CAG Repeat Size Influences the Progression Rate of Spinocerebellar Ataxia Type 3.

OBJECTIVE: In spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), the expanded cytosine adenine guanine (CAG) repeat in ATXN3 is the causal mutation, and its length is the main factor in determining the age at onset (AO) of clinical symptoms. However, the contribution of the expanded CAG repeat length to the rate of disease progression after onset has remained a matter of debate, even though an understanding of this factor is crucial for experimental data on disease modifiers and their translation to clinical trials and their design. METHODS: Eighty-two Dutch patients with SCA3/MJD were evaluated annually for 15 years using the International Cooperative Ataxia Rating Scale (ICARS). Using linear growth curve models, ICARS progression rates were calculated and tested for their relation to the length of the CAG repeat expansion and to the residual age at onset (RAO): The difference between the observed AO and the AO predicted on the basis of the CAG repeat length. RESULTS: On average, ICARS scores increased 2.57 points/year of disease. The length of the CAG repeat was positively correlated with a more rapid ICARS progression, explaining 30% of the differences between patients. Combining both the length of the CAG repeat and RAO as comodifiers explained up to 47% of the interpatient variation in ICARS progression. INTERPRETATION: Our data imply that the length of the expanded CAG repeat in ATXN3 is a major determinant of clinical decline, which suggests that CAG-dependent molecular mechanisms similar to those responsible for disease onset also contribute to the rate of disease progression in SCA3/MJD. ANN NEUROL 2021;89:66-73.

On the distribution of intranuclear and cytoplasmic aggregates in the brainstem of patients with spinocerebellar ataxia type 2 and 3.

The polyglutamine (polyQ) diseases are a group of genetically and clinically heterogeneous neurodegenerative diseases, characterized by the expansion of polyQ sequences in unrelated disease proteins, which form different types of neuronal aggregates. The aim of this study was to characterize the aggregation pathology in the brainstem of spinocerebellar ataxia type 2 (SCA2) and 3 (SCA3) patients. For good recognition of neurodegeneration and rare aggregates, we employed 100 µm PEG embedded brainstem sections, which were immunostained with the 1C2 antibody, targeted at polyQ expansions, or with an antibody against p62, a reliable marker of protein aggregates. Brainstem areas were scored semiquantitatively for neurodegeneration, severity of granular cytoplasmic staining (GCS) and frequency of neuronal nuclear inclusions (NNI). SCA2 and SCA3 tissue exhibited the same aggregate types and similar staining patterns. Several brainstem areas showed statistically significant differences between disease groups, whereby SCA2 showed more severe GCS and SCA3 showed more numerous NNI. We observed a positive correlation between GCS severity and neurodegeneration in SCA2 and SCA3 and an inverse correlation between the frequency of NNI and neurodegeneration in SCA3. Although their respective disease proteins are unrelated, SCA2 and SCA3 showed the same aggregate types. Apparently, the polyQ sequence alone is sufficient as a driver of protein aggregation. This is then modified by protein context and intrinsic properties of neuronal populations. The severity of GCS was the best predictor of neurodegeneration in both disorders, while the inverse correlation of neurodegeneration and NNI in SCA3 tissue implies a protective role of these aggregates.

No parkinsonism in SCA2 and SCA3 despite severe neurodegeneration of the dopaminergic substantia nigra.

See Klockgether (doi:10.1093/awv253) for a scientific commentary on this article.The spinocerebellar ataxias types 2 (SCA2) and 3 (SCA3) are autosomal dominantly inherited cerebellar ataxias which are caused by CAG trinucleotide repeat expansions in the coding regions of the disease-specific genes. Although previous post-mortem studies repeatedly revealed a consistent neurodegeneration of the dopaminergic substantia nigra in patients with SCA2 and with SCA3, parkinsonian motor features evolve only rarely. As the pathophysiological mechanism how SCA2 and SCA3 patients do not exhibit parkinsonism is still enigmatic, we performed a positron emission tomography and a post-mortem study of two independent cohorts of SCA2 and SCA3 patients with and without parkinsonian features. Positron emission tomography revealed a significant reduction of dopamine transporter levels in the striatum as well as largely unaffected postsynaptic striatal D2 receptors. In spite of this remarkable pathology in the motor mesostriatal pathway, only 4 of 19 SCA2 and SCA3 patients suffered from parkinsonism. The post-mortem investigation revealed, in addition to an extensive neuronal loss in the dopaminergic substantia nigra of all patients with spinocerebellar ataxia, a consistent affection of the thalamic ventral anterior and ventral lateral nuclei, the pallidum and the cholinergic pedunculopontine nucleus. With the exception of a single patient with SCA3 who suffered from parkinsonian motor features during his lifetime, the subthalamic nucleus underwent severe neuronal loss, which was clearly more severe in its motor territory than in its limbic or associative territories. Our observation that lesions of the motor territory of the subthalamic nucleus were consistently associated with the prevention of parkinsonism in our SCA2 and SCA3 patients matches the clinical experience that selective targeting of the motor territory of the subthalamic nucleus by focal lesions or deep brain stimulation can ameliorate parkinsonian motor features and is likely to counteract the manifestation of parkinsonism in SCA2 and SCA3 despite a severe neurodegeneration of the dopaminergic substantia nigra.

Ataxin-2 modulates the levels of Grb2 and SRC but not ras signaling.

Ataxin-2 (ATXN2) is implicated mainly in mRNA processing. Some ATXN2 associates with receptor tyrosine kinases (RTK), inhibiting their endocytic internalization through interaction of proline-rich domains (PRD) in ATXN2 with SH3 motifs in Src. Gain of function of ATXN2 leads to neuronal atrophy in the diseases spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS). Conversely, ATXN2 knockout (KO) mice show hypertrophy and insulin resistance. To elucidate the influence of ATXN2 on trophic regulation, we surveyed interactions of ATXN2 with SH3 motifs from numerous proteins and observed a novel interaction with Grb2. Direct binding in glutathione S-transferase (GST) pull-down assays and coimmunoprecipitation of the endogenous proteins indicated a physiologically relevant association. In SCA2 patient fibroblasts, Grb2 more than Src protein levels were diminished, with an upregulation of both transcripts suggesting enhanced protein turnover. In KO mouse embryonal fibroblasts (MEF), the protein levels of Grb2 and Src were decreased. ATXN2 absence by itself was insufficient to significantly change Grb2-dependent signaling for endogenous Ras levels, Ras-GTP levels, and kinetics as well as MEK1 phosphorylation, suggesting that other factors compensate for proliferation control. In KO tissue with postmitotic neurons, a significant decrease of Src protein levels is prominent rather than Grb2. ATXN2 mutations modulate the levels of several components of the RTK endocytosis complex and may thus contribute to alter cell proliferation as well as translation and growth.

Degeneration of the cerebellum in Huntington's disease (HD): possible relevance for the clinical picture and potential gateway to pathological mechanisms of the disease process.

Huntington's disease (HD) is a polyglutamine disease and characterized neuropathologically by degeneration of the striatum and select layers of the neo- and allocortex. In the present study, we performed a systematic investigation of the cerebellum in eight clinically diagnosed and genetically confirmed HD patients. The cerebellum of all HD patients showed a considerable atrophy, as well as a consistent loss of Purkinje cells and nerve cells of the fastigial, globose, emboliform and dentate nuclei. This pathology was obvious already in HD brains assigned Vonsattel grade 2 striatal atrophy and did not correlate with the extent and distribution of striatal atrophy. Therefore, our findings suggest (i) that the cerebellum degenerates early during HD and independently from the striatal atrophy and (ii) that the onset of the pathological process of HD is multifocal. Degeneration of the cerebellum might contribute significantly to poorly understood symptoms occurring in HD such as impaired rapid alternating movements and fine motor skills, dysarthria, ataxia and postural instability, gait and stance imbalance, broad-based gait and stance, while the morphological alterations (ie ballooned neurons, torpedo-like axonal inclusions) observed in the majority of surviving nerve cells may represent a gateway to the unknown mechanisms of the pathological process of HD.

Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions.

Paroxysmal kinesigenic dyskinesia with infantile convulsions (PKD/IC) is an episodic movement disorder with autosomal-dominant inheritance and high penetrance, but the causative genetic mutation is unknown. We have now identified four truncating mutations involving the gene PRRT2 in the vast majority (24/25) of well-characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. PRRT2 encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture, and mutants associated with PKD/IC lead to dramatically reduced PRRT2 levels, leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.

Brain pathology of spinocerebellar ataxias.

The autosomal dominant cerebellar ataxias (ADCAs) represent a heterogeneous group of neurodegenerative diseases with progressive ataxia and cerebellar degeneration. The current classification of this disease group is based on the underlying genetic defects and their typical disease courses. According to this categorization, ADCAs are divided into the spinocerebellar ataxias (SCAs) with a progressive disease course, and the episodic ataxias (EA) with episodic occurrences of ataxia. The prominent disease symptoms of the currently known and genetically defined 31 SCA types result from damage to the cerebellum and interconnected brain grays and are often accompanied by more specific extra-cerebellar symptoms. In the present review, we report the genetic and clinical background of the known SCAs and present the state of neuropathological investigations of brain tissue from SCA patients in the final disease stages. Recent findings show that the brain is commonly seriously affected in the polyglutamine SCAs (i.e. SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17) and that the patterns of brain damage in these diseases overlap considerably in patients suffering from advanced disease stages. In the more rarely occurring non-polyglutamine SCAs, post-mortem neuropathological data currently are scanty and investigations have been primarily performed in vivo by means of MRI brain imaging. Only a minority of SCAs exhibit symptoms and degenerative patterns allowing for a clear and unambiguous diagnosis of the disease, e.g. retinal degeneration in SCA7, tau aggregation in SCA11, dentate calcification in SCA20, protein depositions in the Purkinje cell layer in SCA31, azoospermia in SCA32, and neurocutaneous phenotype in SCA34. The disease proteins of polyglutamine ataxias and some non-polyglutamine ataxias aggregate as cytoplasmic or intranuclear inclusions and serve as morphological markers. Although inclusions may impair axonal transport, bind transcription factors, and block protein quality control, detailed molecular and pathogenetic consequences remain to be determined.

Presence of ATM protein and residual kinase activity correlates with the phenotype in ataxia-telangiectasia: a genotype-phenotype study.

Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with multisystem involvement and cancer predisposition, caused by mutations in the A-T mutated (ATM) gene. To study genotype-phenotype correlations, we evaluated the clinical and laboratory data of 51 genetically proven A-T patients, and additionally measured ATM protein expression and kinase activity. Patients without ATM kinase activity showed the classical phenotype. The presence of ATM protein, correlated with slightly better immunological function. Residual kinase activity correlated with a milder and essentially different neurological phenotype, absence of telangiectasia, normal endocrine and pulmonary function, normal immunoglobulins, significantly lower X-ray hypersensitivity in lymphocytes, and extended lifespan. In these patients, cancer occurred later in life and generally consisted of solid instead of lymphoid malignancies. The genotypes of severely affected patients generally included truncating mutations resulting in total absence of ATM kinase activity, while patients with milder phenotypes harbored at least one missense or splice site mutation resulting in expression of ATM with some kinase activity. Overall, the phenotypic manifestations in A-T show a continuous spectrum from severe classical childhood-onset A-T to a relatively mild adult-onset disorder, depending on the presence of ATM protein and kinase activity. Each patient is left with a tremendously increased cancer risk.

In children with Friedreich ataxia, muscle and ataxia parameters are associated.

AIM: In children with Friedreich ataxia (FRDA), ataxia is assessed using the surrogate marker the International Cooperative Ataxia Rating Scale (ICARS). We aimed to determine whether ICARS scores in children with FRDA are confounded by muscle weakness. METHOD: In 12 children with FRDA (10 males, two females; mean age 13 y 6 mo, SD 2 y 6 mo) and 12 age-matched children without FRDA (nine males; three females), we determined the association between muscle and ataxia parameters (i.e. muscle ultrasound density (MUD), muscle force, sensory evoked potentials, and ICARS scores). Children with FRDA were included on the basis of FXN gene analysis. Children in the comparison group were included on basis of uneventful pregnancy and normal cognitive and neurological development. RESULTS: In children with FRDA, muscle ultrasound density was homogeneously increased in the biceps, quadriceps, and tibialis anterior muscles (median 4SD). FRDA muscle weakness was significantly more pronounced in proximal than in distal muscles (-2SD vs -0.5SD respectively; p=0.004), with a stronger impairment of leg muscles than of arm muscles (-2SD vs -0. SD respectively; p=0.001). Comparing MUD between children with FRDA and an age-matched comparison group revealed a relatively strong increase in MUD in the proximal leg muscles in the FRDA group. Under the condition of persistently absent sensory evoked potentials, leg ICARS subscores in the FRDA group appeared to be positively associated with leg muscle force until a maximal plateau level of ICARS subscores was reached. INTERPRETATION: In children with FRDA, ataxia scales based on ICARS are confounded by muscle weakness. Longitudinal ICARS evaluations in children with FRDA do not necessarily indicate altered ataxia.

HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones.

A small number of heat-shock proteins have previously been shown to act protectively on aggregation of several proteins containing an extended polyglutamine (polyQ) stretch, which are linked to a variety of neurodegenerative diseases. A specific subfamily of heat-shock proteins is formed by the HSPB family of molecular chaperones, which comprises 10 members (HSPB1-10, also called small HSP). Several of them are known to act as anti-aggregation proteins in vitro. Whether they also act protectively in cells against polyQ aggregation has so far only been studied for few of them (e.g. HSPB1, HSPB5 and HSPB8). Here, we compared the 10 members of the human HSPB family for their ability to prevent aggregation of disease-associated proteins with an expanded polyQ stretch. HSPB7 was identified as the most active member within the HSPB family. It not only suppressed polyQ aggregation but also prevented polyQ-induced toxicity in cells and its expression reduces eye degeneration in a Drosophila polyQ model. Upon overexpression in cells, HSPB7 was not found in larger oligomeric species when expressed in cells and-unlike HSPB1-it did not improve the refolding of heat-denatured luciferase. The action of HSPB7 was also not dependent on the Hsp70 machine or on proteasomal activity, and HSPB7 overexpression alone did not increase autophagy. However, in ATG5-/- cells that are defective in macroautophagy, the anti-aggregation activity of HSPB7 was substantially reduced. Hence, HSPB7 prevents toxicity of polyQ proteins at an early stage of aggregate formation by a non-canonical mechanism that requires an active autophagy machinery.

Axonal inclusions in spinocerebellar ataxia type 3.

Protein aggregation is a major pathological hallmark of many neurodegenerative disorders including polyglutamine diseases. Aggregation of the mutated form of the disease protein ataxin-3 into neuronal nuclear inclusions is well described in the polyglutamine disorder spinocerebellar ataxia type 3 (SCA3 or Machado-Joseph disease), although these inclusions are not thought to be directly pathogenic. Neuropil aggregates have not yet been described in SCA3. We performed a systematic immunohistochemical study of serial thick sections through brains of seven clinically diagnosed and genetically confirmed SCA3 patients. Using antibodies against ataxin-3, p62, ubiquitin, the polyglutamine marker 1C2 as well as TDP-43, we analyzed neuronal localization, composition and distribution of aggregates within SCA3 brains. The analysis revealed widespread axonal aggregates in fiber tracts known to undergo neurodegeneration in SCA3. Similar to neuronal nuclear inclusions, the axonal aggregates were ubiquitinated and immunopositive for the proteasome and autophagy associated shuttle protein p62, indicating involvement of neuronal protein quality control mechanisms. Rare TDP-43 positive axonal inclusions were also observed. Based on the correlation between affected fiber tracts and degenerating neuronal nuclei, we hypothesize that these novel axonal inclusions may be detrimental to axonal transport mechanisms and thereby contribute to degeneration of nerve cells in SCA3.

New insights into the pathoanatomy of spinocerebellar ataxia type 3 (Machado-Joseph disease).

PURPOSE OF REVIEW: This review summarizes recent neuropathological findings in spinocerebellar ataxia type 3 and discusses their relevance for clinical neurology. RECENT FINDINGS: The extent of the spinocerebellar ataxia type 3 related central nervous neurodegenerative changes has been recently systematically investigated in a series of pathoanatomical studies. These studies showed that the extent of the central nervous degenerative changes of spinocerebellar ataxia type 3 has been underestimated so far. The newly described pattern of central nervous neurodegeneration includes the visual, auditory, vestibular, somatosensory, ingestion-related, dopaminergic and cholinergic systems. These pathological findings were correlated with clinical findings and explain a variety of the spinocerebellar ataxia type 3 symptoms observed in clinical practice. SUMMARY: Systematic pathoanatomical analysis of spinocerebellar ataxia type 3 brains helps to understand the structural basis of this neurodegenerative disease and offers explanations for a variety of disease symptoms. This better understanding of the neuropathology of the condition has implications for the treatment of spinocerebellar ataxia type 3 patients and represents a basis for further biochemical and molecular biological studies aimed at deciphering the pathomechanisms of this progressive ataxic disorder.

Asian origin for the worldwide-spread mutational event in Machado-Joseph disease.

BACKGROUND: Machado-Joseph disease is the most frequent dominant ataxia worldwide. Despite its frequency and presence in many populations, only 2 founder mutations have been suggested to explain its current geographic distribution. OBJECTIVES: To trace back in history the main mutational events in Machado-Joseph disease, we aimed to assess ancestral haplotypes and population backgrounds, to date the mutations, and to trace the routes and time of introduction of the founder haplotypes in different populations. DESIGN, SETTING, AND PARTICIPANTS: We studied 264 families with Machado-Joseph disease from 20 different populations. Six intragenic single-nucleotide polymorphisms were used to determine ancestral mutational events; 4 flanking short tandem repeats were used to construct extended haplotypes and measure accumulation of genetic diversity over time within each lineage. RESULTS: The worldwide-spread lineage, TTACAC, had its highest diversity in the Japanese population, where we identified the ancestral short tandem repeat-based haplotype. Accumulated variability suggested a postneolithic mutation, about 5774 +/- 1116 years old, with more recent introductions in North America, Germany, France, Portugal, and Brazil. As to the second mutational event, in the GTGGCA lineage, only 7 families (of 71 families) did not have Portuguese ancestry, although gene diversity was again smaller in Portuguese families (0.44) than in non-Portuguese families (0.93). CONCLUSIONS: The worldwide-spread mutation may have first occurred in Asia and later been diffused throughout Europe, with a founder effect accounting for its high prevalence in Portugal; the other Machado-Joseph disease lineage is more recent, about 1416 +/- 434 years old, and its dispersion may be explained mainly by recent Portuguese emigration.

Consistent affection of the central somatosensory system in spinocerebellar ataxia type 2 and type 3 and its significance for clinical symptoms and rehabilitative therapy.

The spinocerebellar ataxias type 2 (SCA2) and type 3 (SCA3) are progressive, currently untreatable and ultimately fatal ataxic disorders, which belong to the group of neurological disorders known as CAG-repeat or polyglutamine diseases. Since knowledge regarding the involvement of the central somatosensory system in SCA2 and SCA3 currently is only fragmentary, a variety of somatosensory disease signs remained unexplained or widely misunderstood. The present review (1) draws on the current knowledge in the field of neuroanatomy, (2) describes the anatomy and functional neuroanatomy of the human central somatosensory system, (3) provides an overview of recent findings regarding the affection of the central somatosensory system in SCA2 and SCA3 patients, and (4) points out the underestimated pathogenic role of the central somatosensory system for somatosensory and somatomotor disease symptoms in SCA2 and SCA3. Finally, based on recent findings in the research fields of neuropathology and neural plasticity, this review supports currently applied and recommends further neurorehabilitative approaches aimed at maintaining, improving, and/or recovering adequate somatomotor output by enforcing and changing somatosensory input in the very early clinical stages of SCA2 and SCA3.

Polarised asymmetric inheritance of accumulated protein damage in higher eukaryotes.

Disease-associated misfolded proteins or proteins damaged due to cellular stress are generally disposed via the cellular protein quality-control system. However, under saturating conditions, misfolded proteins will aggregate. In higher eukaryotes, these aggregates can be transported to accumulate in aggresomes at the microtubule organizing center. The fate of cells that contain aggresomes is currently unknown. Here we report that cells that have formed aggresomes can undergo normal mitosis. As a result, the aggregated proteins are asymmetrically distributed to one of the daughter cells, leaving the other daughter free of accumulated protein damage. Using both epithelial crypts of the small intestine of patients with a protein folding disease and Drosophila melanogaster neural precursor cells as models, we found that the inheritance of protein aggregates during mitosis occurs with a fixed polarity indicative of a mechanism to preserve the long-lived progeny.

Spinocerebellar ataxia type 3 (SCA3): thalamic neurodegeneration occurs independently from thalamic ataxin-3 immunopositive neuronal intranuclear inclusions.

In the last years progress has been made regarding the involvement of the thalamus during the course of the currently known polyglutamine diseases. Although recent studies have shown that the thalamus consistently undergoes neurodegeneration in Huntington's disease (HD) and spinocerebellar ataxia type 2 (SCA2) it is still unclear whether it is also a consistent target of the pathological process of spinocerebellar ataxia type 3 (SCA3). Accordingly we studied the thalamic pathoanatomy and distribution pattern of ataxin-3 immunopositive neuronal intranuclear inclusions (NI) in nine clinically diagnosed and genetically confirmed SCA3 patients and carried out a detailed statistical analysis of our findings. During our pathoanatomical study we disclosed (i) a consistent degeneration of the ventral anterior, ventral lateral and reticular thalamic nuclei; (ii) a degeneration of the ventral posterior lateral nucleus and inferior and lateral subnuclei of the pulvinar in the majority of these SCA3 patients; and (iii) a degeneration of the ventral posterior medial and lateral posterior thalamic nuclei, the lateral geniculate body and some of the limbic thalamic nuclei in some of them. Upon immunocytochemical analysis we detected NI in all of the thalamic nuclei of all of our SCA3 patients. According to our statistical analysis (i) thalamic neurodegeneration and the occurrence of ataxin-3 immunopositive thalamic NI was not associated with the individual length of the CAG-repeats in the mutated SCA3 allele, the patients age at disease onset and the duration of SCA3 and (ii) thalamic neurodegeneration was not correlated with the occurrence of ataxin-3 immunopositive thalamic NI. This lack of correlation may suggest that ataxin-3 immunopositive NI are not immediately decisive for the fate of affected nerve cells but rather represent unspecific and pathognomonic morphological markers of SCA3.

Accumulation of aberrant ubiquitin induces aggregate formation and cell death in polyglutamine diseases.

Polyglutamine diseases are characterized by neuronal intranuclear inclusions (NIIs) of expanded polyglutamine proteins, indicating the failure of protein degradation. UBB(+1), an aberrant form of ubiquitin, is a substrate and inhibitor of the proteasome, and was previously reported to accumulate in Alzheimer disease and other tauopathies. Here, we show accumulation of UBB(+1) in the NIIs and the cytoplasm of neurons in Huntington disease and spinocerebellar ataxia type-3, indicating inhibition of the proteasome by polyglutamine proteins in human brain. We found that UBB(+1) not only increased aggregate formation of expanded polyglutamines in neuronally differentiated cell lines, but also had a synergistic effect on apoptotic cell death due to expanded polyglutamine proteins. These findings implicate UBB(+1) as an aggravating factor in polyglutamine-induced neurodegeneration, and clearly identify an important role for the ubiquitin-proteasome system in polyglutamine diseases.

Caspase-mediated proteolysis of the polyglutamine disease protein ataxin-3.

Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease, is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated disease genes. Polyglutamine disorders are characterized by disease protein misfolding and aggregation; often within the nuclei of affected neurons. Although the precise mechanism of polyglutamine-mediated cell death remains elusive, evidence suggests that proteolysis of polyglutamine disease proteins by caspases contributes to pathogenesis. Using cellular models we now show that the endogenous spinocerebellar ataxia type-3 disease protein, ataxin-3, is proteolyzed in apoptotic paradigms, resulting in the loss of full-length ataxin-3 and the corresponding appearance of an approximately 28-kDa fragment containing the glutamine repeat. Broad-spectrum caspase inhibitors block ataxin-3 proteolysis and studies suggest that caspase-1 is a primary mediator of cleavage. Site-directed mutagenesis experiments eliminating three, six or nine potential caspase cleavage sites in the protein suggest redundancy in the site(s) at which cleavage can occur, as previously described for other disease proteins; but also map a major cleavage event to a cluster of aspartate residues within the ubiquitin-binding domain of ataxin-3 near the polyglutamine tract. Finally, caspase-mediated cleavage of expanded ataxin-3 resulted in increased ataxin-3 aggregation, suggesting a potential role for caspase-mediated proteolysis in spinocerebellar ataxia type-3 pathogenesis.

Gene expression profiling in ataxin-3 expressing cell lines reveals distinct effects of normal and mutant ataxin-3.

Spinocerebellar ataxia type 3 (SCA3) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the gene product, ataxin-3. We have previously shown that mutant ataxin-3 causes upregulation of inflammatory genes in transgenic SCA3 cell lines and human SCA3 pontine neurons. We report here a complex pattern of transcriptional changes by microarray gene expression profiling and Northern blot analysis in a SCA3 cell model. Twenty-three differentially expressed genes involved in inflammatory reactions, nuclear transcription, and cell surface-associated processes were identified. The identified corresponding proteins were analyzed by immunohistochemistry in human disease and control brain tissue to evaluate their implication in SCA3 pathogenesis. In addition to several inflammatory mediators upregulated in mutant ataxin-3 expressing cell lines and pontine neurons of SCA3 patients, we identified a profound repression of genes encoding cell surface-associated proteins in cells overexpressing normal ataxin-3. Correspondingly, these genes were upregulated in mutant ataxin-3 expressing cell lines and in pontine neurons of SCA3 patients. These findings identify for the first time target genes transcriptionally regulated by normal ataxin-3 and support the hypothesis that both loss of normal ataxin-3 and gain of function through protein-protein interacting properties of mutant ataxin-3 contribute to SCA3 pathogenesis.