type huntingtin influences motor dysfunction and survival in the Zuccato, C., Ciammola, A., Rigamonti, D. et al. (2001) Loss of huntingtin- YAC128 mouse model of Huntington disease, Human Molecular mediated BDNF gene transcription in Huntington’s disease, Science, Genetics, 14 (10), 1379–1392. 293 (5529), 493–498. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Does loss of the normal protein function contribute to the pathogenesis of Huntington's disease?

Paine, Heidi
Bioscience Horizons , Volume 8 – Oct 13, 2015
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BioscienceHorizons Volume 8 2015 10.1093/biohorizons/hzv005 Review article Does loss of the normal protein function contribute to the pathogenesis of Huntington’s disease? Heidi Paine Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, England *Corresponding author: St Mary’s Hospital, Praed Street, London W2 1NY, England. Email: heidipaine@doctors.org.uk Supervisor: Nigel Hooper, Institute of Brain, Behaviour and Mental Health, Faculty of Medical and Human Sciences, University of Manchester, Manchester, M13 9PT, England. Neurodegenerative disorders such as Huntington’s, Alzheimer’s, Parkinson’s and prion diseases are progressive and without a cure. A common finding is one of misfolded protein aggregates, conventionally believed to underlie pathogenesis via a toxic gain of function. Recently, a potential contribution of loss of normal protein function has come under the spotlight. With a focus on huntingtin, the protein involved in Huntington’s disease, this review examines the evidence for the conventional ‘gain of function’ model, before considering the hypothesis that a loss of function contributes to pathogenesis. In support of a primarily toxic gain of function are findings that huntingtin aggregates are neurotoxic in vitro. Additionally, aggregates of mutant huntingtin pro- teins have been detected prior to neuropathological changes, supporting a causal role. However, a dissociation between the neurons containing mutant huntingtin aggregates and those that are most vulnerable in Huntington’s disease indicates the pos- sibility of a contribution from a loss of protein function. Evidence suggests a neuroprotective role for huntingtin; loss of its func- tions could feasibly lead to neurodegeneration. An exclusive role of loss of function is contradicted by the finding that genetic ablation of huntingtin protein does not cause Huntington’s disease, but a contribution from loss of function is supported by simi- larities between neuropathological and behavioural phenotypes in animal models of Huntington’s Disease and those produced by loss of the normal functions of huntingtin. Perhaps, therefore, both loss and gain of function are necessary processes in Huntington’s pathogenesis, with neither one sufficient to cause the disease alone. Review of the current evidence fails to eluci- date an exact role for loss of function in Huntington’s disease pathogenesis. More information is required on the extent to which depletion of the normal protein causes, rather than accompanies, disease. In the meantime, attempts at drug discovery should be mindful of the possibility of a contribution from loss of function when designing treatments and interpreting trial results. Key words: Huntington’s disease, huntingtin, neurodegenerative, striatal neurons, gain of function, loss of function Submitted on 4 January 2015; accepted on 22 July 2015 Introduction death of a cell population; the location or function of this population impacts upon phenotype. Because neurons are not Neurodegenerative disorders, which affect the central nervous a dividing cell population, those lost in disease cannot be system, include Alzheimer’s, Parkinson’s and Huntington’s renewed or replaced. These disorders are therefore progressive diseases (HDs), amyotrophic lateral sclerosis and the prion and currently have no cure. diseases. They can be inherited or sporadic, with infectious forms additionally seen in prion disease. Though aetiology A finding common to many of these diseases is that of mis- and pathogenesis of each disease differs, a common feature is folded protein aggregates (Taylor, Hardy and Fischbeck, © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Review article Bioscience Horizons • Volume 8 2015 2002) such as amyloid-β and α-synuclein in Alzheimer’s and and Clark, 2009, p. 1149). Most cases show classical autoso- Parkinson’s diseases, respectively. A large body of evidence mal dominant inheritance, with ∼3% due to de novo muta- support the hypothesis that these aggregates confer a toxic tions. HD is caused by a mutation in the Huntingtin gene on gain of function that underlies disease (mechanisms of toxicity chromosome 4, which codes for huntingtin protein (htt). The reviewed in Ross and Poirier (2004)), and the common fea- mutation is a dynamic CAG repeat expansion in the coding tures shared by neurodegenerative disorders imply successful region of the gene causing expansion of the N-terminus of the treatment of different diseases may involve parallel mecha- huntingtin protein, with 41 or greater CAG repeats seen in nisms. As such, drug discovery has targeted these large protein affected individuals. HD shows anticipation, whereby age of aggregates. Disappointing results from trials of some drugs, onset decreases between generations. This is due to meiotic which theoretically seem mechanistically sound, raise two key instability, which causes an increase in the number of CAG questions. Firstly, are the large protein aggregates toxic or is repeats. toxicity conferred by an intermediate? Support for the latter is The clinical features typically manifest around the fourth emerging, particularly in the case of Alzheimer’s disease, decade, beginning with memory loss, personality changes and where evidence now suggests that oligomers, rather than amy- chorea. Bradykinesia and rigidity occur later, and cognitive loid fibrils, are the toxic species, though definitive character - impairment progresses to dementia. Death usually occurs ization of specific oligomers involved in Alzheimer’s disease is within 15–20 years of symptom onset, often as a consequence lacking (reviewed in Benilova, Karran and De Strooper of heart failure or aspiration pneumonia (Gil and Rego, (2012)). Despite this, the principle is supported by the finding 2008). Gross atrophy of the striatum is commonly cited as the in HD that neurons containing large huntingtin aggregates pathological hallmark of HD. The most severely afflicted neu- show improved survival compared with surrounding neurons rons are the GABAergic medium-sized spiny striatal neurons, devoid of inclusions (Arrasate et al., 2004). The need for fur- which constitute around 95% of the striatal neuronal popula- ther characterization of the toxic species in these diseases is tion. Specific subpopulations of striatal neurons are affected therefore indicated. at different stages of disease, which is reflected by the tempo- Secondly, is the disease exclusively caused by a gain of ral cascade of symptoms. Current therapies provide only function or does a loss of the normal protein function contrib- symptomatic relief, and despite improving quality of life, they ute? The latter would certainly help to explain the failure of fall short of halting or reversing the disease. some drugs to show efficacy in trials. Research has therefore seen a shift towards the identification of the normal protein Huntingtin protein (htt) functions and whether their loss contributes to, or is purely a secondary finding of, neurodegenerative diseases. With par - Huntingtin protein (Fig.  1) is ubiquitously expressed, but ticular focus on HD, this work will discuss the normal protein its concentration is highest in the central nervous system and functions before examining the hypothesis that a loss of the the testes. normal protein function contributes to the disease process. Huntingtin in development Also considered in this work are the subsequent implications of this upon current and future therapeutic avenues. Huntingtin is critical for embryonic development in mice, and generation of nullizygous huntingtin mice (Hdh−/−) causes embryonic death between Day 8.5 and 10.5 (Zeitlin et  al., Huntington’s disease 1995). However, the same study found that mice heterozygous HD is the most common polyglutamine neurodegenerative for the null mutation did not die during embryogenesis and disorder, with a worldwide prevalence of 5 in 100 000 (Kumar were histopathologically and phenotypically indistinguishable Figure 1. Schematic diagram of huntingtin protein. The polyglutamine repeat region begins at amino acid 18, spaning up to 34 glutamine residues in unaffected individuals. Red squares indicate the three main clusters of HEAT repeats that are involved in protein–protein interactions. These are conserved in vertebrates, indicating that huntingtin interacts with similar proteins across vertebrates. Adapted by permission from Macmillan Publishers Ltd: [NATURE REVIEWS NEUROSCIENCE] (Cattaneo et al., 2005), copyright (2015). Accessed at: http://www.nature.com/nrn/index.html (3 January 2015). 2 Bioscience Horizons • Volume 8 2015 Review article from control mice, indicating a single htt allele is sufficient to physical interaction between caspase-3 and wild-type htt in carry out the developmental function of huntingtin in mice. mouse N2a neuroblastoma cells and found an inverse correla- Furthermore, evidence suggests that mutant huntingtin can tion between wild-type htt expression and caspase-3 activa- carry out this developmental role. Expression of human hun- tion. A role for htt in anti-apoptosis has also been demonstrated tingtin with a pathological polyglutamine expansion (72 CAG in vivo. Overexpression of full-length wild-type htt in yeast repeats) rescued Hdh−/− mice from embryonic lethality artificial chromosome (YAC18) transgenic mice conferred (Leavitt et al., 2001); moreover, patients homozygous for the protection against apoptosis triggered by NMDA receptor- mutation in HD express no wild-type htt but are born with no induced excitotoxicity in a gene dose-dependent manner apparent developmental defects (Wexler et  al., 1987). This (Leavitt et al., 2006). suggests that mutant htt can perform the developmental func- tion of htt, and thus, loss of this function is not implicated in BDNF transcription and vesicle trafficking Huntington’s disease. Brain-derived neurotrophic factor (BDNF) is important for Anti-apoptosis and neuroprotection survival of striatal neurons and the activity of cortico-striatal synapses, and is found co-localized with htt in cortical neu- Rigamonti et al. (2000) exposed in vitro ST14A striatal cells rons that project to the striatum (Cattaneo, Zuccato and transfected with wild-type htt to pro-apoptotic conditions and Tartari, 2005). Wild-type huntingtin promotes cortico-striatal found that any decrease in cell viability correlated with a BDNF expression by enhancing transcription of exon II of the reduction in the level of the exogenous protein; conversely, BDNF gene (Fig. 2A and B). In vivo mouse studies show that overexpression of wild-type htt increased cell viability. this function is exclusive to wild-type huntingtin; mice over- Importantly, cells transfected with mutant htt showed a faster expressing wild-type huntingtin showed a significant increase decline in cell viability and mitochondrial activity compared in cortical production and striatal levels of BDNF compared with those transfected with normal htt under pro-apoptotic with control littermates, whereas those overexpressing mutant conditions, indicating the reduced ability of mutant htt to htt demonstrated a decrease compared with control (Zuccato carry out htt’s neuroprotective role. This study also revealed et al., 2001). that wild-type htt directly influences cell survival and acts as an anti-apoptotic protein in neural cells, partially by preven- A further finding reported by Cattaneo, Zuccato and tion of caspase-3 activation. Zhang et  al. (2006) showed a Tartari (2005) was one of the role of htt in BDNF vesicle Figure 2. Huntingtin activity: BDNF transcription and trafficking. (A) Wild-type huntingtin interacts with and sequesters Repressor element 1-silencing transcription factor (REST ) in the cytoplasm. This prevents binding of REST to the neuron-restrictive silencer element (NRSE) site and the BDNF gene is transcribed. (B) In Huntington’s disease, mutant huntingtin is less able to retain REST in the cytoplasm, allowing it to enter the nucleus where it binds to NRSE and represses transcription of BDNF. (C) Wild-type huntingtin binds to huntingtin-associated protein 1 (HAP1) and indirectly regulates the assembly of p150 (grey) with the dyenin (blue) and dynactin (pink) complexes that ultimately control the microtubule transport of BDNF vesicles (yellow). Arrows indicate the movement of BDNF vesicles. (D) Mutant huntingtin binds more tightly to HAP1, reducing the transport of BDNF vesicles along microtubules. Adapted by permission from Macmillan Publishers Ltd: [NATURE REVIEWS NEUROSCIENCE] (Cattaneo et al., 2005), copyright (2015). Accessed at: http://www.nature.com/nrn/index.html (3 January 2015). 3 Review article Bioscience Horizons • Volume 8 2015 trafficking. BDNF vesicle transport along microtubules is observations strongly support a largely toxic gain of function, enhanced in the presence of wild-type htt and reduced either they do not rule out an increased dominant-negative effect on in the presence of mutant htt (Fig. 2C and D) or when levels the remaining wild-type allele (if the patient is heterozygous), of wild-type htt are reduced using RNA interference (Gauthier whereby increased mutant htt toxicity would cause increased et al., 2004). loss of wild-type protein function. HD mouse models suggest a predominant Gain of function in Huntington’s GOF disease Initial experiments involving depletion of wild-type htt, in the presence of mutant htt, have produced results indicative of a The conventional model of neurodegenerative diseases such as predominantly toxic gain of function in HD pathogenesis. Huntington’s has been one of the predominantly gain of toxic Van Raamsdonk et al. (2005b) generated YAC128−/− mice, function, and efforts have focused on identifying and character- which express mutant htt without wild-type htt. The behav- izing the toxic species in each disease. Evidence underpinning ioural and histopathological phenotypes of these mice were such a concept in Huntington’s disease is considered below. compared with that of YAC128+/+ mice, which express the same level of mutant htt with a background of normal levels Expanded CAG is toxic of wild-type htt. Since wild-type htt is neuroprotective over An expanded CAG sequence is itself toxic: expression of an striatal neurons, a reasonable prediction states that its loss in expanded CAG sequence alone, or in the context of a small YAC128−/− mice should predispose to more severe striatal part of the huntingtin protein, causes neurological symptoms pathology, specifically a more pronounced loss of striatal neu- in animals (Cattaneo et al., 2001). In addition, Ordway et al. rons. However, although both YAC128−/− and +/+ mice (1997) introduced a 146-unit CAG repeat into the mouse showed a decrease in striatal volume and striatal neuron num- hypoxanthine phosphoribosyltransferase gene (HPRT). The ber compared with control (as expected), there was no differ- HPRT gene is not involved in any known CAG repeat disor- ence between the former two groups. This finding suggests ders, and inactivation of this gene alone does not have any that loss of striatal neurons, a pathological hallmark of HD, is deleterious effects in mice. The mice developed a late-onset primarily due to a toxic gain of function of mutant huntingtin. neurological phenotype and premature death. Interestingly, this model showed the same inverse relationship between Different gene sets are affected by CAG CAG repeat number and age of onset of symptoms as HD. The expansion and wild-type htt knockout fact that a neurological phenotype can be produced by inser- tion of an expanded CAG fragment into a gene unrelated to Jacobsen et al. (2011) examined genome-wide gene expres- HD, a phenotype that therefore does not require inactivation sion changes correlated with CAG size across an allelic series or depletion of wild-type htt, lends great support to a toxic of heterozygous CAG knock-in mouse embryonic stem cells gain of function in HD. The fact that this model does not pro- and compared them with genes differentially expressed duce the phenotype specific to HD, however, does suggest that between huntingtin-null and wild-type parental embryonic reduced function of wild-type huntingtin might guide the pat- stem cells. Any model in which the expanded CAG tract con- tern of neuronal dysfunction and death seen in HD. In this tributes to a pathogenic loss of htt function (a dominant-neg- sense, similarities between the nine known human CAG ative effect, for example) would predict an extensive overlap expansion disorders could in part be explained by an empiri- in the molecular responses observed; something a simple gain cal gain of toxic function attributable to CAG expansion, of function hypothesis does not require. They found that the while the differences in neuronal populations affected and CAG-correlated gene set was distinct from the huntingtin-null subsequent clinical manifestations may be governed by the gene set and that the pathways that were specifically corre- wild-type proteins upon which each disease impacts. lated with CAG size were not predictive of the pathways altered by knocking out wild-type htt; observations that fail to CAG repeat length correlates to age of support a model of HD involving loss of normal function. onset and severity of neuropathology Cleavage of mutant htt produces fragments The fact that CAG repeat number is inversely proportional to that mediate toxicity in HD animal models age of onset of HD symptoms in humans (Imarisio et  al., 2008) provides further support to a toxic gain of function, Huntingtin is susceptible to cleavage by caspases 2, 3 and 6. whereby larger CAG repeats cause enhanced toxicity. Though N-terminal fragments of huntingtin have been found Accordingly, greatly expanded CAG sequences lead to juvenile in both control and HD brains, Wellington et al. (2002) report onset forms of HD, and length of CAG repeat is positively that cleavage of mutant huntingtin leads to the release of a correlated with neuropathological severity and disease pro- toxic N-terminal fragment. To ascertain the role of these frag- gression (Furtado et  al., 1996). Accordingly, patients with ments, the brains of YAC72 mice (express mutant htt with 72 juvenile onset HD demonstrate a greater degree of pathologi- CAG repeats) were compared with those of controls cal severity than those with adult-onset HD. Though these (Wellington et al., 2002). Detection of fragments derived from 4 Bioscience Horizons • Volume 8 2015 Review article mutant htt occurred several months prior to onset of behav- Similarities between wild-type htt knockout ioural or neuropathological changes, suggesting a possible and HD mouse models causal role. Adding significance to these results was the find- ing that the staining pattern of the fragments closely paralleled In further support of an involvement of loss of function is the that found in post-mortem human HD samples. Similar tem- finding that conditional inactivation of wild-type htt in mice poral proximity between detection of mutant htt fragments has revealed a striking similarity between the loss of htt func- and onset of behavioural abnormalities was reported by tion and the HD mouse model phenotypes (Dragatsis, Levine Van Raamsdonk et al. (2005a) in YAC128 HD mice; the addi- and Zeitlin, 2000). The wild-type htt knockout mice showed tional observation that the nuclear localization of the htt frag- progressive behavioural and motor deficits, as well as decreased ments occurred earliest and to the greatest extent in the striatum, survival, that were similar to findings in HD mice. Histological the area most affected in HD, further implicates a causal role for examination revealed tissue degeneration in the striatum and these fragments in disease. Indeed, further work has implicated cortex, again paralleling that seen in HD mouse models. these fragments as a critical step in HD pathogenesis, whereby Loss of wild-type huntingtin worsens prevention of cleavage of by specific caspases rescues the HD phenotype in mice. Graham et al. (2006) showed that expres- clinical disease progression sion of caspase-6-resistant mutant htt in HD mice was found to Discussed earlier were the findings from Van Raamsdonk et al. prevent striatal volume loss, and the mice showed the same level (2005b) that in mouse models of HD, the presence of wild-type of motor performance as controls, unlike HD mice with htt htt has no effect on striatal neuron number and striatal volume, amenable to caspase-6 cleavage. Nuclear translocation of htt arguing against a role for loss of function in histopathological fragments did occur in these mice, albeit at a later time point, changes in HD. However, the same study found that HD mice suggesting other htt cleavage mechanisms do exist. However, lacking wild-type htt were hypoactive at 2 months compared the absence of any neuropathological or behavioural deficits in with both those with wild-type htt and control. Accordingly, the these mice implicates caspase-6-mediated huntingtin cleavage as YAC128−/− mice performed less well on the rotarod test of a critical rate-limiting step in the pathogenesis of HD, lending motor coordination from 2 to 12 months of age. These findings further support to a gain of function hypothesis. were echoed by a later study in a Drosophila model, which showed that removal of endogenous huntingtin accelerated the neurodegenerative phenotype associated with polyglutamine Loss of function in Huntington’s toxicity (Zhang et al., 2009). Therefore, though loss of normal disease protein function does not seem to worsen the histopathology in the HD models, perhaps it is a factor in the timing and severity Though a loss of some of the aforementioned functions of hun- of the clinical manifestations of HD. tingtin could conceivably lead to reduced neuroprotection, less clear is whether a loss of function in HD is a causative factor, an Depletion of wild-type huntingtin parallels exacerbating factor or an epiphenomenon. Attempts to clarify disease progression in HD mice this have involved genetically manipulating wild-type htt levels and comparing phenotypes to those of HD animal models. R6/2 mice express exon 1 of human huntingtin with an expanded CAG repeat and develop a progressive HD-like phe- Not a simple loss of function, but loss of notype. Zhang et al. (2003) evaluated levels of wild-type htt at different stages of disease in R6/2 mice and compared them function may contribute with the levels of unaffected mice. Unaffected mice showed an It is logical to firstly consider a hypothesis of an exclusive loss increase in wild-type htt at 5, 7 and 12 weeks in comparison of function in HD. Compelling evidence against this however to at 1 week. The same was seen in R6/2 mice at 5 weeks, but comes from Wolf–Hirschhorn Syndrome: a disease character- at 7 and 12 weeks these mice showed a progressive reduction ized by a deletion of the short arm of chromosome 4, which in wild-type htt. This reduction was not due to reduced pro- contains the Huntingtin gene. These patients do not develop duction, suggesting increased protein metabolism or seques- HD, despite the loss of 50% of the normal levels of huntingtin tration, indicating a dominant-negative effect of the mutant protein (Gottfried, Lavine and Roessmann, 1981). Lending fur- huntingtin. Importantly, disease progression in R6/2 mice, ther support to this is the case of one phenotypically normal evaluated using measures of weight loss and tests of motor individual possessing a balanced translocation bisecting the HD coordination, closely paralleled the depletion of wild-type htt, gene locus (Ambrose et al., 1994), which indicates that inacti- supporting a causal role for loss of function. vation of one huntingtin allele is not sufficient to cause HD. However, this does not rule out a pathogenic contribution from Mice lacking BDNF show similarities to loss of function in conjunction with a toxic gain of function. HD mouse models Accordingly, the finding that mutant htt can recruit wild-type htt into insoluble aggregates both in vitro and in vivo (Cattaneo Wild-type htt promotes transcription of BDNF. If a loss of et al., 2001) suggests that the toxic effect might at least partially function was implicated in HD pathogenesis, one might lie in a dominant-negative effect on wild-type htt. expect to find a reduction in levels of BDNF in the brains of 5 Review article Bioscience Horizons • Volume 8 2015 those with HD. Accordingly, Zhang et al. (2003) found a sig- cause of HD and suggesting a dissociation between mutant nificant reduction in BDNF concentration in the brains of protein aggregates and toxicity, lending support to a contribu- symptomatic HD mice, which paralleled wild-type htt deple- tion from loss of function. However, recent suggestions that tion and disease progression. This is supported by observa- large mutant protein aggregates are not the toxic species in tions by Zuccato et al. (2001) of reduced BDNF protein in the neurodegenerative disorders propose a mechanism whereby cortices of HD patients. Though this evidence is consistent smaller intermediate species are instead pathogenic. Therefore, with a role for reduced BDNF in HD, the fact that the mice further study is required to clarify the implications of this dis- were symptomatic at time of analysis means it is difficult to sociation between NIIs and vulnerable neurons. elucidate whether reduced BDNF is a cause or effect of HD. Complementing the above studies are some comparing Implications upon treatment of BDNF knockout mice with HD mouse models; similarities in Huntington’s disease the histological or clinical pictures between the two would suggest that BDNF depletion contributes to at least those Preventing expression of the mutant htt allele is an attractive aspects of HD pathogenesis, rather than being an incidental or target, and mice with only one working copy of the Huntingtin secondary finding. BDNF cortical knockout mice show gene show no overt abnormalities (Imarisio et  al., 2008). reduced cross-sectional area, dendritic diameter and spine Though direct intraventricular injections of short interfering density of medium striatal neurons (Baquet, Gorski and Jones, RNA against mutant htt in HD mice have shown efficacy in 2004), changes seen in both HD mice and htt knockout mice. delaying onset of motor symptoms and prolonging survival In addition, behavioural phenotypes observed in BDNF (Wang et al., 2005), this technique may not be safe or practical knockout mice parallel those of HD mice, such as progressive in humans. Additionally, it would not be applicable to homozy- hind limb clasping. These similarities suggest a causal role in gous HD patients if huntingtin has critical functions. A more both the histopathological and clinical manifestations of HD. acceptable target may be to up-regulate autophagy, the cell’s Interestingly, however, these mice do not show poor rotarod own mechanism of mutant huntingtin clearance. Ravikumar performance compared with wild-type controls, unlike HD et  al. (2004) found that rapamycin enhanced clearance of mice. On face value, this discrepancy between models ques- mutant huntingtin exon 1 fragments in a cell model and slowed tions the role of BDNF depletion in HD. The explanation for neurodegeneration in fly and mouse models expressing mutant this may however lie in the design of the mouse model. The htt. Although no improvement was seen in the lifespan of mice mice used in this study were cortical BDNF knockout mice, treated with rapamycin, the delayed onset of symptoms was yet ∼15% of medium striatal neurons receive BDNF input found to be signic fi ant. If onset could be delayed beyond the from the substantia nigra (Johnston et al., 1990). In the corti- normal life expectancy, the disease would effectively be ‘cured’. cal BDNF knockout mice, some striatal neurons may still have been receiving BDNF from the substantia nigra, thus An advantage of the above treatment strategies is that as perhaps not truly recapitulating the global effects on BDNF of gain of function almost certainly contributes to HD pathogen- loss of huntingtin function. Comparison of rotarod perfor- esis, indicated by evidence supporting gain of function in con- mance of cortical BDNF knockout mice and htt knockout junction with the conclusion that loss of function alone does mice is therefore indicated, but certainly this apparent dis- not lead to HD, targeting the toxic species should, at the very crepancy cannot currently rule out a causal role for loss of least, slow progression. However, the fact that rapamycin did BDNF in HD pathogenesis. Accordingly, supporting a proac- not halt disease progression provides indirect support of a loss tive rather than reactive role for a reduction in BDNF in HD, of function contribution to disease pathogenesis, suggesting Strand et al. (2007) found that of all HD-related mouse mod- the need to address this contribution when designing thera- els, BDNF knockout mice have striatal mRNA expression peutic options. A logical strategy in parallel with those men- patterns that best recapitulate those seen in human HD, sug- tioned above would therefore be to restore normal functioning gesting that the striatal neurons in Huntington’s disease and of wild-type htt, either by replacing the protein itself or by BDNF-deprived environments suffer from similar insults. modifying its downstream targets. A potential problem with the former may arise in the event that loss of function occurs Huntingtin inclusions do not correlate via a dominant-negative effect. This may largely be circum- with neuronal vulnerability vented by the latter, though this would first require identifica- tion of the function, if any, whose loss contributes to disease. A classical finding in HD is neuronal intracellular inclusions (NIIs) and protein aggregates in dystrophic neurites in striatal Though theoretically promising, further studies are needed and cortical neurons. While the number of cortical inclusions to test efficacy of these strategies in treating HD rather than has been found to correlate well with the length of CAG simply reducing mutant or increasing wild-type protein levels. repeat expansion and age of disease onset (Becher et  al., Alongside this, other treatment options, largely focusing on 1998), neurons bearing NIIs do not correspond to the most more generalized neuroprotective strategies, have been sug- vulnerable ones. Kuemmerle et  al. (1999) found that some gested (Fig. 3). Ultimately, any approach to treating HD will interneurons with high numbers of NIIs were spared in the benefit from a further clarification on the specific pathological processes underlying HD. disease, indicating that NIIs may be a marker rather than 6 Bioscience Horizons • Volume 8 2015 Review article Figure 3. Therapeutic strategies for Huntington’s disease. Drug discovery has focused on removal of mutant htt or restoration of the normal htt functions. Additionally, more generalized neuroprotective strategies have been tested, such as attempts to decrease excitotoxic cell death or enhance neurotrophin release. Adapted from Imarisio et al. (2008). Huntington’s disease: from pathology and genetics to potential therapies. Biochemical Journal, 412(2), 191–209. Portland Press. Accessed at: doi: 10.1042/BJ20071619 (3 January 2015). seems a reasonable conclusion that loss of function can Conclusion worsen phenotype, but does that mean it truly contributes to pathogenesis; if any contribution from loss of function were There are three main scenarios regarding pathogenesis of neu- removed, would the disease follow a similar pattern of neuro- rodegenerative disorders. Firstly, disease is caused by a gain of pathology and clinical features? That question remains unan- toxic function, perhaps conferred by mutant protein aggre- swered, and until more is understood about the normal gates. Though evidence supports a correlation between protein functions, as well as the true impact of their loss under mutant proteins, neuropathology and clinical symptoms, as disease rather than normal conditions, it is likely to remain so, yet none definitively rules out a contribution from loss of leaving the contribution of loss of function to pathogenesis function. Secondly, a loss of normal protein function is the ambiguous. What the evidence reviewed here does suggest, cause, and aggregations of mutant protein are either second- however, is that loss of function has a role in directing toxicity, ary to disease or incidental findings. Conditions such as Wolf– whereby reduced BDNF transcription as a result of loss of Hirschhorn Syndrome largely contradict an exclusive role for huntingtin function may contribute to the selective vulnerabil- loss of function, however. A final option is that disease is ity of striatal neurons. caused by a combination of loss and gain of function. An increasing repertoire of studies supports this, and certainly the It is useful to consider any potential relevance of issues dis- proteins involved in these diseases carry functions that, if lost, cussed here within the wider arena of neurodegenerative dis- could conceivably lead to neurodegeneration. eases; interestingly, several parallels can be drawn. With regard A key question regarding the hypothesis addressed in this to loss of function directing toxicity, a similar state may be true review is what is meant by a loss of function contributing to in prion diseases, whereby loss of the neuroprotective signalling disease? Evidence from animal models of Huntington’s dis- role of prion protein at synapses (Zanata et  al., 2002) ease suggests that a loss of function accelerates disease pro- corresponds to the early loss of synapses in the disease (Jeffrey gression. In this way, and others discussed in this review, it et  al., 2000). Similarly, this review discusses a dissociation 7 Review article Bioscience Horizons • Volume 8 2015 Cattaneo, E., Rigamonti, D., Goffredo, D. et al. (2001) Loss of normal hun- between neuronal intracellular inclusions of mutant huntingtin tingtin function: new developments in Huntington’s disease and vulnerable neurons; a well-characterized observation in research, Trends in Neurosciences, 24 (3), 182–188. Alzheimer’s disease is that distribution of the toxic species, amyloid-β plaques, correlates poorly with the pattern and Cattaneo, E., Zuccato, C. and Tartari, M. (2005) Normal huntingtin func- severity of dementia (Terry et al., 1991). tion: an alternative approach to Huntington’s disease, Nature Reviews Neuroscience, 6 (12), 919–930. Armed with the knowledge that further characterization of the role of normal huntingtin protein in health and disease Dragatsis, I., Levine, M. S. and Zeitlin, S. (2000) Inactivation of Hdh in the is required, one must be mindful when developing treat- brain and testis results in progressive neurodegeneration and steril- ments of the possible detrimental effects of compromising ity in mice, Nature Genetics, 26 (3), 300–306. normal functions, as well as the potential beneficial effects of restoring these. In turn, failures and successes in trials involv- Furtado, S., Suchowersky, O., Rewcastle, N. B. et al. (1996) Relationship ing replacement of particular protein functions may feed between trinucliotide repeats and neuropathological changes in back into the understanding of the involvement of loss of Huntington’s diease, Annals of Neurology, 39 (1), 132–136. function in the disease. Encouragingly, similarities can be Gauthier, L. R., Charrin, B. C., Borrell-Pagès, M. et al. (2004) Huntingtin consistently drawn between several neurodegenerative disor- controls neurotrophic support and survival of neurons by enhancing ders, and thus, the principles discussed may impact upon the BDNF vesicular transport along microtubules, Cell, 118 (1), 127–138. direction of future research and drug discovery programmes in several neurodegenerative disorders in an attempt to suc- Gil, J. M. and Rego, A. C. (2008) Mechanisms of neurodegeneration in cessfully treat these increasingly prevalent and devastating Huntington’s disease, European Journal of Neuroscience, 27 (11), diseases. 2803–2820. Gottfried, M., Lavine, L. and Roessmann, U. (1981) Neuropathological Author’s biography findings in Wolf-Hirschhorn (4p-) syndrome, Acta Neuropathologica, 55 (2), 163–165. This review was adapted from Heidi’s BSc dissertation, which Graham, R. K., Deng, Y., Slow, E. J. et al. (2006) Cleavage at the caspase-6 studied loss of protein function in the context of both site is required for neuronal dysfunction and degeneration due to Huntington’s and Prion disease. She obtained a First Class mutant huntingtin, Cell, 125 (6), 1179–1191. Honours BSc in Neuroscience as an intercalated degree and was awarded the Neuroscience Scientifica University prize for Imarisio, S., Carmichael, J., Korolchuk, V. et al. (2008) Huntington’s dis- the highest overall degree mark. She subsequently completed ease: from pathology and genetics to potential therapies, her MBChB at the University of Leeds, graduating with Biochemical Journal, 412 (2), 191–209. Honours. 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Published: Oct 13, 2015

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