SUMOylation by SUMO2 is implicated in the degradation of misfolded ataxin-7 via RNF4 in SCA7 models.

Perturbation of protein homeostasis and aggregation of misfolded proteins is a major cause of many human diseases. A hallmark of the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7) is the intranuclear accumulation of mutant, misfolded ataxin-7 (polyQ-ATXN7). Here, we show that endogenous ATXN7 is modified by SUMO proteins, thus also suggesting a physiological role for this modification under conditions of proteotoxic stress caused by the accumulation of polyQ-ATXN7. Co-immunoprecipitation experiments, immunofluorescence microscopy and proximity ligation assays confirmed the colocalization and interaction of polyQ-ATXN7 with SUMO2 in cells. Moreover, upon inhibition of the proteasome, both endogenous SUMO2/3 and the RNF4 ubiquitin ligase surround large polyQ-ATXN7 intranuclear inclusions. Overexpression of RNF4 and/or SUMO2 significantly decreased levels of polyQ-ATXN7 and, upon proteasomal inhibition, led to a marked increase in the polyubiquitination of polyQ-ATXN7. This provides a mechanism for the clearance of polyQ-ATXN7 from affected cells that involves the recruitment of RNF4 by SUMO2/3-modified polyQ-ATXN7, thus leading to its ubiquitination and proteasomal degradation. In a SCA7 knock-in mouse model, we similarly observed colocalization of SUMO2/3 with polyQ-ATXN7 inclusions in the cerebellum and retina. Furthermore, we detected accumulation of SUMO2/3 high-molecular-mass species in the cerebellum of SCA7 knock-in mice, compared with their wild-type littermates, and changes in SUMO-related transcripts. Immunohistochemical analysis showed the accumulation of SUMO proteins and RNF4 in the cerebellum of SCA7 patients. Taken together, our results show that the SUMO pathway contributes to the clearance of aggregated ATXN7 and suggest that its deregulation might be associated with SCA7 disease progression.

Deregulation of autophagy in postmortem brains of Machado-Joseph disease patients.

Autophagy, the major pathway for protein turnover, is critical to maintain cellular homeostasis and has been implicated in neurodegenerative diseases. The aim of this research was to analyze the expression of autophagy markers in postmortem brains from Machado-Joseph disease (MJD) patients. The expression of autophagy markers in the cerebellum and the oculomotor nucleus from MJD patients and age-matched controls with no signs of neuropathology was inspected postmortem by immunohistochemistry (IHC) and Western blot. Furthermore, autophagy was examined by means of transmission electron microscopy (TEM). Western blot and IHC revealed nuclear accumulation of misfolded ataxin-3 (ATXN3) and the presence of ubiquitin- and p62-positive aggregates in MJD patients as compared to controls. Moreover, the autophagic proteins, autophagy-related gene (Atg) protein (ATG)-7, ATG-12, ATG16L2 and autophagosomal microtubule-associated protein light chain 3 (LC3) were significantly increased in MJD brains relative to controls, while beclin-1 levels were reduced in MJD patients. Increase in the levels of lysosomal-associated membrane protein 2 (LAMP-2) and of the endosomal markers (Rab7 and Rab1A) were observed in MJD patients relatively to controls. In addition, these findings were further confirmed by TEM in brain tissue where large vesicles accumulating electron-dense materials were highly enriched in MJD patients. Postmortem brains with MJD exhibit increased markers of autophagy relative to age-matched control brains, therefore suggesting strong dysregulation of autophagy that may have an important role in the course of MJD pathogenesis.

Control of Huntington's Disease-Associated Phenotypes by the Striatum-Enriched Transcription Factor Foxp2.

Alteration of corticostriatal glutamatergic function is an early pathophysiological change associated with Huntington's disease (HD). The factors that regulate the maintenance of corticostriatal glutamatergic synapses post-developmentally are not well understood. Recently, the striatum-enriched transcription factor Foxp2 was implicated in the development of these synapses. Here, we show that, in mice, overexpression of Foxp2 in the adult striatum of two models of HD leads to rescue of HD-associated behaviors, while knockdown of Foxp2 in wild-type mice leads to development of HD-associated behaviors. We note that Foxp2 encodes the longest polyglutamine repeat protein in the human reference genome, and we show that it can be sequestered into aggregates with polyglutamine-expanded mutant Huntingtin protein (mHTT). Foxp2 overexpression in HD model mice leads to altered expression of several genes associated with synaptic function, genes that present additional targets for normalization of corticostriatal dysfunction in HD.

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.

Cross-talking noncoding RNAs contribute to cell-specific neurodegeneration in SCA7.

What causes the tissue-specific pathology of diseases resulting from mutations in housekeeping genes? Specifically, in spinocerebellar ataxia type 7 (SCA7), a neurodegenerative disorder caused by a CAG-repeat expansion in ATXN7 (which encodes an essential component of the mammalian transcription coactivation complex, STAGA), the factors underlying the characteristic progressive cerebellar and retinal degeneration in patients were unknown. We found that STAGA is required for the transcription initiation of miR-124, which in turn mediates the post-transcriptional cross-talk between lnc-SCA7, a conserved long noncoding RNA, and ATXN7 mRNA. In SCA7, mutations in ATXN7 disrupt these regulatory interactions and result in a neuron-specific increase in ATXN7 expression. Strikingly, in mice this increase is most prominent in the SCA7 disease-relevant tissues, namely the retina and cerebellum. Our results illustrate how noncoding RNA-mediated feedback regulation of a ubiquitously expressed housekeeping gene may contribute to specific neurodegeneration.

The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice.

There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-D were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene ATG12 was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders.

Interferon ß induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.

SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7.

Post-translational modification by SUMO (small ubiquitin-like modifier) was proposed to modulate the pathogenesis of several neurodegenerative diseases. Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder, whose pathology is caused by an expansion of a polyglutamine stretch in the protein ataxin-7 (ATXN7). Here, we identified ATXN7 as new target for SUMOylation in vitro and in vivo. The major SUMO acceptor site was mapped to lysine 257, which is part of an evolutionarily conserved consensus SUMOylation motif. SUMOylation did not influence the subcellular localization of ATXN7 nor its interaction with components of the TFTC/STAGA complex. Expansion of the polyglutamine stretch did not impair the SUMOylation of ATXN7. Furthermore, SUMO1 and SUMO2 colocalized with ATXN7 in a subset of neuronal intranuclear inclusions in the brain of SCA7 patients and SCA7 knock-in mice. In a COS-7 cellular model of SCA7, in addition to diffuse nucleoplasmic staining we identified two populations of nuclear inclusions: homogenous or non-homogenous. Non-homogenous inclusions showed significantly reduced colocalization with SUMO1 and SUMO2, but were highly enriched in Hsp70, 19S proteasome and ubiquitin. Interestingly, they were characterized by increased staining with the apoptotic marker caspase-3 and by disruption of PML nuclear bodies. Importantly, preventing the SUMOylation of expanded ATXN7 by mutating the SUMO site increased both the amount of SDS-insoluble aggregates and of caspase-3 positive non-homogenous inclusions, which act toxic to the cells. Our results demonstrate an influence of SUMOylation on the multistep aggregation process of ATXN7 and implicate a role for ATXN7 SUMOylation in SCA7 pathogenesis.

PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins.

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, beta-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders.

Polyglutamine and polyalanine expansions in ataxin7 result in different types of aggregation and levels of toxicity.

Spinocerebellar ataxia type 7 (SCA7) is caused by expansion of a (CAG)n repeat in the ataxin7 gene, resulting in an abnormally long polyglutamine polyQ tract in the translated protein that aggregates in the form of neuronal intranuclear inclusions. Polyalanine (polyA) stretches, implicated in several genetic disorders, also appear to aggregate. To investigate the role of the aggregates in the pathologies, we compared the effects of ataxin7 containing a polyA (ataxin7 - 90A) or polyQ (ataxin7 - 100Q) expansion in HEK 293 cells and in primary cultures of rat mesencephalon. Both proteins formed nuclear and perinuclear aggregates that contained molecular chaperones and components of the ubiquitin-proteasome system, suggesting that they were abnormally folded. Ataxin-90A aggregates differed morphologically from ataxin7 - 100Q aggregates, consisted of small and amorphous rather than fibrillar inclusions and were more toxic to mesencephalic neurons, suggesting that toxicity was determined by the type of aggregate rather than the cellular misfolding response.