From pathways to targets: understanding the mechanisms behind polyglutamine disease.

The history of polyglutamine diseases dates back approximately 20 years to the discovery of a polyglutamine repeat in the androgen receptor of SBMA followed by the identification of similar expansion mutations in Huntington's disease, SCA1, DRPLA, and the other spinocerebellar ataxias. This common molecular feature of polyglutamine diseases suggests shared mechanisms in disease pathology and neurodegeneration of disease specific brain regions. In this review, we discuss the main pathogenic pathways including proteolytic processing, nuclear shuttling and aggregation, mitochondrial dysfunction, and clearance of misfolded polyglutamine proteins and point out possible targets for treatment.

Cerebellar soluble mutant ataxin-3 level decreases during disease progression in Spinocerebellar Ataxia Type 3 mice.

Spinocerebellar Ataxia Type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominantly inherited neurodegenerative disease caused by an expanded polyglutamine stretch in the ataxin-3 protein. A pathological hallmark of the disease is cerebellar and brainstem atrophy, which correlates with the formation of intranuclear aggregates in a specific subset of neurons. Several studies have demonstrated that the formation of aggregates depends on the generation of aggregation-prone and toxic intracellular ataxin-3 fragments after proteolytic cleavage of the full-length protein. Despite this observed increase in aggregated mutant ataxin-3, information on soluble mutant ataxin-3 levels in brain tissue is lacking. A quantitative method to analyze soluble levels will be a useful tool to characterize disease progression or to screen and identify therapeutic compounds modulating the level of toxic soluble ataxin-3. In the present study we describe the development and application of a quantitative and easily applicable immunoassay for quantification of soluble mutant ataxin-3 in human cell lines and brain samples of transgenic SCA3 mice. Consistent with observations in Huntington disease, transgenic SCA3 mice reveal a tendency for decrease of soluble mutant ataxin-3 during disease progression in fractions of the cerebellum, which is inversely correlated with aggregate formation and phenotypic aggravation. Our analyses demonstrate that the time-resolved Förster resonance energy transfer immunoassay is a highly sensitive and easy method to measure the level of soluble mutant ataxin-3 in biological samples. Of interest, we observed a tendency for decrease of soluble mutant ataxin-3 only in the cerebellum of transgenic SCA3 mice, one of the most affected brain regions in Spinocerebellar Ataxia Type 3 but not in whole brain tissue, indicative of a brain region selective change in mutant ataxin-3 protein homeostasis.

Calpain-mediated ataxin-3 cleavage in the molecular pathogenesis of spinocerebellar ataxia type 3 (SCA3).

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

Automated behavioral phenotyping reveals presymptomatic alterations in a SCA3 genetrap mouse model.

Characterization of disease models of neurodegenerative disorders requires a systematic and comprehensive phenotyping in a highly standardized manner. Therefore, automated high-resolution behavior test systems such as the homecage based LabMaster system are of particular interest. We demonstrate the power of the automated LabMaster system by discovering previously unrecognized features of a recently characterized atxn3 mutant mouse model. This model provided neurological symptoms including gait ataxia, tremor, weight loss and premature death at the age of 12 months usually detectable just 2 weeks before the mice died. Moreover, using the LabMaster system we were able to detect hypoactivity in presymptomatic mutant mice in the dark as well as light phase. Additionally, we analyzed inflammation, immunological and hematological parameters, which indicated a reduced immune defense in phenotypic mice. Here we demonstrate that a detailed characterization even of organ systems that are usually not affected in SCA3 is important for further studies of pathogenesis and required for the preclinical therapeutic studies.

N-terminal ataxin-3 causes neurological symptoms with inclusions, endoplasmic reticulum stress and ribosomal dislocation.

Mutant ataxin-3 is aberrantly folded and proteolytically cleaved in spinocerebellar ataxia type 3. The C-terminal region of the protein includes a polyglutamine stretch that is expanded in spinocerebellar ataxia type 3. Here, we report on the analysis of an ataxin-3 mutant mouse that has been obtained by gene trap integration. The ataxin-3 fusion protein encompasses 259 N-terminal amino acids including the Josephin domain and an ubiquitin-interacting motif but lacks the C-terminus with the polyglutamine stretch, the valosin-containing protein binding region and part of the ubiquitin-interacting motif 2. Homozygous ataxin-3 mutant mice were viable and showed no apparent anatomical defects at birth. However, at the age of 9 months, homozygous and heterozygous mutant mice revealed significantly altered behaviour and progressing deficits of motor coordination followed by premature death at ∼12 months. At this time, prominent extranuclear protein aggregates and neuronal cell death was found in mutant mice. This was associated with disturbances of the endoplasmic reticulum-mediated unfolded protein response, consistent with the normal role of ataxin-3 in endoplasmic reticulum homeostasis. Thus, the ataxin-3 gene trap model provides evidence for a contribution of the non-polyglutamine containing ataxin-3 N-terminus, which mimics a calpain fragment that has been observed in spinocerebellar ataxia type 3. Consistent with the disease in humans, gene trap mice develop cytoplasmic inclusion bodies and implicate impaired unfolded protein response in the pathogenesis of spinocerebellar ataxia type 3.

Polyglutamine-induced neurodegeneration in SCA3 is not mitigated by non-expanded ataxin-3: conclusions from double-transgenic mouse models.

A crucial question in polyQ-induced neurodegeneration is the influence of wild type protein on the formation of aggregates and toxicity. Recently it was shown that non-expanded ataxin-3 protein mitigated neurodegeneration in a Drosophila and mouse model of SCA3. We now explored the effects of overexpressing non-expanded ataxin-3 with 15Q in a SCA3 transgenic mouse model with 70 polyglutamine repeats. These double-transgenic mice (dt) developed neurological symptoms with premature death at the age of 6 months comparable to the single-transgenic (st) SCA3 disease model. Furthermore, immunohistochemistry revealed similar localization and distribution of nuclear aggregates in dt- and st-mutant SCA3 mice. In a second dt-mutant mouse model, coexpression of ataxin-3 with 148Q attached to a nuclear export signal, which usually diminishes the phenotype, did even reinforce toxic effects of mutant expanded ataxin-3. We therefore conclude that overexpressing wild type ataxin-3 or mutant ataxin-3 with NES are not striking suppressors of polyglutamine-induced neurodegeneration and have thus no potential for future gene therapeutic interventions in SCA3.

Familial thrombocytosis caused by the novel germ-line mutation p.Pro106Leu in the MPL gene.

Familial thrombosis (FT) has been described as a rare autosomal-dominant disorder, mostly caused by activating mutations of the thrombopoietin gene (THPO). Other cases of FT have been linked to one of two different germline mutations in the myeloproliferative leukaemia virus oncogene gene (MPL), which codes for the thrombopoietin receptor MPL. We studied an Arab family with two siblings with severe thrombocytosis by linkage analysis and obtained evidence for linkage to MPL. Sequencing revealed homozygosity for the novel MPL germline mutation p.Pro106Leu (c.317C > T) in the two siblings. Subsequently, homozygosity for p.Pro106Leu was identified in six further FT patients from three other Arab families. Of 18 heterozygous carriers, 14 had normal platelet counts, while four had mild thrombocytosis. Strong support for association of the novel MPL mutation p.Pro106Leu with development of familial thrombocytosis has been obtained. Overall, p.Pro106Leu was absent on 386 alleles of 193 healthy German controls and present on 14 of 426 alleles (3.3%) of 213 unrelated Arabs, which was statistically significantly different (P < 0.001, Fisher's exact test). We assume that p.Pro106Leu is a frequent MPL mutation in the Arab population, leading to severe thrombocytosis in homozygotes and occasionally to mild thrombocytosis in heterozygotes. In the families described the mode of inheritance could be regarded as autosomal-recessive with possible mild heterozygote manifestation rather than autosomal-dominant with high penetrance as usually seen in FT.

Gene expression changes in a transgenic mouse model overexpressing human wildtype and mutant torsinA.

Primary torsion dystonia is an autosomal-dominantly inherited, neurodevelopmental movement disorder caused by a GAG deletion (ΔGAG) in the DYT1 gene, encoding torsinA. This mutation is responsible for approximately 70% of cases of early-onset primary torsion dystonia. The function of wildtype torsinA is still unknown, and it is unsolved how the deletion in the DYT1 gene contributes to the development of the disease. To better understand the molecular processes involved in torsinA pathology, we used genome-wide oligonucleotide microarrays to characterize gene expression patterns in the striatum of mouse models overexpressing the human wildtype and mutant torsinA. By this approach we were able to detect gene expression changes that seem to be specific for torsinA pathology. We found an impact of torsinA, independent from genotype, on vesicle trafficking, exocytosis, and neurotransmitter release in our mouse model. In addition, we were able to identify several new pathways and processes involved in the development of the nervous system that are affected by wildtype and mutant torsinA. Furthermore, we have striking evidence from our gene expression data that glutamate receptor mediated synaptic plasticity in the striatum is the affected underlying cellular process for impaired motor learning in human ΔGAG torsinA transgenic mice.

Nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3: in vivo evidence.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat in the MJD1 gene resulting in an expanded polyglutamine repeat in the ataxin-3 protein. To study the course of the disease, we generated transgenic mice for SCA3 using full-length ataxin-3 constructs containing 15, 70, or 148 CAG repeats, respectively. Control mice (15 CAGs) were phenotypically normal and had no neuropathological findings. However, mice transgenic for ataxin-3 with expanded polyglutamine repeats were severely affected by a strong neurological phenotype with tremor, behavioral deficits, strongly reduced motor and exploratory activity, a hunchback, and premature death at 3 to 6 months of age. Neuropathological examination by immunohistochemical staining revealed ubiquitin- and ataxin-3-positive intranuclear inclusion bodies in a multitude of neurons. Directing ataxin-3 with 148 CAGs to the nucleus revealed an even more pronounced phenotype with more inclusions and earlier death, whereas mice transgenic with the same construct but attached to a nuclear export signal developed a milder phenotype with less inclusions. These studies indicate that nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3 in vivo.