Therapeutic prospects for spinocerebellar ataxia type 2 and 3

Abstract

Spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3) are autosomal-dominant neurodegenerative disorders. SCA2 primarily affects cerebellar Purkinje neurons. SCA3 primarily affects dentate and pontine nuclei and substantia nigra. Both disorders belong to a class of polyglutamine (polyQ) expansion disorders. SCA2 is caused by a polyQ expansion in the amino-terminal region of a cytosolic protein ataxin-2 (Atxn2). SCA3 is caused by a polyQ expansion in the carboxy-terminal portion of a cytosolic protein ataxin-3 (Atxn3). Both disorders are found worldwide, but SCA2 is common among people of Cuban decent and SCA3 is common among people of Portuguese decent. No effective treatment exist for SCA2, SCA3 or any other polyQ-expansion disorder. Based on anecdotal evidence, a number of small scale clinical trials have been attempted previously for SCA2 and SCA3. These trials were underpowered and did not yield any promising results so far. A number of pathogenic mechanisms have been proposed to explain neuronal dysfunction and degeneration in SCA2 and SCA3. Knockdown of mutant Atxn2 and Atxn3 protein by RNAi or similar approach is most promising avenue of therapeutic development in the long term, but translation of this approach to clinic faces very serious technical challenges. Recent preclinical studies in SCA2 and SCA3 genetic mouse model suggested that abnormal neuronal calcium (Ca²⁺) signaling may play an important role in SCA2 and SCA3 pathology. These studies also suggested that dantrolene and other Ca²⁺ signaling inhibitors and stabilizers may have a therapeutic value for treatment of SCA2 and SCA3. Controlled clinical evaluation of dantrolene, memantine, riluzole, dihydropyridines, CoQ10, creatine or other Ca²⁺ blockers and stabilizers in SCA2 and SCA3 patients is necessary to test clinical importance of these ideas. The EUROSCA consortium provides a potential framework for such clinical evaluation.

Spinocerebellar ataxias (SCAs) – clinical overview

The SCAs are a genetically and clinically heterogeneous group of autosomal dominantly inherited progressive ataxia diseases. Up to now, almost 30 different gene loci have been found [1]. Of the mutations that have been identified, SCA1,2,3,6,7,17 are translated CAG repeat expansions that code for an elongated polyglutamine tract within the respective proteins [2-9]. These diseases belong to a larger group of polyglutamine disorders that also include Huntington's disease, dentatorubro-pallidoluyisian atrophy and spinobulbar muscular atrophy [10,11]. Three SCAs, SCA8,10,12 are caused by untranslated repeat expansions in non-coding regions of the respective genes [12-14]. In SCA5 (beta-III spectrin, SPTBN2) [15], SCA11 (tau tubulin kinase-2, TTBK2) [16], SCA13 (voltage-gated potassium channel, KCNC3) [17], SCA14 (protein kinase Cγ, PKCγ) [18], SCA15 (inositol 1,4,5-triphosphate receptor type 1, ITPR1) [19], SCA27 (fibroblast growth factor 14, FGF14) [20], SCA28 (AFG3L2) [21] and 16q22-linked ADCA (puratrophin) [22] non-repeat mutations have been found in the respective genes. In all other SCAs, the affected genes and mutations have not yet been identified. There are only few prevalence studies of SCAs. According to these studies, the prevalence ranges between 3.0 and 4.0 for 100,000 inhabitants [23,24]. Due to founder effects, the prevalence may be much higher in certain regions. For example, the prevalence of SCA2 is 43 for 100,000 in the Cuban province Holguin [25]. The worldwide most frequent SCA is SCA3 [26].

Spinocerebellar ataxia type 2 (SCA2)

Autopsy studies of SCA2 patients consistently show olivopontocereballar atrophy with marked reduction of Purkinje cells, degeneration of the inferior olives, pontine nuclei, and pontocerebellar fibers [27,28]. Atrophy of the cerebellum and brainstem can be nicely visualized in vivo by MRI [29]. In most cases, there is additional degeneration of posterior columns and spinocerebellar pathways, and cell loss in the substantia nigra. Ubiquitinated nuclear inclusions have not been observed in brains of SCA2 patients. SCA2 patients suffer from a progressive cerebellar syndrome with ataxia of gait and stance, ataxia of limb movements, and dysarthria [1,28,30]. Saccade slowing is a highly characteristic feature that is observed in the majority of SCA2 patients. About half of the patients have vertical or horizontal gaze palsy. Cerebellar oculomotor abnormalities are rarely found in SCA2. Typically, tendon reflexes are absent or decreased. Pyramidal tract signs are present in less than 20% of the patients. Vibration sense is decreased in most patients, while sensation is otherwise normal [29,31,32]. Atypical SCA2 phenotypes with prominent dementia, an ALS-like presentation and levodopa-responsive parkinsonism are also encountered. Disease onset in SCA2 varies between childhood and adulthood with an average around the age of 30 years. As in other polyglutamine disorders, there is an inverse correlation between CAG repeat length and age of onset. Anticipation is present in many SCA2 families, in particular if the disease is inherited from the father. Median latency to become wheelchair-bound after disease onset is 15 years, median survival after onset of symptoms 21 years, and median age at death 68 years [28,33].

Spinocerebellar ataxia type 3 (SCA3)

SCA3 is a multi systemic disorder characterized by degeneration of spinocerebellar tracts, dentate nucleus, pontine and other brainstem nuclei, substantia nigra and pallidum [34]. In contrast to most other SCA, the cerebellar cortex and the inferior olives are widely spared. MRIs show cerebellar atrophy which is less pronounced than in SCA2 in combination with brainstem atrophy. Nuclear inclusions containing expanded ataxin-3 have been found in neurons of affected brain regions. The clinical picture of SCA3 is characterized by a wide range of clinical manifestations, the precise nature of which partly depends on repeat length. All SCA3 patients suffer from a progressive syndrome with ataxia of gait and stance, ataxia of limb movements, and dysarthria [35]. Vertical or horizontal gaze palsy are frequent additional findings which occur independently of age of onset. At least 40% of SCA3 patients have a levodopa-responsive restless legs syndrome. Saccade velocity is usually normal. Patients with very long repeats, usually of more than 74 have an early disease onset, and clinical features of pyramidal tract and basal ganglia involvement. Most of these patients have increased tendon reflexes, extensor plantar responses, spasticity, and dystonia. Patients with an intermediate repeat length have a disease onset in middle age and show mainly ataxia and gaze palsy. In patients with shorter repeats and a later disease onset, signs of peripheral neuropathy with loss of tendon reflexes, amyotrophy, and decreased vibration sense are often prominent. However, the boundaries between these clinical syndromes are vague, and the clinical phenotype of an individual may change with progression of the disease [32,36]. The onset in SCA3 varies between adolescence and late adulthood with an average around the age of 42 years. As in other polyglutamine disorders, there is an inverse correlation between CAG repeat length and age of onset. Median latency to become wheelchair-bound after disease onset is 15 years, median survival after onset of symptoms 25 years, and median age at death 72 years [33,34].

2. Spinocerebellar ataxias (SCAs) – molecular genetics and pathogenic mechanisms

It is generally assumed that many polyQ-expansion disorders share a common pathogenic mechanism [11,37,38]. Huntington's disease (HD) is the most extensively studied among these disorders. As a result of these studies it was concluded that polyQ expanded Huntingtin protein (Htt^exp) acquires a “toxic gain of function” [39]. A number of toxic functions have been assigned to Htt^exp, including formation of toxic aggregates, effect on gene transcription, induction of apoptosis, and disruption of key neuronal functions such as proteosomal function, ubiquitination, axonal transport, endocytosis, synaptic transmission and Ca²⁺ signaling [40-46]. Many of the proposed mechanisms suggest that the mutant Htt^exp is involved in pathological interactions with other signaling proteins in cells, leading to neuronal dysfunction and death. As discussed in the following sections, similar ideas have been expanded from HD to other polyglutamine expansion disorders including SCA2 and SCA3.

Spinocerebellar ataxia type 2 (SCA2)

At the molecular level, the cause of SCA2 is the expansion of an unstable trinucleotide CAG repeat in the ATXN2 gene, which encodes a polyglutamine (polyQ) track in the ataxin-2 protein (Atxn2). In normal individuals, ATXN2 alleles contain between 14 and 31 CAG repeats, with the 22 CAG repeat allele predominant in the normal population [3-5]. The pathological alleles of ATXN2 found in SCA2 patients contain ≥32 CAG repeats [3-5,47]. The Atxn2 is a cytosolic ubiquitously expressed protein composed of 1,312 amino acid residues with a molecular mass of approximately 140 kDa [48] (Fig 1A). Atxn2 contains an RNA-binding Lsm domain [49,50] (Fig 1A) and has been implicated in RNA translation and splicing [49,51,52]. The role in endocytosis [53], and actin-cytoskeleton organization [54] were also postulated for Atxn2. Genetic knockouts of ATXN2 orthologs in fly and worm resulted in embryonic lethality [54,55]. Atxn2 knockout mice were viable but displayed a late-onset obesity phenotype [56]. Mice deficient in Atxn2 did not show Purkinje cell loss or marked changes in the Purkinje cell dendritic tree [56]. Non-essential role of Atxn2 in rodents is most likely related to the presence of orthologs and redundancy in its function [56].

Fig 1. Domain structure of ataxin-2 and ataxin-3.

Based on [48]

A. Ataxin-2 is a 1312 aa protein. The polyQ-expansion region (Q) is in the amino-terminal portion of the protein (aa 166-187). Two globular domains in the context of ataxin-2 are Lsm domain (Like Sm, aa 254-345) and LsmAD domain (Lsm-associated domain, aa 353-475) of unknown function. The rest of the protein is unstructured but contain PAM2 motif (aa 908-925) which binds to polyA-binding protein.

B. Ataxin – 3 is 376 aa protein. The polyQ-expansion region (Q) is in the carboxy-terminal portion of the protein (aa 296-317). Ataxin-3 consists of amino-terminal Josephin domain (aa 1 – 170) which has deubiquitinating activity. The rest of the protein is unstructured but contains 3 ubiquitin-interacting motifs (UIMs).

The connection between polyQ-expansion in Atxn2 and neuronal degeneration is poorly understood. The SCA2 mouse model was developed which express cDNA for a human Atxn2-58Q protein under control of Purkinje cell–specific promoter [57]. These mice displayed age-dependent progressive phenotype in motor coordination assays and approximately 15% loss of cerebellar Purkinje cells by 12 months of age [57,58]. Analysis of these mice revealed that Atxn2-58 remained cytosolic in Purkinje neurons and did not form nuclear inclusions or was heavily ubiquitinated [57]. More recently SCA2 mouse model expressing human Atxn2-75Q gene under control of self promoter was established [59]. These mice also demonstrated rotarod deficit and Purkinje cell degeneration when compared with the wild type mice [59]. Genetic analysis of age of onset modifiers in SCA2 families revealed a connection with polymorphisms in CACNA1A voltage-gated Ca²⁺ channel gene [47]. The connection between SCA2 and neuronal Ca²⁺ signaling was further strengthened by discovery that polyQ-expanded Atxn2 but not wild type Atxn2 directly binds to and activates type 1 inositol (1,4,5)-trisphosphate receptor (InsP₃R1), an intracellular Ca²⁺ release channel [58]. In the same study it was demonstrated that dantrolene, a clinically relevant inhibitor of intracellular Ca²⁺ release, protects Purkinje cells from SCA2-58Q mice model from cell death both in vitro and in vivo [58]. These results provided strong experimental support to the “Ca²⁺ hypothesis of SCA2 pathogenesis” [58] (Fig 2).

Fig 2. Ca²⁺ hypothesis if SCA2 and SCA3.

Based on [58,83].

Pathological interactions between polyQ-expanded Atxn2 or Atxn3 (Atx^exp) and InsP₃R1 carboxy-terminal result in supranormal Ca²⁺ release from the endoplasmic reticulum (ER) in response to stimulation of mGluR receptors and InsP₃ generation. Resulting excessive Ca²⁺ release causes activation of calpains and promotes mitochondrial Ca²⁺ overload and mitochondrial permeability pore (MPTP) opening, induction of apoptosis and apoptotic cell death. Potential points of therapeutic intervention include partial inhibition of Ca²⁺ release from the ER by dantrolene, block of NMDAR-mediated Ca²⁺ influx by memantine (MMT), block of Ca²⁺ influx mediated by voltage-gated Ca²⁺ channels by dihydropyridines (DHP), inhibition of glutamate signaling by riluzole and stabilization of mitochondrial function by CoQ10 and creatine.

Spinocerebellar ataxia type 3 (SCA3)

At molecular level the cause of SCA3 is a polyQ expansion in the carboxy-terminal of ataxin-3 (Atxn3) protein [60,61]. The ataxin-3 is a 43 kDa cytosolic protein of 316 amino acids which contains amino-terminal Josephin domain and 3 ubiquitin-interactions motifs (UIMs) [48,62] (Fig 1B). The solution structure of Atxn3 Josephin domain was solved by NMR [63]. Consistent with the presence of Josephin domain Atxn3 functions as deubiquitinating enzyme (DUB) [64]. Atxn3 binds both K48- and K63-linked ubiquitin chains, yet preferentially cleaves K63-linked Ub chains in vitro [65]. Therefore, Atxn3 appear to function as a novel DUB that edits topologically complex short, mixed-linkage ubiquitin chains. The polyQ-expansion has no effect on DUB activity of Atxn3 [64,65], so it is not likely that neurodegeneration in SCA3 develops due to impaired normal function of Atxn3. Ataxin-3 has been shown to act as a potent transcriptional repressor, a function that is probably related to its DUB activity [66]. Knockout of the ATXN3 gene in mice increases protein ubiquitination but does not result in an obvious behavioral phenotype [67].

The initial pathogenic mechanism suggested for SCA3 was focused on nuclear aggregates formed by mutant Atxn3 [68,69] and on impairment in the ubiquitin-proteasome pathway [70]. Further support for these ideas was provided by studies of transgenic flies expressing mutant human Atxn3 [71] and by recent findings that genetic reduction or elimination of cochaperone and ubiquitin ligase CHIP (C-terminus of Hsp70-interacting protein) accelerated progression of the phenotype in transgenic mice expressing mutant human Atxn3 [72]. Nuclear localisation of ataxin-3 is essential for the pathogenesis of SCA3, and the phosphorylation state critically determines the intracellular localisation and toxicity of ataxin-3 [73]. All these results suggested that aggregates of expanded Atxn3 exert pathological influence on neurons, most likely by causing impairment of ubiquitin-proteasome system function and by affecting gene transcription.

Transgenic invertebrate models of SCA3 have been developed by expressing an expanded human Atxn3 protein in Caenorhabditis elegans [74] and in Drosophila melanogaster [75]. Analysis of these models supported an important role played by ubiquitination and proteasomal systems in SCA3 pathogenesis [71,74-76]. In addition, potential importance of Atxn3 cleavage by caspases [77] and RNA toxicity mechanisms [78] have been implicated in SCA3 pathogenesis based on the analysis of fly model of SCA3.

Several transgenic mouse models of SCA3 have been developed. In most of these models expanded human Atxn3 protein has been expressed under control of a mouse prion promoter (PrP) [79]. [80,81] These mice develop severe phenotype that includes progressive postural instability, gait and limb ataxia, weight loss and premature death [79] [80,81]. In another mice human Atxn3-84Q expressed in the context of endogenous gene regulatory elements using yeast artificial chromosome (YAC) technology [82]. These mice develop much more subtle and delayed motor phenotype [82], which is more closely resembles disease manifestation in SCA3 patients. Ageing SCA3-YAC-84Q mice display impaired performance in beam walk and gait walk assays and stereological analysis revealed significant neuronal loss in pontine nuclei (Pn) and substantia nigra (SN) regions of these mice at 14 months of age [83]. Recently a mouse expressing human Atxn3-148 transgenic construct under control of huntigtin promoter (HDProm) have been developed and characterized [84]. These mice developed late-onset (12 months) motor phenotype without prominent intranuclear aggregate formation [84]

Abnormal neuronal Ca²⁺ signaling has also been implicated in SCA3 pathogenesis. It was discovered that inhibition of Ca²⁺-dependent protease calpain suppressed aggregation of polyglutamine-expanded Atxn3 in transfected cells [85]. Similar to mutant Htt and mutant Atxn2, mutant Atxn3 but not wild type Atxn3 binds to and activates InsP₃R1, an intracellular Ca²⁺ release channel [83]. Moreover, long-term feeding of SCA3-YAC-84Q transgenic mouse with Ca²⁺ stabilizer dantrolene alleviated age-dependent motor coordination deficits in this mice and prevented neuronal loss in SNc and PN [83]. These results provided strong experimental support to the “Ca²⁺ hypothesis of SCA3 pathogenesis” [83] (Fig 2).

3. Previous clinical trials in SCA2 and SCA3 patients

There have been several attempts to pharmacologically improve ataxia with centrally acting drugs such as physostigmine, L-5-hydroxytryptophan, buspirone, thyreotropin releasing hormone and others [86,87]. The trials in which these compounds were tested were performed in mixed groups of ataxia patients with variable diagnoses, and the antiataxic efficacy of the tested drugs was not proven. To date, there are only a limited number of clinical trials in SCA3 and no trials in SCA2. The trials that have been completed in SCA3 were performed in small number of patients and often had an inadequate study design. A major critique of all published studies is the short study period often not exceeding a few weeks. In none of the studies, there was a strong scientific rationale for selecting the respective study compound. Based on the observation that treatment of an SCA3 patient with the antibiotic sulfamethoxazole/trimethoprim improved ataxia, Correira et al. (1995) tested sulfamethoxazole/ trimethoprim in a double-blind, placebo-controlled crossover trial in 8 SCA3 patients using timed tests as an outcome parameter. There was a modest improvement of undetermined clinical relevance [88]. Sakai et al. (1995) used a very similar design and also found a small improvement which the authors attributed to an increase of brain biopterins caused by the treatment [89]. These effects were not confirmed in a 26 week, double-blind, placebo-controlled crossover trial in 22 SCA3 patients [90]. In another double-blind, placebo-controlled crossover trial, Sakai et al. (1996) tested tetrahydobiopterin and found a weak short-term effect [91]. More recent studies, which were all performed in an open-label design, tested taltireline hydrate, fluoxetin, tandospirone and lamotrigine [92-95]. Positive results were reported for all compounds except fluoxetine, but due to the lack of control, the significance of these findings is limited.

4. Perspectives for clinical intervention in SCA2 and SCA3 patients

There are limited targets for therapeutic intervention available for developing SCA2 and SCA3 treatment. The most obvious targets are mutant Atxn2 and Atxn3 proteins themselves. Expression of mutant Htt and mutant ataxin-1 (Atxn1) was reduced by injection of RNAi-encoding viruses, resulting in benefical effects in mouse models of HD and SCA1 [96,97]. Similar RNAi knockdown approach could also be expanded to SCA2 and SCA3. Selective knockdown of mRNA encoding mutant Htt and mutant Atxn3 proteins was recently demonstrated using peptide nucleic acid (PNA) and locked nucleic acid (LNA) antisense oligomers targeting expanded CAG repeats [98]. These are very attractive approaches, but their utility at the moment is limited by the absence of RNAi or antisense brain delivery system that can be used in humans.

In addition to reducing expression of polyQ-expanded proteins at the mRNA level, another potential therapeutic strategy is based on developing agents that selectively bind to mutant forms of these proteins. A polyQ-containing protein can exist in multiple conformations, some of which are nontoxic and some of which are aggregation-prone and toxic [99]. One possibility to reduce the amount of toxic conformations of Atxn2 and Atxn3 proteins is to increase the levels of chaperons which control protein folding. Although these approaches are being developed for polyQ-expansion disorders [100-102], they have not produced clinical leads yet. Several small molecules were isolated in screens as inhibitors of polyQ aggregation. Some of these molecules were able to reduce polyQ aggregation and toxicity in cellular and animal models [103-108]. A specific polyQ binding peptide (QBP1) was isolated in the phage library screen [109]. It has been reported that QBP1 peptide prevented toxic conformational transition within polyQ-expanded proteins [109] and exerted neuroprotective effects in cellular and animal models of polyQ toxicity [110-112]. Despite excellent scientific rationals, so far none of these candidates has successfully advanced into clinical trials due to problems related to finding an appropriate mode of delivery, poor pharmacokinetics and low efficacy in vivo. Recent solution of the crystal structure of Htt exon 1 containing polyQ region [113] opens perspective for rational design of novel polyQ-binding therapeutic agents.

There is very limited number of candidates that can be considered for SCA2 and SCA3 clinical trials. Treatment with lithium carbonate resulted in beneficial effects in SCA1 mouse model, apparently due to modulation of glycogen synthase kinase 3 (GSK3) activity [114]. Although lithium has not been directly tested in SCA2 and SCA3 mouse models, it is likely that lithium can produce beneficial effects in these models as well. Lithium is widely used for treatment of bipolar disease and has a long history of use in humans. Another potential candidate is Ca²⁺ stabilizer dantrolene, which has been evaluated with beneficial effects in both SCA2 and SCA3 mouse models [58,83] (Fig 2) Dantrolene also has a long history of human use for treatment of malignant hyperthermia and for neurological indications. In considering design of the clinical trial potential side effects of long term exposure to lithium or dantrolene must be taken into consideration.

Although never directly tested in the context of SCA models, additional therapeutic intervenations can be potentially considered for treatment of SCA2 and SCA3 on the basis of proposed “Ca²⁺ hypothesis” (Fig 2). NMDAR inhibitor memantine and anti-glutamate agent riluzole were neuroprotective in experiments with primary MSN cultures from YAC128 HD mouse model, with memantine being more effective [115]. Memantine was also effective in 3-NP model of HD [116] and in YAC128 genetic HD model [117]. Memantine demonstrated some beneficial effects in small scale pilot evaluation in HD patients [118] and is currently being tested soon in phase IV HD clinical trial. Riluzole was tested in phase III HD clinical trial, but it was not successful [119] (Table 2). It is possible that memantine and riluzole may have a beneficial effect in SCA2 and SCA3 patients (Fig 2). In addition to NMDAR, voltage-gated Ca²⁺ channels constitute another potential target for SCA treatment. In recent studies L-type Ca²⁺ channel inhibitor isradipine significantly protected SNc neurons in animal models of Parkinson's disease [120]. Moreover, a recent retrospective epidemiological study has found that treatment of hypertension with Ca²⁺ channel antagonists significantly diminished the risk of developing PD [121]. It is possible that isradipine and other dihydropyridines (DHP) may also be useful for treatment of SCA2 and SCA3 patients (Fig 2). More conservative potential strategy involves use of mitochondrial stabilizers and energizers, such as creatine, CoQ10, and MitoQ (Fig 2). In previous neurodegenerative trials these compounds resulted in modest benefit [122] and it is likely that similar result will be obtained for SCA2 and SCA3 patients as well. Recent clinical research of the EUROSCA consortium demonstrated the feasibility of large-scale clinical trials in SCAs. The EUROSCA consortium devised and validated a novel rating scale for ataxias, the Scale for the Assessment and Rating of Ataxia (SARA) [123] that is now considered standard in clinical ataxia studies. A European SCA registry was created that currently contains entries of approximately 4000 SCA patients. The registry is connected to a web-based electronic data capture system that is designed to run and monitor observational and interventional trials in SCAs. A large cross-sectional study of more than 500 SCA1, SCA2, SCA3 and SCA6 patients led to the identification of factors that determine disease severity and extracerebellar involvement in these disorders [32]. Longitudinal observation of this cohort allowed to calculate the required sample sizes for future interventional trials [124]. The EUROSCA provides a potential framework for controlled clinical evaluation of lithium, dantrolene, memantine, riluzole, dihydropyridines, creatine, CoQ10 or other potential therapeutic agents in ataxia patients in the near future.