Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE79020 | GEO | 2016/10/28
SECONDARY ACCESSION(S): PRJNA314719
REPOSITORIES: GEO
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Project description:Spinocerebellar ataxia type 2 (SCA2) is among the progressive neurodegenerative polyglutamine (polyQ) diseases. It is caused by a CAG repeat expansion in an encoded region of the ATXN2 gene giving rise to an expanded polyQ domain in the encoded ATXN2 protein. SCA2 is an autosomal dominant disorder characterized by symptoms resulting from neurodegeneration of cerebellar Purkinje cells. To molecularly characterize SCA2 disease progression, we analyzed RNA-sequencing data produced using the cerebella of ATXN2Q127 mice collected at three distinct time points. The ATXN2Q127 mouse model contains 127 CAG repeats in the full-length ATXN2 cDNA under the control of the PC targeted Pcp2 promoter.
2017-12-09 | E-MTAB-6293 | biostudies-arrayexpress
Project description:Spinocerebellar ataxia type 1 (SCA1) is a polyglutamine (polyQ) repeat neurodegenerative disease in which a primary site of pathogenesis are cerebellar Purkinje cells. In addition to polyQ expansion of ataxin-1 protein (ATXN1), phosphorylation of ATXN1 at the serine 776 residue (ATXN1-pS776) plays a significant role in protein toxicity. Utilizing a biochemical approach, pharmacological agents and cell-based assays, including SCA1 patient iPSC-derived neurons, we examine the role of Protein Kinase A (PKA) as an effector of ATXN1-S776 phosphorylation. We further examine the implications of PKA-mediated phosphorylation at ATXN1-S776 on SCA1 through genetic manipulation of the PKA catalytic subunit Cα in Pcp2-ATXN1[82Q] mice. Here we show that pharmacologic inhibition of S776 phosphorylation in transfected cells and SCA1 patient iPSC-derived neuronal cells lead to a decrease in ATXN1. In vivo, reduction of PKA-mediated ATXN1-pS776 results in enhanced degradation of ATXN1 and improved cerebellar-dependent motor performance. These results provide evidence that PKA is a biologically important kinase for ATXN1-pS776 in cerebellar Purkinje cells.
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Project description:The molecular mechanism(s) leading to Purkinje neuron loss in the neurodegenerative disorder Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) are limited by the complex morphology of this cell type. Purkinje neurons are notoriously difficult to isolate and maintain in culture presenting considerable difficultly to identify molecular changes in response to riboCGG repeat-containing mRNA that induces neurotoxicity in FXTAS. Several studies have uncovered a number of RNA binding proteins involved in translation that aberrantly interact with the toxic RNA; however, whether these interactions alter the translational profile of cells has not been investigated. Here we employ bacTRAP translational profiling to demonstrate that Purkinje neurons ectopically expressing 90 CGG repeats exhibit a dramatic change in their translational profile even prior to the onset of riboCGG-induced phenotypes. This approach identified nearly 500 transcripts that are differentially associated with ribosomes in r(CGG)90-expressing mice. Functional annotation cluster analysis revealed broad ontologies enriched in the r(CGG)90 list, including RNA binding and response to stress. Intriguingly, a transcript for the Tardbp gene, implicated in a number of other neurodegenerative disorders, exhibits altered association with ribosomes in the presence of r(CGG)90 repeats. We therefore tested and showed that reduced association of Tardbp mRNA with the ribosomes results in a loss of TDP-43 protein expression in r(CGG)90–expressing Purkinje neurons. Furthermore, we showed that TDP-43 could modulate the rCGG repeat-mediated toxicity in a Drosophila model that we developed previously. These findings together suggest translational dysregulation may be an underlying mechanism of riboCGG-induced neurotoxicity and provide insight into the pathogenicity of FXTASBAC-trap studies of Purkinje cels in normal and mutant mice Looking for cell type specific gene expression differences caused by mutation in FMR1 using BAC-Trap RNA capture assay. A cutoff value of 4.5 for mean probe_set mean across all 18 hybridizations was used to determine a cutoff for expression in Purkinje cells. This 16040 corresponded to a mean value of at least 3.4323 in the 4week time point. L7CGG90Fmr1:Pcp2 BAC (r(CGG)90 BAC) and wild-type:L7/Pcp2 BAC (WT BAC) transgenic mice used in this study have a 50% C57B6 and 50% FVB genetic background and were derived from the same parents by crossing L7CGG90Fmr1 heterozygote transgenics (100% C57B6 background) with L7/Pcp2eEGFPL10a BAC homozygous transgenics (100% FVB background). All mice reported were F1 progeny and were therefore hemizygous for one or both transgenes. Littermate wild-type mice were used for all experiments whenever possible.
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Project description:The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis we have identified Collapsin Response Mediator Protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)- mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wildtype CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wildtype CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.
2022-02-24 | PXD020124 | Pride
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