A computerized database-scan to identify c-MYC targets

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the protein ataxin-7, a protein of unknown function. In order to analyze the expression pattern of wild type ataxin-7 in detail, the murine SCA7 gene homolog was cloned and the expression pattern in mice analyzed. The SCA7 mouse and human gene exhibit a high degree of identity at both DNA (88.2%) and protein (88.7%) level. The CAG repeat region, known to be polymorphic in man, is conserved in mouse but contained only five repeats in all mouse strains analyzed. The arrestin homology domain and the nuclear localization signal found in human ataxin-7 is also conserved in the murine homolog. Expression of ataxin-7 was detected during mouse embryonic development and in all adult mouse tissues examined by northern and western blots. In brain, immunohistological staining revealed an ataxin-7 expression pattern similar to that in human, with ataxin-7 expression in cerebellum, several brainstem nuclei, cerebral cortex and hippocampus. Our data show high conservation of ataxin-7 both structurally and at the level of expression, suggesting a conserved role for the protein in mice and humans.     

SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in short-term plasticity

We targeted 266 CAG repeats (a number that causes infantile-onset disease) into the mouse Sca7 locus to generate an authentic model of spinocerebellar ataxia type 7 (SCA7). These mice reproduced features of infantile SCA7 (ataxia, visual impairments, and premature death) and showed impaired short-term synaptic potentiation; downregulation of photoreceptor-specific genes, despite apparently normal CRX activity, led to shortening of photoreceptor outer segments. Wild-type ataxin-7 was barely detectable, as was mutant ataxin-7 in young animals; with increasing age, however, ataxin-7 staining became more pronounced. Neurons that appeared most vulnerable had relatively high levels of mutant ataxin-7; it is interesting, however, that marked dysfunction occurred in these neurons weeks prior to the appearance of nuclear inclusions. These data demonstrate that glutamine expansion stabilizes mutant ataxin-7, provide an explanation for selective neuronal vulnerability, and show that mutant ataxin-7 impairs posttetanic potentiation (PTP).     

Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins

Background

We used lentiviral vectors (LVs) to generate a new SCA7 animal model overexpressing a truncated mutant ataxin-7 (MUT ATXN7) fragment in the mouse cerebellum, in order to characterize the specific neuropathological and behavioral consequences of the genetic defect in this brain structure.

Results

LV-mediated overexpression of MUT ATXN7 into the cerebellum of C57/BL6 adult mice induced neuropathological features similar to that observed in patients, such as intranuclear aggregates in Purkinje cells (PC), loss of synaptic markers, neuroinflammation, and neuronal death. No neuropathological changes were observed when truncated wild-type ataxin-7 (WT ATXN7) was injected. Interestingly, the local delivery of LV-expressing mutant ataxin-7 (LV-MUT-ATXN7) into the cerebellum of wild-type mice also mediated the development of an ataxic phenotype at 8 to 12 weeks post-injection. Importantly, our data revealed abnormal levels of the FUS/TLS, MBNL1, and TDP-43 RNA-binding proteins in the cerebellum of the LV-MUT-ATXN7 injected mice. MUT ATXN7 overexpression induced an increase in the levels of the pathological phosphorylated TDP-43, and a decrease in the levels of soluble FUS/TLS, with both proteins accumulating within ATXN7-positive intranuclear inclusions. MBNL1 also co-aggregated with MUT ATXN7 in most PC nuclear inclusions. Interestingly, no MBNL2 aggregation was observed in cerebellar MUT ATXN7 aggregates. Immunohistochemical studies in postmortem tissue from SCA7 patients and SCA7 knock-in mice confirmed SCA7-induced nuclear accumulation of FUS/TLS and MBNL1, strongly suggesting that these proteins play a physiopathological role in SCA7.

Conclusions

This study validates a novel SCA7 mouse model based on lentiviral vectors, in which strong and sustained expression of MUT ATXN7 in the cerebellum was found sufficient to generate motor defects.

     

Spinocerebellar ataxia 7 (SCA7)

Spinocerebellar ataxia 7 (SCA7) is a progressive autosomal dominant neurodegenerative disorder characterized clinically by cerebellar ataxia associated with progressive macular dystrophy. The disease affects primarily the cerebellum and the retina, but also many other CNS structures as the disease progresses. SCA7 is caused by expansion of an unstable trinucleotide CAG repeat encoding a polyglutamine tract in the corresponding protein, ataxin-7. Normal SCA7 alleles contain 4-35 CAG repeats, whereas pathological alleles contain from 36-306 CAG repeats. SCA7 has a number of features in common with other diseases with polyglutamine expansions: (i) the appearance of clinical symptoms above a threshold number of CAG repeats (>35); (ii) a correlation between the size of the expansion and the rate of progression of the disease: the larger the repeat, the faster the progression; (iii) instability of the repeat sequence (approximately 12 CAG/transmission) that accounts for the marked anticipation of approximately 20 years/generation. The CAG repeat sequence is particularly unstable and de novo mutations can occur during paternal transmissions of intermediate size alleles (28-35 CAG repeats). This can explain the persistence of the disease in spite of the anticipation that should have resulted in its extinction.     

RNA Interference-Based Therapy for Spinocerebellar Ataxia Type 7 Retinal Degeneration

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disease characterized by loss of motor coordination and retinal degeneration with no current therapies in the clinic. The causative mutation is an expanded CAG repeat in the ataxin-7 gene whose mutant protein product causes cerebellar and brainstem degeneration and retinal cone-rod dystrophy. Here, we reduced the expression of both mutant and wildtype ataxin-7 in the SCA7 mouse retina by RNA interference and evaluated retinal function 23 weeks post injection. We observed a preservation of normal retinal function and no adverse toxicity with ≥50% reduction of mutant and wildtype ataxin-7 alleles. These studies address an important safety concern regarding non-allele specific silencing of ataxin-7 for SCA7 retinal therapy.     

Histone deacetylase-3 interacts with ataxin-7 and is altered in a spinocerebellar ataxia type 7 mouse model

Spinocerebellar ataxia type 7 (SCA7) is caused by a toxic polyglutamine (polyQ) expansion in the N-terminus of the protein ataxin-7. Ataxin-7 has a known function in the histone acetylase complex, Spt/Ada/Gcn5 acetylase (STAGA) chromatin-remodeling complex. We hypothesized that some histone deacetylase (HDAC) family members would impact the posttranslational modification of normal and expanded ataxin-7 and possibly modulate ataxin-7 function or neurotoxicity associated with the polyQ expansion. Interestingly, when we coexpressed each HDAC family member in the presence of ataxin-7 we found that HDAC3 increased the posttranslational modification of normal and expanded ataxin-7. Specifically, HDAC3 stabilized ataxin-7 and increased modification of the protein. Further, HDAC3 physically interacts with ataxin-7. The physical interaction of HDAC3 with normal and polyQ-expanded ataxin-7 affects the toxicity in a polyQ-dependent manner. We detect robust HDAC3 expression in neurons and glia in the cerebellum and an increase in the levels of HDAC3 in SCA7 mice. Consistent with this we found altered lysine acetylation levels and deacetylase activity in the brains of SCA7 transgenic mice. This study implicates HDAC3 and ataxin-7 interaction as a target for therapeutic intervention in SCA7, adding to a growing list of neurodegenerative diseases that may be treated by HDAC inhibitors.     

Hsp70 and Hsp40 chaperones do not modulate retinal phenotype in SCA7 mice

Nine neurodegenerative diseases, including spinocerebellar ataxia type 7 (SCA7), are caused by the expansion of polyglutamine stretches in the respective disease-causing proteins. A hallmark of these diseases is the aggregation of expanded polyglutamine-containing proteins in nuclear inclusions that also accumulate molecular chaperones and components of the ubiquitin-proteasome system. Manipulation of HSP70 and HSP40 chaperone levels has been shown to suppress aggregates in cellular models, prevent neuronal death in Drosophila, and improve to some extent neurological symptoms in mouse models. An important issue in mammals is the relative expression levels of toxic and putative rescuing proteins. Furthermore, overexpression of both HSP70 and its co-factor HSP40/HDJ2 has never been investigated in mice. We decided to address this question in a SCA7 transgenic mouse model that progressively develops retinopathy, similar to SCA7 patients. To co-express HSP70 and HDJ2 with the polyglutamine protein, in the same cell type, at comparable levels and with the same time course, we generated transgenic mice that express the heat shock proteins specifically in rod photoreceptors. While co-expression of HSP70 with its co-factor HDJ2 efficiently suppressed mutant ataxin-7 aggregation in transfected cells, they did not prevent either neuronal toxicity or aggregate formation in SCA7 mice. Furthermore, nuclear inclusions in SCA7 mice were composed of a cleaved mutant ataxin-7 fragment, whereas they contained the full-length protein in transfected cells. We propose that differences in the aggregation process might account for the different effects of chaperone overexpression in cellular and animal models of polyglutamine diseases.     

Proteasome function is inhibited by polyglutamine-expanded ataxin-1, the SCA1 gene product

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. Using d2EGFP, a short-lived enhanced green fluorescent protein, we investigated whether polyglutamine-expanded ataxin-1 affects the function of the proteasome, a cellular multicatalytic protease that degrades most misfolded proteins and regulatory proteins. In Western blot analysis and immunofluorescence experiments, d2EGFP was less degraded in HEK 293T cells transfected with ataxin-1(82Q) than in cells transfected with lacZ or empty vector controls. To test whether the stability of the d2EGFP protein was due to aggregation of ataxin-1, we constructed a plasmid carrying ataxin-1-Delta114, lacking the self-association region (SAR), and examined degradation of the d2EGFP. Both the level of ataxin-1-Delta114 aggregates and the amount of d2EGFP were drastically reduced in cells containing ataxin-1-Delta114. Furthermore, d2EGFP localization experiments showed that polyglutamine-expanded ataxin-1 inhibited the general function of the proteasome activity. Taken together, these results demonstrate that polyglutamine-expanded ataxin-1 decreases the activity of the proteasome, implying that a disturbance in the ubiquitin-proteasome pathway is directly involved in the development of spinocerebellar ataxia type1.     

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.     

Polyglutamine-expanded ataxin-7 activates mitochondrial apoptotic pathway of cerebellar neurons by upregulating Bax and downregulating Bcl-x(L)

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by polyglutamine-expanded ataxin-7. In the present investigation, we expressed disease-causing mutant ataxin-7-Q75 in the primary neuronal culture of cerebellum with the aid of recombinant adenoviruses. Subsequently, this in vitro cellular model of SCA7 was used to study the molecular mechanism by which mutant ataxin-7-Q75 induces neuronal death. TUNEL staining studies indicated that polyglutamine-expanded ataxin-7-Q75 caused apoptotic cell death of cultured cerebellar neurons. Mutant ataxin-7-Q75 induced the formation of active caspase-3 and caspase-9 without activating caspase-8. Polyglutamine-expanded ataxin-7-Q75 promoted the release of apoptogenic cytochrome-c and Smac from mitochondria, which was preceded by the downregulation of Bcl-x(L) protein and upregulation of Bax protein expression in cultured cerebellar neurons. Further real-time TaqMan RT-PCR assays showed that mutant ataxin-7-Q75 upregulated Bax mRNA level and downregulated Bcl-x(L) mRNA expression in the primary neuronal culture of cerebellum. The present study provides the evidence that polyglutamine-expanded ataxin-7-Q75 activates mitochondria-mediated apoptotic cascade and induces neuronal death by upregulating Bax expression and downregulating Bcl-x(L) expression of cerebellar neurons.