Molecular Neurobiology

, Volume 47, Issue 1, pp 90–104

Spinocerebellar Ataxia Type 2: Clinical Presentation, Molecular Mechanisms, and Therapeutic Perspectives

Authors

  • J. J. Magaña
    • Department of GeneticsNational Rehabilitation Institute (INR)
  • L. Velázquez-Pérez
    • Center for the Research and Rehabilitation of the Hereditary Ataxias (CIRAH)
    • Department of Genetics and Molecular BiologyCINVESTAV-IPN
Article

DOI: 10.1007/s12035-012-8348-8

Cite this article as:
Magaña, J.J., Velázquez-Pérez, L. & Cisneros, B. Mol Neurobiol (2013) 47: 90. doi:10.1007/s12035-012-8348-8

Abstract

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant genetic disease characterized by cerebellar dysfunction associated with slow saccades, early hyporeflexia, severe tremor of postural or action type, peripheral neuropathy, cognitive disorders, and other multisystemic features. SCA2, one of the most common ataxias worldwide, is caused by the expansion of a CAG triplet repeat located in the N-terminal coding region of the ATXN2 gene, which results in the incorporation of a segment of polyglutamines in the mutant protein, being longer expansions associated with earlier onset and more sever disease in subsequent generations. In this review, we offer a detailed description of the clinical manifestations of SCA2 and compile the experimental evidence showing the participation of ataxin-2 in crucial cellular processes, including messenger RNA maturation and translation, and endocytosis. In addition, we discuss in the light of present data the potential molecular mechanisms underlying SCA2 pathogenesis. The mutant protein exhibits a toxic gain of function that is mainly attributed to the generation of neuronal inclusions of phosphorylated and/or proteolytic cleaved mutant ataxin-2, which might alter normal ataxin-2 function, leading to cell dysfunction and death of target cells. In the final part of this review, we discuss the perspectives of development of therapeutic strategies for SCA2. Based on previous experience with other polyglutamine disorders and considering the molecular basis of SCA2 pathogenesis, a nuclei-acid-based strategy focused on the specific silencing of the dominant disease allele that preserves the expression of the wild-type allele is highly desirable and might prevent toxic neurodegenerative sequelae.

Keywords

Spinocerebellar ataxia type 2Ataxin-2Molecular mechanismsPolyglutaminesTrinucleotide repeatsGene therapy

Abbreviations

SCA2

Spinocerebellar ataxia type 2

polyQ

Polyglutamine

SBMA

Spinal bulbar muscular atrophy

HD

Huntington’s disease

SCA

Spinocerebellar ataxia

SCA1

Spinocerebellar ataxia type 1

SCA3

Spinocerebellar ataxia type 3

SCA6

Spinocerebellar ataxia type 6

SCA7

Spinocerebellar ataxia type 7

SCA17

Spinocerebellar ataxia type 17

SCA10

Spinocerebellar ataxia type 10

OPCA

Olivopontocerebellar atrophy

REM

Rapid eye movement

ALS

Amyotrophic lateral sclerosis

PD

Parkinson disease

MSA

Multiple system atrophy

Lsm

Like Sm domain

LsmAD

Lsm-associated domain

PAM2

PABPC interacting motif-2

A2D

Ataxin-2 domain protein

PABP

Polyadenylate-binding protein

Pbp1

Ataxin-2 homolog in yeast

Pab1

PABP homolog in yeast

A2BP1

Ataxin-2-binding protein

RNP

Ribonucleoprotein

LTP

Long-term potentiation

RBM9

RNA-binding motif protein 9

RBPMS

RNA-binding protein with multiple splicing

InsP3R1

Type 1 inositol (1,4,5)-triphosphate receptor

RyanR1

Ryanodine receptor

DRPLA

Dentatorubral–pallidoluysian atrophy

UPS

Ubiquitin–proteasome system

shRNA

Short-hairpin RNA

AAV1

Adeno-associated virus serotype 1

RNAi

RNA interference

AON

Antisense oligonucleotide

CNS

Central nervous system

Introduction

The abnormal increase of repeat triplets has been related with the development of multiple genetic diseases, principally neuronal and muscular diseases [1]. To date, >30 genetic disorders have been reported as attributed to trinucleotide expansions; among them, polyglutamine (polyQ) disorders comprise one of the most common group of inherited neurodegenerative conditions. This category of diseases is characterized by the pathological expansion of a CAG trinucleotide repeat in the translated regions of unrelated genes. At least nine polyQ-related disorders have been characterized so far, including spinal bulbar muscular atrophy, Huntington’s disease (HD), and different types of spinocerebellar ataxia (SCA) [13].

SCA conforms a group of neurodegenerative disorders that display clinical, genetic, and neuropathological heterogeneity. The distinctive clinical characteristics of this family of disorders include gait ataxia, cerebellar dysarthria, dysmetria, adiadochokinesia, and postural tremor, among other manifestations. Within extracerebellar manifestations, it could be distinguished cognitive dysfunction, pyramidal and extrapyramidal signs, ophthalmoplegia, peripheral neuropathy, and sleep and dysautonomia [47]. Initially, autosomal dominant cerebellar ataxias were classified into three different types based on their clinical characteristics [1, 4]; however, recent identification of different genes for each pathology type allowed the description of more than 30 varieties of SCA [8, 9]. The genetic mutation that gives rise to SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17 consists of the abnormal expansion of repetitive units of the trinucleotide CAG, localized in the translated region of their respective genes [9]. Trinucleotide repeats are highly polymorphic in human population and might present instability during processes of mitosis and/or meiosis, originating expansions of the number of repeated units [2, 10]. There are a specific number of repeats that represent the threshold between the normal and the pathological state for each of the different SCAs caused by expanded CAG triplets; thus, an increase that exceeds this border gives rise to the appearance of the classical phenotype of these diseases.

The general prevalence of SCA is 5–7/100,000 inhabitants [8]; however, incidence varies depending on ataxia type, and it is even influenced by populational genetic phenomena present in certain geographic regions. In this manner, the diverse studies reported throughout the world show that SCA3 is the most common ataxia with 21 % of total cases, followed by spinocerebellar ataxia type 2 (SCA2) and SCA6 with 15 %. Notwithstanding this, it has been observed that SCA2 is the prevalent ataxia in certain populations, such as those of India and Cuba [11, 12]. Likewise, large SCA2 families have been found in Australia, Martinique, Tunisia, Germany, Italy, Poland, and Southern Brazil [13, 14]. In the case of Cuba, high incidence of SCA2 was specifically reported in the Holguín province (40.18/100,000 inhabitants), a phenomenon caused by the presence of a founder effect [11, 15]. Interestingly, it was determined recently that SCA2 is the most common ataxia in Mexican population (45.4 %), followed at a distance by SCA10 (13.9 %) [16]. Thus, the study of SCA2 is of prime interest in Hispano–American populations.

Historical Background and Clinical Features of SCA2

SCA2 was initially reported by Wadia and Swami in India in the year 1971; therefore, it is also known as Wadia–Swami-type ataxia. These authors described it as a heredo-degenerative ataxia characterized by slow saccadic eye velocity and limited ocular movements [17, 18]. Other authors have denominated SCA2 as Menzel-type ataxia or olivopontocerebellar atrophy. The most frequently found clinical characteristics of SCA2 included progressive cerebellar syndrome, ataxic gait, cerebellar dysarthria, dysmetria, dysdiadochokinesia associated with slow saccadic movements [18, 19], peripheral neuropathies [20, 21], fasciculations, painful muscle contractures, sleep disorders, and dysphagia [13, 22]. The main oculomotor alteration in SCA2 is the slowing of horizontal saccadic movements; this feature that appears before the disease onset and which severity depends on the CAG repeats’ size [13, 19] is electrooculographically detectable in 99 % of the patients and in several presymptomatic subjects [18]. Interestingly, postmortem studies have documented a dramatic reduction in pontine excitatory burst neurons, which may account for the altered saccadic system of SCA2 patients [23]. Velazquez-Perez et al. assessed maximal saccade velocity (MSV) in 82 SCA2 patients and 80 controls and demonstrated that MSV was strongly influenced by the polyglutamine expansion size and less by disease duration and conclude that saccade velocity is a sensitive, quite specific, and objective endophenotype [18]. The most frequent abnormality of the examination of the deep-tendon reflexes in the SCA2 patients is areflexia or hyporeflexia. However, there are important clinical variations; while some patients show severe hyperreflexia in the lower limbs concurred with normal reflexes and/or areflexia in upper limbs, others have areflexia in all limbs. Hyperreflexia in the lower limbs, clonus, and Babinski reflex are frequent and prominent at the start of the disease but are soon followed by hypo- and areflexia, while pyramidal signs such as the Babinski reflex remain infrequent [11, 24].There is a subgroup of SCA in which parkinsonian manifestations predominate [25, 26]. Cardiac, gastrointestinal, and urinary affectations, olfactory dysfunction, as well as exocrine gland dysfunction and malnutrition have also been described in association with SCA2 [13, 27, 28]. In some cases, the following SCA-associated cognitive affections have been reported: fronto-executive dysfunction, altered short-term memory, lack of attention, visuomotor-learning alterations, and psychological alterations that lead to insomnia, depression, and suicidal impulses [28, 29].

Although SCA2 is a late-onset disease with initial symptoms usually appearing when affected subjects are in their 30s, pediatric onset SCA2, which is associated with expansion of 130 to more than 200 CAG repeats, displays symtomatology during the first months or years of life. The main clinical features of pediatric-onset SCA2 include rare symptoms such as retinitis pigmentosa, myoclonus epilepsy, tetraparesis, developmental delay, dysphagia, cognitive impairment, facial dysmorphism, and infantile spasms [3035]. Ramocki and coworkers describe a female child who met all developmental milestones until the age of 3 years, deterioration of expressive language, comprehension, memory, graphomotor skills, and dysarthria [36]. Cranial nerve examination showed bilaterally restricted lateral gaze with oculomotor apraxia [36]. Abdel-Aleem and Zakiwith reported a male child with progressive extrapyramidal manifestations, developmental delay, slow eye movements and cognitive impairment, trophic changes, vasomotor instability, and dysphagia [37].

Microscopic examination of the cerebellum shows significant atrophy with a reduction in its weight, as well as loss of Purkinje neurons along both hemispheres and the vermis, but mainly in the paleocortex and neocerebellum [38]. A diminution of the dendritic tree, especially in distal branches, can also be appreciated, as well as torpedo-shaped formations, and poor density in medial and inferior perpendicular fibers [38]. In more advanced disease stages, a diminution is produced in granulose-layer neurons, cerebellar cortex, and dentate nucleus. It has recently been proven that the degenerative process additionally extends to the cerebellar flocculus [39, 40]. In the brainstem, volume and weight diminution has been identified, as well as the loss of pontine fibers of the medial cerebellar peduncle and a marked decrease in the neurons of the lower olivary complex [41, 42]. The degenerative process of the brainstem in patients with SCA2 is also characterized by the reduction of olivocerebellar fibers, pontine neurons, and pre-cerebellar brainstem nuclei, affecting also the locus coeruleus, cranial-nuclei motoneurons, and cervical motoneurons [13, 38]. Another highly relevant anatomopathological finding is the degeneration of thalamic nuclei, including the anterior ventral, the lateral ventral, the posterior lateral ventral, and the posterior medial ventral nucleus, as well as the reticular nucleus and the posterior geniculate body [43].The spinal cord of these patients shows both moderate and severe demyelination in the dorsal spinocerebellar sheaths and posterior cord, respectively [39, 43, 44]. Additionally, diminution of anterior shaft motoneurons and of the columns of Clarke may be present, as well as partial demyelination of the posterior and anterior intraganglion-fiber roots [45]. A concomitant increase of astrocytes and microglia was noted in affected areas such as globus pallidus, thalamus, subthalamus, and periaqueductal region [45]. Furthermore, moderate diminution of dorsal-column-level sensitive neurons was identified, which was higher in Goll than in Burdach nuclei [21]. In conclusion, the main neuropathological marker of SCA2 is early onset olivopontocerebellar atrophy, which is accompanied by degeneration in somatosensorial pathways, thalamus, substantia nigra, frontal lobe, medulla oblongata, and cranial nerves. In fact, a loss of >70 % of neurons in the substantia nigra of mesencephalon have been reported [39, 4347]. From the neuropathological viewpoint, all these findings indicate that SCA2 represents one of the most severe forms of hereditary ataxias.

Genetics of SCA2

In 1996, the mutation that produces SCA2 was identified; it consists of an abnormal expansion of the CAG trinucleotide localized in exon 1 of the gene denominated thereafter ATXN2 (Fig. 1a), which mapped in the 12q23-24 chromosomal region [4850]. Due to that SCA2 shares practically the same symptomatology with the other ataxias, its accurate diagnosis is based on the identification of its genetic mutation. Moreover, molecular diagnosis allows for early identification of SCA2, which is fundamental for offering genetic advice and psychological help to the families of affected individuals [51]. ATXN2 gene is constituted of 25 exons (4,500 bp) (Fig. 1a), and its product, ataxin-2, is a 140-kDa protein (1,312 amino acids) that contains a variable-length glutamine-rich region in the amino-terminal domain that is encoded by CAG triplets [52] (Fig. 1b). Alleles of the ATXN2 gene that carry 13–31 CAG-trinucleotide repeats are present in normal individuals. At the worldwide level, it has been demonstrated that the 22 CAG repeats allele is present in >90 % of individuals analyzed [53]. This range of repeats is stably inherited because the repeats present a relatively low mutation rate. The genetic stability of normal-range repeats appears to be conferred by the presence of one or two CAA-type interruptions between repeated units, following the (CAG)8-CAA-(CAG)4-CAA-(CAG)8 sequence pattern. Contrariwise, alleles with a CAG-triplet repeat number of >31 and up to approximately 200 (there is a reported case of 500) are present in patients with SCA2 and are highly unstable on being inherited, which correlates with the absence of CAA interruptions [17, 53, 54].
https://static-content.springer.com/image/art%3A10.1007%2Fs12035-012-8348-8/MediaObjects/12035_2012_8348_Fig1_HTML.gif
Fig. 1

a Structure of the ATXN2 gene localized in the human chromosome 12. Wide and narrow boxes represent the 25 exons encoding ataxin-2 protein. The arrow denotes the transcription initiation site. A number >31 CAG repeats in exon 1 of the gene produces the clinical manifestations of SCA2. b Scheme of ataxin-2 protein. The protein is constituted by 1,312 amino acids. Protein domains and the amino acid range comprises in each region are indicated

Different from other diseases produced by abnormal trinucleotide expansion, SCA2 does not present alleles within the “premutation” range, that is, intermediate alleles without clinical significance but that are genetically unstable and that can lead to repeat expansion in subsequent generations to reach the pathological range [55, 56]. Thus, the length of wild-type and mutant alleles does not overlap, which has permitted accurate DNA-based diagnosis of the disease in populations analyzed to date [57, 58]. Expansions occur in 89 % and contractions in 11 % of the offspring of affected patients. There is a higher variability in repeat lengths in the paternal transmission, compared with the maternal transmission [11].

Noteworthy, SCA2 presents the “anticipation” phenomenon, which consists of earlier disease onset and more severe symptomatology in subsequent generations of an affected genealogy, which correlates with an increase in the number of CAG repeats [48, 58]. The fact that a number of CAG repeats and age at onset of the pathology are highly variable between affected individuals suggests that other factors can affect the development of the disease [59]. Therefore, it is probable that early onset of SCA2 is influenced by genomic variations present in the ATXN2 gene itself, in adjacent genes, and/or in genes associated with polyQ disorders. In this respect, Pulst et al. described that alleles of the CACNA1A (SCA6) gene that carry a high number of CAG repeats are associated with early onset of SCA2 [59]. Other identified modifying genetic factors that exert an influence on age at onset of SCA2 include the RaI1 gene and a polymorphic variant of the mitochondrial complex I gene [60, 61].

Moreover, ATXN2 trinucleotide repeats have been reported associated with non-ataxic phenotypes, such as amyotrophic lateral sclerosis (ALS) and parkinsonism, including Parkinson disease (PD), and multiple system atrophy. Distinct observations from European, Chinese, and North American populations have indicated that ATXN2 intermediate expansions below the threshold of SCA2 manifestation enhance ALS risk, modulating the manifestation and age at onset of motor neuron degeneration [6268]. In PD usually, the ATXN2 CAG repeats tract of these patients is characterized by short repeats range (from 32 to 43 units) and the presence of CAA interruptions [2569].

Ataxin-2 Function

Ataxin-2 is a 140-kDa protein (1,312 amino acid residues) that exhibits no sequence similarity with other polyglutamine-rich proteins associated with neurological diseases, with the exception of the glutamine-rich domain, which increases its molecular mass according to the length of the CAG-repeat expansion [70]. Despite being a basic protein (isoelectric point, 9.6), ataxin-2 has an acid region comprised of 456 amino acids that corresponds to exons 2–7 of the gene, which forms the Like Sm domain (Lsm domain) and the Lsm-associated domain [70, 71] (Fig. 1b). Additionally, ataxin-2 presents two regions localized next to the carboxyl-terminal region, which are denominated PABPC1-interacting motif-2 (PAM2) and ataxin-2 domain protein [72, 73] (Fig. 1b). Identification of the normal function of ataxin-2 is an essential step for understanding the molecular basis of SCA2 [74]. In this section, we review the experimental evidence that has allowed for defining the function of ataxin-2.
  1. (a)
    Participation of ataxin-2 in posttranscriptional and translational regulation. One way of inferring ataxin-2 function has been comparison of its protein domains with others that have been previously characterized. For instance, ataxin-2′s Lsm domain is shared with proteins involved in RNA posttranscriptional modifications [70, 71, 75]; therefore, ataxin-2 might be involved in these regulatory process. Alternatively, protein’s function can be discerned through identification of the proteins with which it associates. In this regard, by means of immunofluorescence and immunoprecipitation studies, the interaction of the ataxin-2 PAM2 domain with the PAB6 domain of polyadenylate-binding protein (PABP) was determined [76]. This protein participates in RNA-messenger stability and translation regulation. PABP activation takes place through interaction in its PAB6 domain with proteins PAIP1, PAIP2, and RF3 [77]. Interestingly, Pbp1 (an ataxin-2 homolog in yeast) binds to Pab1 (a PABP homolog in yeast) to regulate RNA-messenger polyadenylation [76]. In terms of the ataxin-2 carboxyl-terminal region, its interaction with an ataxin-2-binding protein (A2BP1) has been reported; A2BP1 contains a ribonucleoprotein motif, characteristic of RNA-binding proteins [78]. Recently, it has been demonstrated that A2BP1 regulates the RNA splicing of the NMDAR1 gene receptor, a receptor that modulates excitatory synaptic transmission in the hippocampus, participating in this manner in long-term potentiation and learning [79, 80]. On the other hand, by means of protein–protein interaction assays employing the double-hybrid system in yeast, the association of ataxin-1 and ataxin-2 with RNA-binding motif protein 9 (RBM9) and RNA-binding protein with multiple splicing (RBPMS) was revealed; both proteins are involved in RNA splicing [81]. Altogether, these lines of evidence described above suggest that ataxin-2 might modulate RNA alternative splicing by binding to RBM9 and RBPMS, as well as to RNA, PABP, and A2BP1 (Fig. 2a). If both ataxin-1 and ataxin-2 participate in alternative RNA splicing, this could explain the similarity between SCA1 and SCA2 symptomatologies [11]. Furthermore, it has been demonstrated in a Drosophila model as well as in human cells that the Lsm, LsmAD, and PAM2 domains of ataxin-2 interact independently with polyribosomes [71]. The ataxin-2 LsmAD domain contains both an endoplasmic reticulum export signal and a clathrin-mediated signal sequence to be transported to the Golgi apparatus [70]. In agreement, the endogenous ataxin-2 and an exogenously expressed recombinant variant that carries 32 glutamine residues were found localized in the rough endoplasmic reticulum bound to polyribosomes in neuronal cells [71]. Therefore, it is tempting to speculate that ataxin-2 binds to polyribosomes to regulate in concert with PABP protein translation (Fig. 2b).
    https://static-content.springer.com/image/art%3A10.1007%2Fs12035-012-8348-8/MediaObjects/12035_2012_8348_Fig2_HTML.gif
    Fig. 2

    Participation of ataxin-2 in different cellular processes. a Interaction of ataxin-2 with proteins involved in mRNA alternative splicing (PABP, A2BP1, RBM9, and RBPMS) implies a role for ataxin-2 in posttranscriptional modifications. b Association of ataxin-2 and PABP with polyribosomes suggests their participation in regulating protein translation. As stress granules inhibit the translation of certain proteins via blockage of the translation initiator factor eIF4F and as ataxin-2 and PABP are recruited to these cellular compartments, it is thought that ataxin-2 is involved in this negative regulation of translation. c Association of ataxin-2 with members of the ubiquitin-regulated endocytic machinery, including endophilins A1 and A3 and parkin, suggests a role for ataxin-2 in endocytosis and actin-based cytoskeleton remodeling.d Ataxin-2 localizes to the endoplasmic reticulum, where it interacts with the calcium channel type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1), and by this way, it might modulate cell signaling

     
  2. (b)

    Role of ataxin-2 in stress-granule formation. Studies conducted in Caenorhabditis elegans have demonstrated that ataxin-2 and its homologs, QKI and A2BP1, are associated with stress granules for regulating translation in certain transcripts [8289]. These structures, which are formed when cells are subjected to adverse conditions, inhibit the translation of certain proteins by blocking the translation initiator factor eIF4F function [76, 83, 85]; therefore, it is thought that ataxin-2 participates in this negative regulation of translation (Fig. 2b). Supporting this hypothesis, it was recently demonstrated that Pbp1, ataxin-2′s homolog protein in yeast, participates in the formation of stress granules [70, 86, 87]. In fact, the absence of Pbp1 provokes considerable diminution in the number of these specialized compartments, finally affecting the normal function of the cell.

     
  3. (c)

    Participation of ataxin-2 in endocytosis and cytoskeleton reorganization. Association of ataxin-2 with the endoplasmic reticulum and its participation in the formation of stress granules suggest that this protein could interact with the plasma membrane, performing some particular function. It has been reported that ataxin-2 interacts with endophilins A1 and A3, proteins that are implicated in the formation of plasmatic membrane curvature at endocytotic sites, through the activation of an ubiquitination-regulated protein complex coupled with actin filaments [88]. Likewise, it has been reported that ataxin-2 is associated with parkin, a protein with ubiquitin E3-ligase function [89, 90]. Thus, this series of data suggests that ataxin-2 might be part of the endocytosis machinery and could participate in ubiquitination-mediated reorganization of the actin cytoskeleton [8892] (Fig. 2c).

     
  4. (d)

    Role of ataxin-2 in calcium-mediated signaling. The presence of ataxin-2 in the endoplasmic reticulum suggests its participation in calcium-mediated cell-signaling cascades. In support of this hypothesis, it was recently determined in the SCA2-transgenic mouse denominated Q58 [93, 94] that mutant ataxin-2, but not wild-type ataxin-2, interacts with the carboxyl-terminal region of the type 1 inositol (1,4,5)-triphosphate receptor of the calcium channel, thus affecting the intracellular signaling pathway, which leads to an increase of glutamate and apoptosis in neuronal cells [94] (Fig. 2d). Interestingly, inhibition of the Ryanodine receptor (RyanR1) impedes calcium release and consequently reduces glutamate levels. Hence, this strategy could function as a therapeutic treatment for SCA2 patients [94].

     

Other polyglutamine diseases have been directly associated with alteration of diverse cellular processes, including apoptotic pathway activation, mitochondrial modifications, interference of axonal transport, rupture of subcellular organelles, proteasome dysfunction, autophagia, and excitotoxicity [95]. Thus, greater knowledge of the participation of both wild-type and mutant ataxin-2 in cellular processes will lead to better understanding of the pathological mechanisms underlying SCA2 and, finally, to the development of therapies against this disease.

Molecular Bases of SCA2

As mentioned above, the CAG-triplet expansion in the ATXN2 gene gives rise to an abnormal polyglutamine region in the ataxin-2 protein, which results in a gain of a toxic function that affects specific groups of neurons, such as Purkinje cells, striated Pontus nuclei, and anterior shaft motoneurons of the spinal medulla and of cranial-nerve motor nuclei [13, 74]. The degree of neuronal degeneration is related with the length of the polyglutamine expansion and is expressed phenotypically as motor and cognitive alterations.

Experimental advances obtained in different cell-based and animal models have allowed for definition of the molecular mechanisms underlying polyQ diseases’ pathogenesis. These are related to altered behavior of mutant polyQ proteins, including generation of proteolytic cleavage products and protein misfolding and aggregation [3]. These alterations seem to be valid to SCA2 because polyQ diseases shared a common toxic effect related to the polyQ expansion.

Aggregation of Ataxin-2

A hallmark of most of polyglutamine diseases is the cytoplasmic and intranuclear accumulation of aggregated polyQ proteins inside neurons [96, 97]. Ataxin-2 is a cytoplasmic protein with higher expression in Purkinje cells, the most affected neurons in SCA2 [45, 98]. However, mutant ataxin-2 presents abnormal folding that gives rise to the formation of aggregates, which might trigger a series of events that lead to programmed cell death and consequently to the degeneration of central and peripheral neuronal structures [95]. Ubiquitinated intranuclear inclusions have been found only in few pontine neurons of SCA2 patients but not in Purkinje cells [99]. Interestingly, recruitment of ataxin-1, ataxin-3, and TATA box-binding protein was found into neuronal intranuclear inclusions in SCA1, SCA2, and SCA3 human brains [100], raising the possibility that nuclear aggregates alter the transcriptional process, as widely reported for huntingtin (reviewed in Bauer and Nukina 2009) [3]. It is thought that cytoplasmic and/or nuclear aggregation of ataxin-2 is involved in SCA2 pathology; however, this issue is controversial, because aggregation may merely represent end products of the upstream toxic events. In fact, several cell and animal models for polyglutamine disorders show the discrepancy between inclusion formation and cell death [101106]. Furthermore, it has been observed in necropsies of SCA1, SCA2, SCA3, and dentatorubral–pallidoluysian atrophy patients the absence of intranuclear inclusions in cerebellar Purkinje cells, which are targets of neurodegeneration in these polyQ disorders [107]. Recently, it has been proposed that oligomeric species such as protofibrils and microaggregates are the direct source of polyQ toxicity and that larger aggregates are cytoprotective [41, 107]. Future studies in recent developed SCA2 transgenic mice models [94, 108] would help to define the precise participation of intracellular ataxin-2 aggregates in SCA2 pathogenesis.

Posttranslational Modification of Ataxin-2

Posttranslational modification extends the range of functions of a given protein, changing its biochemical properties and modifying its interactions as well as its subcellular localization. Ataxin-2 suffers different posttranslational modifications including proteolytic cleavage phosphorylation, and ubiquitination [99, 109], which might alter the characteristics and consequently the function of both wild-type and mutant variants [70, 71].

Proteolytic Cleavage of Ataxin-2

Pathogenesis of various polyglutamine diseases, such as HD and SCA3, appears to be linked to proteolytic cleavage that results in the production of toxic polyQ-containing fragments. It has been suggested that truncated proteins may serve as a nidus for the formation of intranuclear aggregates and in turn may recruit full-length proteins. Consistent with this, truncated polyQ proteins appeared more prone than full-length proteins to form inclusions or cause apoptosis [110112]. Caspase-mediated cleavage sites have been identified or predicted in huntingtin, atrophin-1, and ataxin-3 [113]. In fact, it has been reported that cleavage of mutant huntingtin at the 586 amino acid by caspase 6 is crucial for the development of HD pathogenesis in a HD mouse model expressing the whole human huntingtin with 120 glutamine residues (YAC128) [114]. Moreover, it has been observed that ataxin-3 is a target for caspase 1 and that the SCA3 transgenic mice brain contained a cleaved C-terminal fragment [115117]. Regarding SCA2, truncated 42-kDa N-terminal fragments were found to be increased in the Purkinje cells of SCA2 patients, compared with normal brains, although it is not yet known whether this truncation event is related to SCA2 pathogenesis [94]. Furthermore, ataxin-2 truncated C-terminal fragments of 70 and 230 kDa were found in rat PC12 cells [109]. Proteolytic cleavage of mutant ataxin-2 may result in the production of smaller neurotoxic N-terminal fragments containing expanded polyQ tracts, while yielding of ataxin-2 C-terminal fragments might alter cellular functions, such as RNA splicing, because the C-terminal of ataxin-2 preferentially binds A2BP1 protein [109].

Phosphorylation and Ubiquitination of Ataxin-2

It is thought that phosphorylation is involved in the initial conversion of mutant polyQ proteins to pathogenic conformation as well as in their nuclear transport by affecting proteolytic cleavage [118]. Phosphorylation of huntingtin has been implicated in different aspects of HD pathogenesis, including neuroprotection against HD cellular toxicity [119, 120], modulation of huntingtin cleavage [121], and nuclear accumulation of both full-length and truncated fragments of huntingtin [118, 122]. Phosphorylation of ataxin-3 by glycogen synthase kinase 3β resulted in reduced mutant ataxin-3 aggregation in vitro [123], while phosphorylation by CK2 kinase modulates its nuclear localization [124]. In this regard, ataxin-2 was found to be phosphorylated in SY5Y cells [109], and in analogy to ataxin-1, the phosphorylation of ataxin-2 may occur at the RXXSXP motif present at amino acids 853–858. Nevertheless, the potential implication of ataxin-2 phosphorylation in its proteolytic cleavage and/or aggregation remains to be addressed.

The ubiquitin–proteasome system (UPS) is a cellular protein degradation pathway that regulates cell functioning serving as detoxification machinery by targeting damage proteins for degradation. Ubiquitination of ataxin-2 was suggested early by immunofluorescence analyses on SCA2 patients showing the colocalization of ataxin-2 expanded polyglutamine with ubiquitin in neuronal intranuclear inclusions in affected brain regions except the cerebellum [99]. Ubiquitination and incorporation of various chaperones and proteasome components into these inclusions likely reflect a decreased ability of the protein degradation machinery to efficiently turn over the mutant protein [125, 126]; however, implication of the UPS in SCA2 remains to be approached.

Therapeutic Perspectives

Current treatments for SCA2 are limited to supportive care that partially alleviates signs and symptoms of the disease but fail to halt the progression of the disease. However, molecular basis underlying SCA2 has started to be unveiled, allowing envision of therapeutic approaches aimed at reversing SCA2 symptomatology.

Pharmacologic Therapy

Pharmacologic treatment for SCA2 has been conducted in small samples of patients who present high variability in clinical stage as well as in the number of CAG repeats, rendering interpretation of the results difficult. Another problem is the absence of quantitative variables to evaluate the effectiveness of applied therapies. Despite this, it has been shown that dopaminergic and anticholinergic treatments achieve reduction of tremor, dystonia, and bradykinesia in patients with SCA2, while painful muscle contractions can be alleviated with magnesium, quinine, or mexiletine, or with high doses of vitamin B [127, 128]. SCA2 patients with parkinsonism exhibit beneficial response to levodopa; specifically, the presence of rigidity/bradykinesia has been alleviated for many years by this treatment [25, 74]. In addition, postural tremor can be treated by deep brain stimulation at the thalamic and subthalamic levels [127].

Recently, the efficacy of riluzole in the treatment of SCA2 was demonstrated [129]; this drug may work by reducing hyperexcitability of neurons in the deep cerebellar nuclei. As reduced concentration of zinc in serum and cerebrospinal fluid has been found in patients with SCA2, and considering the important role of zinc in the nervous system as neurosecretory product or cofactor, a promissory clinical trial based on zinc supplementation in combination with neurorehabilitation was carried out for 6 months in a group of 26 Cuban SCA2 patients. Interestingly, significant increase of Zn levels in the cerebrospinal fluid of SCA2 patients correlated with mild decrease in the ataxia scale subscores for gait, posture, stance, and dysdiadochokinesia; reduction of lipid’s oxidative damage and saccadic latency was also observed, when compared with the placebo group [130].

Gene Silencing

Decreasing the levels of the mutant protein by specific degradation or translational suppression of the corresponding mRNA and thus preventing the downstream pathological consequences appears to be one of the best strategies to fight SCA2. As polyQ disorders involve a dominant-gain-of-function effect of the polyQ expansion tract, numerous transgenic mice have been generated to test molecular therapies by simply expressing a human mutant protein in relevant neuronal population with heterologous promoters. The first in vivo evidence showing the feasibility of gene silencing in a polyQ disorder employed the SCA1 transgenic mouse SCA1-82Q [131]. Xia et al, designed a potent ataxin-1 short-hairpin RNA (shRNA) that was further delivered into the murine cerebella by using the adeno-associated virus serotype 1 (AAV1) as a vector. Interestingly, these authors found an improvement in motor coordination, restoration of cerebellar morphology, as well as significant reduction in the accumulation of the pathogenic ataxin-1 in the Purkinje cell nuclei in the virally transduced transgenic mice. After that, Harper et al. reported that huntingtin gene silencing with an AAV1-huntingtin shRNA resulted in reduced inclusions in the striatum, and alleviation of behavioral and neuropathological abnormalities in the HD transgenic mice N171-82Q HD [132]. Subsequently, there have been many successful studies using RNA interference (RNAi)-based methods in different polyQ disorders, including SCA7 and SCA1 and mainly HD [131, 133138].

In spite of the enticing therapeutic potential of RNAi in treating polyQ diseases, application of this strategy to human patients may not be so straightforward for obvious differences. As shRNA-mediated knockdown is directed selectively to the human polyglutamine transgene introduced in the transgenic mice models, simultaneous knockdown of the normal endogenous ortholog of the disease gene is avoided; however, in the human disease situation, the shRNA being used would reduce expression of both the normal and the disease alleles (Fig. 3a). Although evidence for a dominant gain-of-function effect with polyglutamine expansion is overwhelming, string data also suggest that a concomitant partial loss of normal protein function might contribute to the development of some polyglutamine disorders [139]. At present, different antisense oligonucleotide (AON)-based molecular tools could be applied to target and cleave the mutant ATXN2 RNA, including AON shRNA and self-cleaving hammerhead ribozymes; however, as stated above, such strategies would inevitably destroy also the normal RNA (Fig. 3a). It is likely that silencing of ataxin-2 expression would alter the cellular pathways to which this protein is linked, including posttranscriptional and translational regulation and endocytosis and cytoskeleton reorganization (see previous sections). Therefore, in those cases where wild-type protein is required for cellular function, the specific targeting of the mutant allele would be required. The design of an RNAi that could discriminate between wild-type and mutant alleles is nontrivial, because they are naturally almost identical in sequence apart from the mutation itself. A plausible approach, however, is to take advantage of occasional linked polymorphisms in the disease allele that makes it different from wild-type allele in at least one nucleotide in the same region, allowing then the design of allele-specific silencing strategies. Such strategy has been successfully employed in three different polyglutamine disorders: targeting of single nucleotide polymorphisms (SNPs) linked to ATXN3 and ATXN7 genes [140143] and targeting of a deletion linked to HTT gene [144, 145] were recently reported.
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Fig. 3

Molecular strategies for SCA2 therapy. ATXN2 RNA-targeting strategies: a Non-allele-specific silencing of ATXN2 gene could be achieved by the use of antisense oligonucleotides (AON), short-hairpin RNAs (shRNA), or ribozymes. However, a limitation of this strategy is the fact that both mutant and wild-type ATXN2 mRNAs are recognized and cleavage with similar efficacy by antisense molecules. bATXN2 gene allelic-specific silencing could be attained by using AON or shRNA that target single-nucleotide polymorphism linked exclusively to the mutant allele, which allows cleavage of the mutant RNA while maintaining intact the normal RNA. Ataxin-2 protein-targeting strategies: c Enhancement of ataxin-2 degradation could be achieved by stimulation of autophagia with rapamycin (inhibitor of mTOR signaling pathway) or by using activators (benzamil or Y-27632) of the ubiquitin–proteasome system (UPS). d Prevention of ataxin-2 aggregation could be attained through the use of β-sheet structure destabilizing compounds or by employing small molecules that act as binding competitors to block the interaction between monomers of mutant ataxin-2. Inhibition of ataxin-2 aggregation could also be accomplished by increasing the levels of endogenous chaperones, such as Hsp70

Although allele-specific silencing based on SNP discrimination using AONs is feasible, most researchers have still utilized RNAi-based methods in view of their inherent potency and potential for sustained silencing. In spite of the idea that two alleles with different sizes of CAG repeats cannot be differentially targeted by therapeutic nucleic acids, Hu et al. demonstrated the specific inhibition of mutant huntingtin and ataxin-3 expression with a minimal effect on their respective wild-type proteins by using peptide nucleic acid conjugates and locked nucleic acid oligonucleotides [146]. It is likely that the secondary RNA structure conferred by the CAG expansion lends itself to increase susceptibility to CAG repeat-containing AONs, in comparison to the smaller wild-type repeat region, leading to the preferential inhibition of the mutant protein expression (Fig. 3b). An alternative strategy that is consist of silencing both mutant and wild-type allele with a non-allele-specific RNAi molecule and the further replacement of the full-length gene with an RNAi-resistant codon-optimized copy was successfully employed in a SC6 model system [147].

All these promissory strategies remain to be approached in SCA2 experimental models. Two different transgenic mice have been generated so far, in which the therapeutic potential of RNAi strategies could be tested. The first mouse model in which the murine PcP2 (L7) promoter directs the expression of the human ATXN2 gene with an expanded allele of 58 CAG repeats was developed by Huyng et al. [94]. The second model consists of a transgenic mouse expressing the full-length human ATXN2 gene with 75 CAG units under the control of the human ATXN2 self promoter [108]. Finally, success of the RNAi approach depends on a safe and precise method of delivery to the central nervous system as well as on the sustainability and effectiveness of the therapeutic molecules. The significant advance obtained recently in the development of lentivirus- and AAV-based vectors appears to be promissory for CNS treatment [148]. Although virus-based vectors would provide highly effective and long-term production of therapeutic molecules; immune system problems associated with viral proteins still raise a practical hurdle [149].

Enhancement of Protein Degradation

Apart from elimination of the mutant protein by specific degradation of the corresponding CUG-expanded mRNA by antisense technology, removing of mutant protein could be alternatively attained by enhancing its degradation by means of UPS stimulation and/or autophagy (Fig. 3c) [95]. Autophagy is a major clearance route for intracellular aggregate prone. It is known that the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates by autophagy induction; however, rapamycin has nontrivial side effects. Interestingly, identification and further use of small-molecule enhancers of rapamycin-independent mammalian autophagy allow to induce the autophagic clearance of mutant huntingtin, attenuating its toxicity in mammalian cell, fly, and zebrafish models [150, 151]. With respect to UPS activity, while many UPS inhibitors exist, no chemical activators of UPS activity have been available. Recently, benzamil (amiloride-derivative drug) and Y-27632 (rho-associated kinases inhibitor) compounds have been reported to induce UPS activity and reduce polyQ protein aggregation and toxicity in HD and SCA3 models [152, 153] (Fig. 3c). Previous positive experience of these chemical agents in models of different polyQ disorders would facilitate their employment in SCA2.

Inhibition of Aggregation

Due to that, one of the primary events in the development of polyQ disorders is the formation of cellular aggregates; it is likely that inhibiting their formation delays or stop disease progression. The therapeutic potential of small molecules able to prevent directly the formation of polyQ aggregates, such as β-sheet structure destabilizer molecules, has been shown in several studies (Fig. 3d). For example, treatment of Huntington’s disease mice model R6/2 mice with Congo red or trehalose significantly improved the mice survival [154, 155]. However, in a recent study, the systemic administration of Congo red failed to improve cognitive function in this mice [156], which might be due to the limited ability of this compound to cross the blood–brain barrier. Another, alternative strategy is the competitive inhibition of cell aggregates through the use of compounds that bind specifically to the mutant protein’s monomers impeding the incorporation of new monomers (Fig. 3d). PolyQ-binding peptide 1 was shown to prevent conversion of the expanded polyglutamine into aggregation-prone β-sheet-rich conformation and prevent neurodegeneration in a Droshophila model of HD [157, 158]. Additionally, it is suggested that employment of transglutaminase inhibitors would reduce aggregation levels in the mutant protein, because this enzyme participates in the covalent bond among polyglutamine domains [159161]. Finally, another promissory strategy to reduce misfolding, oligomerization, and aggregation of polyQ proteins is to increase the cellular levels of molecular chaperones such as Hsp70. Recently, it has been reported that Hsp90 inhibitor (17-/allylamino)-17-demethoxygeldanamycin suppressed polyglutamine-induced neurodegeneration in Drosophila models of SCA3 and HD via activation of heat shock factor 1, which in turn upregulated molecular chaperones Hsp40, Hsp70, and Hsp90 (Fig. 3d) [162]. This particular strategy appears to be promissory to SCA2.

Inhibition of Mutant Ataxin-2-Induced Pathogenic Mechanisms

Another promising strategy for fighting SCA2 is modulation of the cellular processes implicated in SCA2. Modulation of energy metabolism through the use of antioxidants such as α-lipoic acid or N-acetylcysteine can ameliorate polyQ protein aggregation and cell death under conditions of oxidative stress [163]. Excitotoxicity has been implicated in diverse polyQ diseases; at this respect, the use of NMDA agonists such as remacemide in combination with coenzyme Q10 in a mouse model of HD resulted in 31.8 % increase in survival [164]; however, the role of excitotoxicity in SCA2 is unclear so far.

Physical Rehabilitation

It is known that the therapeutic exercise constitutes one of the basic pillars in the physical treatment of patients with neurodegenerative affectations, an affirmation that has been supported recently by the concept of neuroplasticity [13]. The most prominent actions carried out with regard to this issue have been performed in carriers of recessive ataxias, such as Friedreich-type ataxia, as well as in patients with multiple sclerosis. There is a positive experience of the rehabilitation in Cuban patients with SCA2. Sixty-eight percent of rehabilitated patients improved with respect to some clinical parameter, obtaining better quality of life by reduction of the disability associated with the disease. However, it is propitious to mention that in order to obtain these benefits, correct dosification and sustainability of rehabilitation sessions are necessary [165]. In view of these results, we consider that neurorehabilitation comprises a very important strategy in the physical treatment of persons with SCA2. Thus, this should always be applied to patients, even when these receive some pharmacological or other treatment.

Conclusions

After identification of SCA2 as a polyQ disorder 16 years ago, there has been a substantial progress in defining ataxin-2 function and the molecular mechanisms underlying SCA2. As SCA2 shares main features with other polyQ disorders, including formation of intracellular aggregates of the mutant protein, and cytotoxicity of certain neuronal subtypes, it is expected that previous experience in other polyQ disorders will help the design of suitable therapeutic approaches for SCA2. A more comprehensive characterization of SCA2 animal models is required to ascertain whether these systems mimic faithfully the molecular defects in SCA2 and would then allow for an accurate assessment of RNAi and AON therapies, including the precise evaluation of gene and delivery specificities. Although it is not yet possible to determine with certainty which therapeutic approach might be suited for SCA2, the putative involvement of ataxin-2 in crucial cell functions, such as posttranscriptional and translational regulation, indicates that a therapy that can retain full or partial endogenous wild-type allele function is the most appropriate and would be well tolerated.

Acknowledgments

This work was supported by CONACyT-México, Grant No. 128418 (B.C.).

Conflict of interest

The authors declare that there are no conflicts of interest.

Copyright information

© Springer Science+Business Media, LLC 2012