Rev. Neurosci. 2015; 26(1): 95–104

Yan Wu, Ying Peng* and Yidong Wang*

An insight into advances in the pathogenesis and therapeutic strategies of spinocerebellar ataxia type 3 Abstract: Spinocerebellar ataxia type 3 (SCA3) is the most common type of spinocerebellar ataxia, which are inherited neurodegenerative diseases. CAG repeat expansions that translate into an abnormal length of glutamine residues are considered to be the disease-causing mutation. The pathological mechanisms of SCA3 are not fully elucidated but may include aggregate or inclusion formation, imbalance of cellular protein homeostasis, axonal transportation dysfunction, translation dysregulation, mitochondrial damage and oxidative stress, abnormal neural signaling pathways, etc. Currently, symptom relief is the only available therapeutic route; however, promising therapeutic targets have been discovered, such as decreasing the mutant protein through RNA interference (RNAi) and antisense oligonucleotides (AONs) and replacement therapy using stem cell transplantation. Other potential targets can inhibit the previously mentioned pathological mechanisms. However, additional efforts are necessary before these strategies can be used clinically. Keywords: AONs; ataxin-3; deubiquitinating enzyme; poly Q disease; RNAi; SCA3. DOI 10.1515/revneuro-2014-0040 Received June 8, 2014; accepted July 30, 2014; previously published online September 12, 2014

Introduction Spinocerebellar ataxia (SCA) is a family of dominant autosomal inherited neurodegenerative diseases whose prevalence is 1/100,000–5/100,000 according to previous reports. Dominant autosomal inherited neurodegenerative diseases share common manifestations with *Corresponding authors: Ying Peng and Yidong Wang, Neurology Department, Sun Yat-sen Memorial Hospital, No. 107 Yanjian West Road, Guangzhou City, Guangdong Province, Guangzhou 510080, China, e-mail: [email protected], [email protected] Yan Wu: Neurology Department, Sun Yat-sen Memorial Hospital, Guangzhou 510080, China

cerebellar ataxia and are accompanied by dysarthria, intention tremor, ophthalmoplegia, and pyramidal and/or extrapyramidal symptoms (Durr, 2010). SCA is subdivided into several types; spinocerebellar ataxia type 3 (SCA3), also called Machado-Joseph disease, is the most common clinically observed SCA. SCA3 usually manifests as cerebellar ataxia with progressive external ophthalmoplegia, dysarthria, dysphagia, pyramidal signs, dystonia, rigidity, and distal muscular atrophies. An indistinct texture and decreasing volume of cerebellum, accompanied by the enlargement of the fourth ventricle, have been revealed in neuroimaging studies. SCA3’s highly variable clinical presentation has led to further subtype classification (Rosenberg, 1992): type 1 begins early in an individual’s life, often before the second decade, evolves more quickly, and is characterized by prominent pyramidal signs (rigidity and spasticity) and extrapyramidal signs (bradykinesia and dystonia) besides ataxia. Type 2 is the most common type, whose onset age is 20–50, and is characterized by ataxia with progressive ophthalmoplegia and extrapyramidal signs. Type 3 has a late onset, age of 40–75, and is characterized by ataxia with motor neuropathy and muscle atrophy. Type 4 is rare and is characterized by core symptoms in addition to Parkinsonism. Recently, a type 5 SCA3 was proposed, which presents as pure spastic paraplegia (Wang et  al., 2009). Other features besides those mentioned above are weight loss, restless leg syndrome, and relatively rare, mild cognitive and behavior disorders.

Hereditary features of SCA3 Like other members of the SCA family, SCA3 is dominant autosomal inherited. SCA3/ATXN3 is located on chromosome 14q32.1 and is composed of 48,240 base pairs, including 11 exons. An abnormal expansion of a CAG repeat (located in exon 10) in the mutant SCA3/ATXN3 3′ terminal encoding region is considered to be the pathogenic mutation. Normal CAG repeats range from 12 to 44 triplets, while in SCA3 patients, CAG repeats may reach

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96      Y. Wu et al.: SCA3 advances of pathology and therapy approximately 60–87 triplets. Trinucleotide numbers that are between those ranges present as disease with incomplete penetrance (Ichikawa et  al., 2001). Ataxin-3, the protein produced by SCA3/ATXN3, contains a poly Q peptide chain at its C-terminus. The mutant poly Q expansions selectively aggregate in cerebellar, brainstem, and spinal neurons and lead to the formation of neuronal nuclear inclusions (NNIs) (Schmidt et  al., 1998). Consequently, SCA3, together with spinal bulbar muscular atrophy, Huntington disease (HD), dentatorubral-pallidoluysian atrophy, Huntington disease-like 2, and six other SCAs (SCA1–3, 6, 7, 17) belong to the poly Q disease family. SCA3 usually has an onset age between 20 and 50, and studies have revealed that the CAG repeat expansion number is negatively correlated with onset age and is positively correlated with disease severity (Jardim et al., 2001). Anticipation is a predominant phenomenon in SCA3 families, where affected offspring tend to manifest the disease earlier than affected progenitors do. This dynamic feature (different lengths of CAG repeats in different generations) is presumed to be a result of CAG repeat intergenerational instability, where large repeats are prone to forming hairpin structures, causing replication to slow and leading to repeat replication, while normal sequences generally do not form hairpins and rarely mutate (Sequeiros and Coutinho, 1993). Moreover, gender is thought to influence SCA3 phenotype. Paternal mutant allele transmission is thought to increase generational instabilities compared with maternal transmission (Igarashi et  al., 1996), but interestingly, anticipation is predominant in the maternal transmission of Chinese SCA3 families. Simultaneously, the somatic mosaicism phenomenon is observed, where different CAG repeat numbers are present in different tissues of the same individual, which is induced by instabilities during mitosis and meiosis. However, there is no direct correlation between CAG repeat number and tissue damage (Maciel et al., 1997).

Advances in the pathogenesis of SCA3 The role of normal ataxin-3 in cellular function Normal ataxin-3 has a molecular weight of approximately 42 kDa, contains 361 amino acids, and is thought to be neuroprotective. Ataxin-3 has a Josephin domain (JD)

residing in the N-terminus and 2–3 ubiquitin-interacting motifs (UIMs) in different isoforms (the 3-UIM ataxin-3 is expressed predominantly in the brain). UIMs prefer specific ubiquitin (Ub) and ubiquinated protein substrates, which are cleaved by the JD in subsequent steps. This cooperation inhibits the formation of polyUb chains and promotes the regeneration of reusable Ubs, rescuing the target proteins from being degraded (Burnett et  al., 2003; Todi et al., 2009). As a result, ataxin-3 is implicated as a deubiquitinating enzyme (DUB) in the Ub proteasome system (UPS) and cellular homeostasis. The UPS is involved in various cellular processes, such as protein degradation, endocytosis, transcriptional regulation, and antigen presentation. The amino acids cysteine 14, histidine 119, and asparagine 134 are indispensable for the isopeptidase activity of JD (Nicastro et al., 2009), while UIMs mediate selective binding to the Ub chain and restrict the type of chains that can be cleaved by the JD (Masino et al., 2013). DUBs are known to interact with numerous Ub ligases (E3), which mediate the covalent attachment of Ub to lysine residues within target proteins. In recent years, the C-terminus of heat shock protein 70-interacting protein (CHIP) and Parkin have been demonstrated to be E3s that interact with ataxin-3. Along with self-regulation, ataxin-3 has been implicated to have the ability to alter the functional activities of CHIP and Parkin. Ataxin-3-mediated deubiquitination does not occur until a critical length of poly-Ub chains has conjugated onto the target substrate. Conversely, CHIP ubiquinates ataxin-3, which enhances the overall deubiquinating activity of ataxin-3. Therefore, through self- and cross-regulation, ataxin-3 opposes CHIP activity and uses its editing function against poly-Ub chains formed on target substrates, ensuring efficient degradation by proteasomes. If CHIP cannot act on its target proteins, ataxin-3 is able to edit the protein independently of CHIP, forming free C-termini that can be recognized by histone deacetylase 6 (HDAC6) and sequester the protein to aggresomes, alleviating toxicity due to misfolded proteins (Scaglione et al., 2011). A subset of SCA3 patients present Parkinsonismlike manifestations, raising the possibility that a Parkinson disease-associated protein may be involved in SCA3 pathogenesis; the most suspected candidate protein for this is Parkin. Unlike CHIP, Parkin self-ubiquitination is inhibited by ataxin-3, and Parkin is unable to ubiquinate ataxin-3 (Durcan et al., 2011). Strikingly, in an SCA3 mouse model, levels of both CHIP and Parkin are significantly decreased when there are more than 51 amino acids in the poly Q tract, which disrupts cellular homeostasis and promotes neuron loss.

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In addition to E3s, ataxin-3 associates with shuttle proteins that assist in the delivery of target proteins to proteasomes. It has been established that the Valosin-containing protein or ATPase p97 (VCP/p97)/ataxin-3 complex participates in this process. Furthermore, this complex also participates in the endoplasmic-reticulum-associated degradation process (Zhong and Pittman, 2006). More­ over, ataxin-3 participates in DNA repair by interacting with HHRB23A/B, a human homologue of yeast RAD23 (Wang et al., 2000). Intriguingly, ataxin-3 can also regulate transcriptional activity. Studies indicate that ataxin-3 is able to repress the transcription of matrix metalloproteinase 2 and that this process is enhanced after ataxin-3 phosphorylation, which increases nuclear localization (Evert et al., 2011). Ataxin-3 inhibits cAMP-response element binding protein (CREB)associated transcription by interacting with CREB-binding protein, p300, and p300/CREBBP-associated factor (Li et al., 2002). Histone acetylation is another transcriptional regulation process influenced by ataxin-3. By interacting with HDAC3 and nuclear receptor co-repressor 1, histone deacetylation is promoted (Burnett and Pittman, 2005). Ataxin-3 has been demonstrated to directly bind with DNA via a putative leucine zipper motif (Landschulz et al., 1988); whether ataxin-3 acts as a classical transcriptional repressor remains unknown. Recently, reports have suggested that ataxin-3 stabilizes the forkhead box O (FOXO) transcription factor FOXO4 during oxidative stress; both proteins translocate into the nucleus and promote the transcription of manganese superoxide dismutase (SOD2), which may protect cells from oxidative damage (Araujo et al., 2011). Ataxin-3 also participates in cytoskeletal organization processes and the formation of local adhesions. In this cellular pathway, ataxin-3 regulates the level of integrin and other protein components during integrin signaling transduction in a DUB activity dependent manner (do Carmo Costa et al., 2010).

Altered properties and functional activities of mutant ataxin-3 Studies suggest that ATXN3 expression levels do not directly correlate with the severity of the involved brain region, which supports the hypothesis that altered mutant ataxin-3 functional activities rather than ataxin-3 accumulation may be more important for SCA3 pathogenesis. NNIs and neuronal cytoplasmic inclusions formed by mutant ataxin-3 aggresomes are thought to be the pathological hallmark of SCA3. There has been wide debate centered upon whether these inclusions are cytotoxic.

However, there is a positive correlation between inclusion quantity, CAG repeats, and the severity of SCA3 manifestation. Moreover, these inclusions consist of ubiquitins, proteasomal components, chaperones, transcription factors, and normal ataxin-3, and deficiencies of these essential elements can disrupt various cellular processes (Chai et  al., 2001). Currently, the mainstream opinion is that these inclusions are biomarkers of cells that are overwhelmed by mutant ataxin-3 degradation. Normal ataxin-3 tends to aggregate in vitro. Aggregation occurs via self-association; the JD forms dimers and then fibrils that are SDS soluble, which is similar to other amyloidgenic proteins. The conformation conversion from an α-helix into a β-sheet causes JD to lose catalytic activity (Masino et al., 2011). Under normal circumstances, an interaction with Ub or αβ-crystallin inhibits JD self-association (Robertson et  al., 2010). However, in pathological conditions, protein-protein interactions are reduced, and SDS-soluble fibrils are assembled more rapidly. More importantly, glutamine side-chain hydrogen bonding in the poly Q region may contribute to the formation of special conformations such as β-helix turns or hairpin structures, producing SDS-insoluble fibrils (Koch et al., 2011). Mutant ataxin-3 aggregation is thought to be enhanced by proteolysis, and caspase and calcium-dependent calpain are thought to be involved in this process. Caspases, e.g., caspases 1 and 3, have been implicated in the pathogenesis of ploy Q diseases, such as HD and DTPLA. Nevertheless, compared with these diseases, caspases appear to have less efficient proteolytic activity in SCA3 (Natalello et al., 2011). Both normal and mutant ataxin-3 proteins are thought to undergo the same types of caspase and calpain cleavage (Jung et al., 2009), but the stability of the generated fragments differ with or without the poly Q tract. The C-terminus fragments are cleaved proximal to amino acid 190 and contain UIMs, the expanded poly Q tract, and nuclear localization signal (NLS), which are thought to act as a seed for aggregation (Teixeira-Castro et al., 2011), a phenomenon known as the ‘toxic fragment hypothesis’. These fragments are more abundant in the nucleus of cells in the affected brain regions, most likely due to a lack of the nuclear export signal rather than the possession of a weak NLS (Antony et  al., 2009), which allows the protein to evade UPS clearance in the cytoplasm (Breuer et al., 2010). In addition, the translocation of both the normal and mutant ataxin-3 proteins from the cytoplasm to nucleus is mediated by casein kinase 2 (Mueller et  al., 2009), which is more often observed during heatshock and oxidative stress conditions (Reina et al., 2010). Various protein quality control systems cooperate to maintain cellular homeostasis. To refold and

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98      Y. Wu et al.: SCA3 advances of pathology and therapy eliminate mutant ataxin-3, molecular chaperones, UPS, and autophagy are mobilized (Chai et al., 1999; Berger et al., 2006). During the earlier stages of the disease, an increase in specific molecular chaperones facilitates mutant ataxin-3 refolding and decreases aggregates (Chou et  al., 2008). One protein that participates in this process is heatshock factor 1 (HSF-1) (Teixeira-Castro et al., 2011). In later stages of the disease, HSP70 and HSP40 down-regulation is observed (Chou et al., 2008), impeding the neuronal ability to manage stress. HSP27 expression has also been observed to be compromised in cell lines (Chang et al., 2009). It has been mentioned above that E3 partners of ataxin-3, CHIP and Parkin, are decreased in pathological conditions, which disrupts their functions in the UPS. The expanded ploy Q tract increases affinity for CHIP, most likely altering the functional relationship between the two and leading to the inevitable degradation of CHIP. An alternative, and perhaps more reasonable, explanation attributes the decrease in CHIP and Parkin to autophagylysosome formation caused by poly Q tract aggregates rather than to interaction alterations. Autophagy eliminates aggresomes and leads to the diminishment of these E3 partners simultaneously, which is a result of clearance (Durcan et  al., 2011). Subsequent studies have indicated that Parkin participates in the removal of damaged mitochondria via the autophagic process, also referred to as ‘mitophagy’ (Narendra et al., 2008). Mitochondrial abnormalities have been identified in various poly Q diseases in addition to SCA3. Interactions between the poly Q tract and mitochondria have been implicated to damage the mitochondria (Kazachkova et al., 2012). Similarly, the aberrant conjugation of mutant-3 and VCP results in a delayed release in VCP from the VCP/E4B complex, impeding subsequent proteasomal degradation. Additionally, the tight binding of mutant ataxin-3 and VCP also compromises the ERAD, disrupting ERAD-related cellular processes (Zhong and Pittman, 2006). Mutant ataxin-3 has been observed to aggregate in the neuronal nucleus and cytoplasm as well as in fiber tracts, leading to swelling and aberrant neural branches in neural processes, which impairs synaptic transmission and initiates neurodegeneration (Seidel et al., 2010). Conversely, normal ataxin-3 interacts with cytoskeleton organizational components, but mutant ataxin-3 does not (do Carmo Costa et  al., 2010), suggesting cytoskeletal transportation dysfunction. Collectively, axonal functional damaged caused by mutant ataxin-3 is potentially crucial to SCA3 onset. As mentioned above, normal ataxin-3 participates in transcriptional regulation, while deacetylated complex

assembly failure due to mutant ataxin-3 represses transcription. Genes that have down-regulated transcription include genes that participate in glutaminergic transmission, intracellular calcium signaling or MAP kinase pathways, GABA A/BA receptor subunits, HSPs, and transcriptional factors regulating neural survival and differentiation (Chou et al., 2008). Up-regulated genes are involved in neural death and inflammation (Chou et al., 2008). For example, ataxin-3 was recently demonstrated to be able to stabilize and act with FOXO4, increasing SOD2 expression (Araujo et al., 2011). Additionally, the detection of neuroinflammatory markers in the pons of SCA3 patients provides evidence that glia contribute to SCA3 pathogenesis (Evert et  al., 2003). However, the precise role of glia in SCA3 pathogenesis remains uncertain. Consistent with the altered transcription of HSPs and SOD2, the clearance of reactive oxygen species is thought to contribute to SCA3 pathogenesis. For instance, decreased antioxidant enzyme activity and increased apoptosis are mediated by the mitochondria in some cellular models (Yu et al., 2009). However, it is remains controversial whether an apoptosis increase exists because no such evidence has been observed in other cellular and animal models of SCA3 (Silva-Fernandes et  al., 2010). Nonetheless, previous studies have suggested that mitochondrial impairment and oxidative stress are indispensable during disease development. In addition to the down-regulation of genes involved in intracellular calcium signaling/mobilization or MAP kinase pathways, intracellular Ca2+ decreases via an abnormal association with the type 1 inositol 1, 4, 5-triphosphate receptor (InsP3R1) (Chen et  al., 2008). Moreover, potassium channel dysfunction and resting membrane potential depolarization have been observed (Shakkottai et al., 2011), suggesting altered physical function and signal transduction. Studies have determined that during the early disease stage, when mice display motor incoordination prior to neural degeneration, repetitively firing Purkinje cells in transgenic mice become inactivated, supporting the hypothesis that the impairment of Purkinje electrophysiological function develops prior to cellular death (Shakkottai et al., 2011). In summary, mutant ataxin-3 causes significant cellular dysfunction and induces neural death via the following pathways: (1) aggresome generation, (2) imbalance of cellular protein homeostasis, (3) transcriptional dysregulation, (4) impairment of axonal transport, (5) mitochondrial damage and oxidative stress, and (6) abnormal neuronal signaling transduction.

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RNA toxicity comes along for the ride Mutant protein toxicity is a wildly accepted concept; however, evidence has emerged that implicates RNA toxicity in pathogenesis. Noncoding CAG expansions introduced in a SCA3 Drosophila model led to neuronal degeneration, while interspersed CAA units resulted in a mild phenotype (Li et al., 2008). This RNA toxicity is thought to be partly due to hairpin formation, sequestering RNA binding proteins and leading to alternative splicing. In addition, bidirectional translation has been observed in other poly Q diseases, such as HD, and the expression of antisense transcripts cannot be ignored. In cooperation with bidirectional transcription, translational reading frame shifting and non-ATGinitiated translation produce various toxic products, including poly alanine (poly A) and poly serine (poly S) sequences (Zu et al., 2011). In addition to transcript alteration, gene silencing mediated by small and microRNAs may occur, which results in neural degeneration (Alves et al., 2010).

Advances in SCA3 therapeutic strategies There are no available treatments for SCA3 and other poly Q diseases to date. Currently, pharmacological and nonpharmacological therapies are used to provide symptom relief. Current studies investigating SCA3 therapeutic strategies focus primarily on the following: first, targeting disease-causative proteins via silencing specific genes and transcripts; and second, targeting crucial events in pathogenesis based on the detailed understanding of pathological mechanisms. There have been some promising advances for both approaches.

Approaches to inhibit mutant ataxin-3 expression The nonallele-specific manner for ATXN3 silencing has been shown to down-regulate levels of both normal and mutant ataxin-3, in both wild-type and transgenic mouse models. Although there appears no detectible side effects in animal studies with silenced or knocked out normal and mutant genes, these results are difficult to translate to humans. With regard to the relatively important roles of normal ataxin-3 in cellular functions, allele-specific

silencing may be a more appropriate approach for SCA3 treatment. The following section details allele-specific or semispecific silencing strategies available to date. The first strategy targets specific single nucleotide polymorphisms (SNPs) within the mutant ATXN-3 sequence to achieve RNA interference (RNAi) (Gaspar et al., 2001). This target SNP located in the 3’-terminus of the ATXN3 gene exists in more than 70% of SCA3 patients (Bilen et al., 2006). Short hairpin RNAs that have specific affinity for the target SNP specifically induce silencing of the mutant ataxin-3 gene, and the protective effect of this approach has been confirmed in animal models (Gaspar et  al., 2001). However, additional research must be performed because there is no optimized delivery system to deliver RNAi into the central nervous system. The second strategy focuses on directly targeting the expanded CAG repeats. Modified antisense oligonucleotides (AONs) successfully bind to expanded CAG repeats in vitro, resulting in the transcriptional blockage of mutant ataxin-3 (Evers et  al., 2011; Hu et  al., 2011). Basic substitutions and single-stranded silencing RNA, based on RNAi principles, can achieve allele-specific mutant ataxin-3 down-regulation in a similar manner (Liu et  al., 2013a,b). A major issue with using these molecules is their inadequate affinity to their intended target sequence, which leads to poor potency. To correct this defect, high concentrations are required, which can lead to ‘off-target’ effects, i.e., the unintended downregulation of endogenous transcripts, such as HD, TATA binding protein, and other ataxins. To date, chemical modifications, such as locked nuclease acid and bridge nuclease acid (BNA), are modification strategies that are utilized to promote affinity and retain potencies, whereas peptide nuclease acid and phosphorodiamidate morpholino oligomers, known as uncharged DNA analogues, are also used to promote efficient binding (Watts and Corey, 2012). Another approach being considered is exon skipping. AONs are designed to mask a single exon within the premRNA, resulting in the deletion of the poly Q sequence in spliced mRNA; normal reading frames are retained, resulting in the translation of normal ataxin-3 (Evers et al., 2013). Recently, this concept was successfully proven in cell lines, where the desired RNA sequence and a normal ataxin-3 chain without a poly Q sequence were obtained. These protein products retained their functional domains and activity and were able to bind Ub molecules without cellular toxicity (Evers et al., 2013). The primary advantage of AONs is their efficient uptake in vivo, and AON application in animal models of other neurodegenerative diseases, such as HD and

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100      Y. Wu et al.: SCA3 advances of pathology and therapy amyotrophic lateral sclerosis (ALS), has been performed. Through intrathecal injection in primate models, AONs were distributed ubiquitously in the brain and were detected in the brain regions most severely involved in SCA3, including the cerebella, pons, midbrain, spinal cord, etc. (Kordasiewicz et al., 2012). The safety of the AON intrathecal injection was confirmed in a phase I clinical trial using AONs to treat ALS patients, where no significant adverse events were observed (Miller et al., 2013). In addition, in Duchenne muscular dystrophy (DMD) clinical trials, the hypodermic injection of AONs decreased dystrophin levels and improved clinical symptoms with acceptable tolerance (Cirak et al., 2011). Despite their superior potency, siRNAs have delivery challenges and evoke native immune responses when delivered in cationic lipid-based formulations (Watts and Corey, 2012). Although these barriers must be overcome before gene silencing strategies are viable drug candidates, previous and ongoing studies have indicated that these therapies will be available in the future.

Approaches focused on d ­ ownstream pathogenic mechanisms Intensive inhibition aimed at disease-causing cellular processes is an indispensable component of SCA3 treatment. Targeting mutant ataxin-3 elimination by up-regulating autophagy and UPS is a promising strategy. Lentiviralmediated overexpression of the autophagy protein beclin-1 has been demonstrated to increase mutant ataxin-3 clearance and decrease aggregates in a SCA3 model (Nascimento-Ferreira et al., 2011). Rapamycin ester (temsirolimus) was proven to decrease both aggregated and soluble mutant proteins in SCA3 mice, whereas normal ataxin-3 proteins remained intact (Menzies et  al., 2010). Temsirolimus has been used to treat renal cell carcinoma via the autophagic pathway; therefore, it is also a candidate for SCA3 treatment. In addition, lithium carbonate was recently tested in a phase 2 clinic trial of SCA3, and a significant inhibition of the progression of gait ataxia was observed (Saute et  al., 2014). However, there was no effect on disease progression because an elevation of the neurological examination score for cerebellar ataxia was not observed, most likely due to limited sample size (Saute et  al., 2014). Additionally, H1152 has been proven to activate the proteasome to eliminate mutant axtaxin-3 in SCA3 mice, alleviating neuronal death and the disease phenotype (Wang et al., 2013).

We can gain knowledge from research studies examining neuronally protective antioxidants in HD. For example, creatine and coenzyme Q10 have produced some benefits in HD studies (Hersch et al., 2006; Hyson et al., 2010) and may be promising pharmaceuticals for SCA3 treatment. The transcriptional down-regulation and histone H3/ H4 hypoacetylation mediated by mutant ataxin-3 in transgenic SCA3 mice can be reversed by the HDAC inhibitor sodium butyrate and consistently improve motor performance (Minamiyama et al., 2004). Another HDAC inhibitor, valproic acid, demonstrated similar effects in SCA3 transgenic Caenorhabditis elegans (Teixeira-Castro et al., 2011). Dantrolene was capable of stabilizing intracellular Ca2+ signaling in SCA3 mice, improving motor performance and decreasing neuronal death (Chen et al., 2008). The short-term administration of SKA-31, an activator of calcium-activated potassium channels, resulted in the partial correction of Purkinje neuron firing and improved motor abilities in SCA3 mice (Chen et al., 2008). As mentioned above, the C-terminus fragments generated by caspase and calpain proteolysis increase aggregation. However, caspase inhibitors are not suitable for administration to SCA3 patients because of their controversial effects on pathogenesis, in which processes including apoptosis, synaptic plasticity, dendrite differentiation, and memory formation are all affected. In contrast, calpain inhibitors are more promising candidates; however, they have unsatisfactory selectivity. Recently, aggregates and nuclear localization of mutant proteins have been reduced via the up-regulation of an endogenous calpain inhibitor, calpastatin, in SCA3 mice (Simões et al., 2012). The elevation of molecular chaperone levels, such as HSP40 and HSP70, enables the elimination of mutant ataxin-3 and decreases the amounts of aggregates in several cell and animal models (Adachi et  al., 2003). Strikingly, HSP104, a powerful protein disaggregase derived from yeast, was recently introduced into a SCA3 Drosophila model and was established as the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression (Cushman-Nick et  al., 2013). Some chemical molecular chaperones cannot be used for SCA3 treatment because of their concentration-dependent cytotoxicity. Congo red, minocycline, and chlorpromazine are inhibitors of aggregation in vitro and have been tested in HD models demonstrating limited effects (Schilling et al., 2004). Intrabodies, vector-encoded small antibodies that can bind the poly Q tract and decrease aggregates, improved the phenotype of HD mice (Snyder-Keller et  al., 2010); however, no intrabodies have been developed for SCA3. Additionally,

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17-AAG and its analog 17-DMAG (HSP90 inhibitor) and Y-27632 (Rho kinase inhibitor) are potential candidates that target aggregate reduction (Bauer et al., 2009; Teixeira-Castro et al., 2011; Silva-Fernandes et al., 2014).

Stem cell transplantation holds promise Moreover, stem cell transplantation may be a new strategy for SCA3 treatment, including embryonic stem cells, neuronal stem cells, mesenchymal stem cells (MSCs), and induced pluripotent stem cells. Stem cells have enormous potential and are expanding our current understanding of the molecular mechanisms of neurodegeneration. Stem cell-based therapies can be beneficial by acting through several mechanisms, such as cell replacement, trophic support, neuroprotection, and immunomodulation (Martínez-Morales et al., 2013). The study of stem cell therapy offers promising solutions for the treatment of incurable poly Q diseases such as SCA1 or HD (Fan et  al., 2014). A phase I clinical trial confirmed that MSC transplantation into the spinal cord of ALS patients is safe (Mazzini et  al., 2010), suggesting that it may be used to preserve brain functions through neuron replacement. However, the selection of suitable cellular grafts remains uncertain, and the factors that control stem cell differentiation, survival, and maturation are also unknown. Currently, there are no studies examining stem cell application in SCA3. In summary, despite the few pharmaceuticals that have been tested in SCA3 clinic trials, research into SCA3 therapeutic approaches has resulted in encouraging improvements, particularly in animal models. It is necessary to develop safer and more effective drugs and treatments (e.g., stem cell transplantation) while enhancing delivery to the central nervous system with low toxicity and adverse effects. Lessons can be learned from other poly Q disorders, such as HD; however, the mechanisms are not interchangeable because SCA3 has unique characteristics. We hope that further research into the pathologic mechanisms of SCA3 will lead to effective SCA3 treatments.

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