Project description:Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear TDP-43. Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-mRNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from sporadic ALS patients and familial ALS patients with expansion in C9orf72, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS/FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability.
Project description:QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs
Project description:We generated total mRNA libraries and ribosome footprint libraries from motor neuron-like cells (MN1) and primary cortical neurons expressing GFPor pure wild type cells as control, human TDP-43 wild type and mutant proteins to identify translational targets of human TDP-43 mutant protein
Project description:To clarify the functional properties of Tdp-43, we established the differentially expressed alternative exons in Tdp-43-silenced primary cortical neurons by using exon-sensitive microarray technology.
Project description:TDP-43 proteinopathies including frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders characterized by aggregation and mislocalization of the nucleic-acid binding protein TDP-43 and subsequent neuronal dysfunction. Here, we developed an endogenous model of sporadic TDP-43 proteinopathy based on the principle that disease-associated TDP-43 acetylation at lysine 145 (K145) alters TDP-43 conformation, impairs its RNA-binding capacity, and induces downstream mis-regulation of target genes. Expression of aberrant acetylation-mimic TDP-43K145Q resulted in stress-induced phase-separated nuclear TDP-43 foci formation and loss-of-TDP-43-function in mouse primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons. Aged mice harboring the single TDP-43K145Q mutation recapitulate several key hallmarks of neurodegenerative proteinopathies, including progressive TDP-43 phosphorylation and insolubility, cytoplasmic mis-localization, widespread transcriptomic and splicing alterations, and cognitive dysfunction. Our study supports a model in which aberrant TDP-43 acetylation drives neuronal dysfunction and cognitive decline through alternative splicing and transcription of genes important in synaptic plasticity and apoptosis, providing a new paradigm to interrogate FTLD disease mechanisms and uncover disease-modifying therapeutics.
Project description:To clarify the functional properties of Tdp-43, we established the differentially expressed alternative exons in Tdp-43-silenced primary cortical neurons by using exon-sensitive microarray technology. We analyzed total RNA of primary motor neuron infected with lentivirus expressing shRNA against mouse Tdp-43 or control. RNA was harvested 11 days after transfection.
Project description:TDP-43 aggregation and redistribution have been recognised as a hallmark of amyotrophic lateral sclerosis, frontotemporal dementia and other neurological disorders. While TDP-43 has been studied extensively in neuronal tissues, TDP-43 inclusions have also been described in the muscle of inclusion body myositis patients, highlighting the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq we performed the first direct comparison of TDP-43-mediated transcription and alternative splicing in muscle (C2C12) and neuronal (NSC34) mouse cells. Our results clearly show that TDP-43 displays a tissue-characteristic behaviour targeting unique transcripts in each cell type. This is not due to variable transcript abundance but rather due to cell-specific expression of RNA-binding proteins, which influences TDP-43 performance. Among splicing events commonly dysregulated in both cell lines, we identified some that are TDP-43-dependent also in human cells and show that inclusion levels of these alternative sequences appear to be altered in affected tissues of FTLD and IBM patients. We therefore propose that TDP-43 dysfunction, reflected in aberrant splicing, contributes to disease development but it does so in a tissue-specific manner.
Project description:MicroRNAs (miRNAs) play important roles in a wide range of cellular processes. Aberrant regulation of miRNA genes contributes to human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a DNA/RNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs whose expression levels are regulated by TDP-43 using RNA-Seq coupled with siRNA-mediated knockdown approach. TDP-43 knocking down affected the expression of a number of miRNAs. Alterations in isomiR patterns and miRNA arm selection after TDP-43 knockdown suggest a role of TDP-43 in miRNA editing. We examined correlation of selected TDP-43 associated miRNAs and their candidate target genes in human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p expression. On the other hand, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Low expression of miR-500a-3p was associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Our experiments reveal that cancer-associated genes LIF and PAPPA may be targets of miR-500a-3p. Together with other studies, our work suggests that TDP-43-regulated miRNAs may play multi-facet roles in the pathogenesis of cancer.