The Alpha-Synuclein Gene (SNCA) is a Genomic Target of Methyl-CpG Binding Protein 2 (MeCP2)—Implications for Parkinson’s Disease and Rett Syndrome

Mounting evidence suggests a prominent role for alpha-synuclein (a-syn) in neuronal cell function. Alterations in the levels of cellular a-syn have been hypothesized to play a critical role in the development of Parkinson’s disease (PD); however, mechanisms that control expression of the gene for a-syn (SNCA) in cis and trans as well as turnover of a-syn are not well understood. We analyzed whether methyl-CpG binding protein 2 (MeCP2), a protein that specifically binds methylated DNA, thus regulating transcription, binds at predicted binding sites in intron 1 of the SNCA gene and regulates a-syn protein expression. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) were used to confirm binding of MeCP2 to regulatory regions of SNCA. Site-specific methylation and introduction of localized mutations by CRISPR/Cas9 were used to investigate the binding properties of MeCP2 in human SK-N-SH neuroblastoma cells. The significance of MeCP2 for SNCA regulation was further investigated by overexpressing MeCP2 and mutated variants of MeCP2 in MeCP2 knockout cells. We found that methylation-dependent binding of MeCP2 at a restricted region of intron 1 of SNCA had a significant impact on the production of a-syn. A single nucleotide substitution near to CpG1 strongly increased the binding of MeCP2 to intron 1 of SNCA and decreased a-syn protein expression by 60%. In contrast, deletion of a single nucleotide closed to CpG2 led to reduced binding of MeCP2 and significantly increased a-syn levels. In accordance, knockout of MeCP2 in SK-N-SH cells resulted in a significant increase in a-syn production, demonstrating that SNCA is a genomic target for MeCP2 regulation. In addition, the expression of two mutated MeCP2 variants found in Rett syndrome (RTT) showed a loss of their ability to reduce a-syn expression. This study demonstrates that methylation of CpGs and binding of MeCP2 to intron 1 of the SNCA gene plays an important role in the control of a-syn expression. In addition, the changes in SNCA regulation found by expression of MeCP2 variants carrying mutations found in RTT patients may be of importance for the elucidation of a new molecular pathway in RTT, a rare neurological disorder caused by mutations in MECP2

ATXN3 controls DNA replication and transcription by regulating chromatin structure

The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease.</p

ATXN3 controls DNA replication and transcription by regulating chromatin structure

The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease.Spanish Agencia Estatal de Investigacion´ [PID2019- 109222RB-I00/AEI/10.13039/501100011033]; European Union Regional Funds (FEDER) (to R.F., V.A.J.S.); Agencia Canaria de Investigacion, ´ Innovacion´ y Sociedad de la Informacion´ [ProID2020010109]; FEDER (to R.F.); Agencia Canaria de Investigacion, ´ Innovacion´ y European Social Fund integrated Operational programme of the Canary Islands Sociedad de la Informacion´ de la Consejer´ıa de Econom´ıa, Industria, Comercio y Conocimiento and the 2014–2020, Eje 3 Tema Prioritario 74 (85%) (to E.H.C.); Medical Research Council Programme [MR/X006409/1 to K.R.]; Breast Cancer Now [2019DecPR1406 to K.R.]. Funding for open access charge: Agencia Canaria de Investigacion, ´ Innovacion´ y Sociedad de la Informacion´ [ProID2020010109]; European Union Regional Funds (FEDER)

Pathological consequences of VCP mutations on human striated muscle

*These authors have contributed equally to this work. Mutations in the valosin-containing protein (VCP, p97) gene on chromosome 9p13-p12 cause a late-onset form of autosomal dominant inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD). We report on the pathological consequences of three heterozygous VCP (R93C, R155H, R155C) mutations on human striated muscle. IBMPFD skeletal muscle pathology is characterized by degenerative changes and filamentous VCP-and ubiquitin-positive cytoplasmic and nuclear protein aggregates. Furthermore, this is the first report demonstrating that mutant VCP leads to a novel form of dilatative cardiomyopathy with inclusion bodies. In contrast to post-mitotic striated muscle cells and neurons of IBMPFD patients, evidence of protein aggregate pathology was not detected in primary IBMPFD myoblasts or in transient and stable transfected cells using wild-type-VCP and R93C-, R155H-, R155C-VCP mutants. Glutathione S-transferase pull-down experiments showed that all three VCP mutations do not affect the binding to Ufd1, Npl4 and ataxin-3. Structural analysis demonstrated that R93 and R155 are both surface-accessible residues located in the centre of cavities that may enable ligand-binding. Mutations at R93 and R155 are predicted to induce changes in the tertiary structure of the VCP protein. The search for putative ligands to the R93 and R155 cavities resulted in the identification of cyclic sugar compounds with high binding scores. The latter findings provide a novel link to VCP carbohydrate interactions in the complex pathology of IBMPFD. Keywords: VCP; p97; myopathy; cardiomyopathy; IBMPFD Abbreviations: GST = glutathione S-transferase; IBMPFD = inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia; PBS = phosphate-buffered saline; SDS = sodium dodecyl sulphate; VCP = valosin-containing protei

Spinocerebellar Ataxia Type 3 Pathophysiology—Implications for Translational Research and Clinical Studies

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado–Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed

Structural modeling of ataxin-3 reveals distant homology to adaptins

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3

High Level Expression of Expanded Full-length Ataxin-3 In Vitro Causes Cell Death and Formation of Intranuclear Inclusions in Neuronal Cells

Spinocerebellar ataxia type 3 (SCA3) is caused by a CAG/polyglutamine repeat expansion in the SCA3 gene. To analyse the pathogenic mechanisms in SCA3, we have generated ataxin-3-expressing rat mesencephalic CSM14.1 cells. In these cells, a post-mitotic neuronal phenotype is induced by temperature shift. The isolated stable cell lines provided high level expression of non-expanded (Q23) or expanded (Q70) human full-length ataxin-3. CSM14.1 cells expressing the expanded full-length ataxin-3 developed nuclear inclusion bodies, strong indentations of the nuclear envelope and cytoplasmic vacuolation. These ultrastructural alterations were present prior to a significantly decreased viability of neuronally differentiated cells expressing expanded ataxin-3. The observed spontaneous cell death did not correlate with formation of intranuclear inclusions and was not apoptotic by ultrastructural analysis. No increased susceptibility to staurosporine-induced apoptosis was found for the expanded or non-expanded ataxin-3-expressing cell lines. These data show that high level expression of expanded full-length ataxin-3 in a neuron-like cell line generates ultrastructural alterations of SCA3 pathogenesis and results in increased spontaneous non-apoptotic cell deat

Reciprocal Regulation between Bifunctional miR-9/9∗ and its Transcriptional Modulator Notch in Human Neural Stem Cell Self-Renewal and Differentiation

Tight regulation of the balance between self-renewal and differentiation of neural stem cells is crucial to assure proper neural development. In this context, Notch signaling is a well-known promoter of stemness. In contrast, the bifunctional brain-enriched microRNA miR-9/9∗ has been implicated in promoting neuronal differentiation. Therefore, we set out to explore the role of both regulators in human neural stem cells. We found that miR-9/9∗ decreases Notch activity by targeting NOTCH2 and HES1, resulting in an enhanced differentiation. Vice versa, expression levels of miR-9/9∗ depend on the activation status of Notch signaling. While Notch inhibits differentiation of neural stem cells, it also induces miR-9/9∗ via recruitment of the Notch intracellular domain (NICD)/RBPj transcriptional complex to the miR-9/9∗_2 genomic locus. Thus, our data reveal a mutual interaction between bifunctional miR-9/9∗ and the Notch signaling cascade, calibrating the delicate balance between self-renewal and differentiation of human neural stem cells

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 line's 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