Project description:Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several pathogenic mutations have been identified, the vast majority of ALS cases have no family history of disease. Thus, for most ALS cases, the disease may be a product of multiple pathways contributing to varying degrees in each patient. Using machine learning algorithms, we stratified the transcriptomes of 148 ALS postmortem cortex samples into three distinct molecular subtypes. The largest cluster, identified in 61% of patient samples, displayed hallmarks of oxidative and proteotoxic stress. Another 19% of the samples showed predominant signatures of glial activation. Finally, a third group (20%) exhibited high levels of retrotransposon expression and signatures of TARDBP/TDP-43 dysfunction. We further demonstrated that TDP-43 (1) directly binds a subset of retrotransposon transcripts and contributes to their silencing in vitro, and (2) pathological TDP-43 aggregation correlates with retrotransposon de-silencing in vivo.
Project description:We report the enrichment of mRNAs with motor neuron specific TDP-43 and tagged ribosomes in control as well as multiple models of TDP-43 induced neurodegeneration. Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. The goal of this study is to understand the translational consequences of TDP-43 pathology. Using the GAL4-UAS system, we expressed TDP-43WT and TDP-43G298S in the motor neurons of our drosophila to induce ALS-like neurodegeneration. TDP-43 was immunoprecipitated from the larvae at the 3rd instar stage; TDP-43 associated mRNAs, the whole larvae input, and ventral nerve cords of the same genotype were then sequenced. To identify translational changes we conducted TRAP (translating ribosome affinity purifications) in 3rd instar larvae by immunoprecipitating the motor neuron specific tagged ribosomal subunit RpL10-GFP in both ALS models as well as a RpL10-GFP control. The RpL10-GFP associated mRNAs, the whole larval input, and ventral nerve cords of the same genotype. From this data, we identified both compensatory alterations to translation induced by TDP-43 pathology and direct targets of TDP-43 mediated translational inhibition.
Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from fibroblasts grown from neurologically healthy controls (n=6) and 3 groups of patients with ALS: 1) sporadic cases (n=6); 2) cases due to mutations of SOD1 (n=4); 3) cases due to mutations of TARDBP (n=3). The three ALS groups were compared to the controls.
Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from lower motor neurons obtained by laser capture microdissection from autopsy material from neurologically healthy controls (n=6) and cases of sporadic ALS (n=3) and ALS due to C9ORF72 mutations (n=3).
Project description:Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in glia vs neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.
Project description:Mutations in the TDP-43 gene (also called TARDBP), which encodes transactive response DNA-binding protein 43 (TDP-43), have been found in 1–5% of sporadic and familial cases of the devastating adult-onset neurodegenerative disorder amyotrophic lateral sclerosis (ALS). TDP-43 is a predominantly nuclear RNA/DNA-binding protein that has diverse roles in RNA processing, but in diseased motor neurons it is depleted from the nucleus and sequestered into cytoplasmic protein aggregates that are a hallmark of the disease. How the loss of the RNA/DNA-binding activity of TDP-43 contributes to ALS pathogenesis is largely unknown. Using a large-scale RNAi screening approach, we recently identified TDP-43 as a gene required for maintaining genomic stability. We showed that induced pluripotent stem cells (iPSCs) and neurons derived from ALS patients harboring TDP-43 mutations are defective in two major pathways for repair of DNA double-strand breaks, non-homologous end joining and homologous recombination. Accordingly, TDP-43-mutant ALS iPSCs and neurons display increased DNA damage, which we also observed in brain tissues from ALS mice harboring a knock-in ALS-linked Tdp-43 mutation and from human ALS patients. To gain insight into the mechanistic basis by which ALS-associated TDP-43 mutants result in increased DNA damage, we performed transcriptome profiling (RNA-seq) in TDP-43-mutant ALS iPSCs, and found aberrant alternative pre-mRNA splicing of a number of genes involved in DNA repair. Our results suggest a role for TDP-43 in DNA repair and reveal a previously unknown mechanism that likely contributes to the pathogenesis of ALS harboring TDP-43 mutations.
Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from NSC34 motor neuronal cells depleted of TDP-43 by shRNA (n=4), treated with control shGFP (n=4), and treated with control shLuciferase (n=3).
Project description:Transactive Response DNA-binding protein 43 (TDP-43) is a multifunctional nuclear protein ubiquitously expressed in all tissues. Although it is primarily nuclear, Amyotrophic Lateral Sclerosis (ALS) patients frequently present with cytoplasmic aggregates of TDP-43 in motor neurons on post-mortem examination. Although only about 5% of all ALS patients have a TARDBP mutation (Ghasemi and Brown 2018; Ingre et al. 2015), which is known to cause these insoluble cytoplasmic aggregates, 97% of ALS patients will develop cytoplasmic, insoluble TDP-43 aggregates regardless of whether they have a mutation in TARDBP or not (Ling et al. 2013). Overexpression of wild-type TDP-43 has previously been shown to be a valid model of inducing neurotoxicity as well as a limited amount of cytoplasmic re-localization and aggregation characteristic of ALS (Elden et al. 2010; Wils et al. 2010). In the search for modifiers of toxicity as conferred by TDP-43 overexpression in these models, knockout of Ataxin-2 (ATXN2) was proposed as a protective intervention in the context of TDP-43 perturbation in non-human model systems (Elden et al. 2010, Becker et al. 2017). This promising finding had not previously been validated in a human motor neuron system, nor had a thorough investigation been conducted to determine the mechanism of neuroprotection that ATXN2 knockout employs to drive the therapeutic effect. This sequencing study examines both ATXN2 intact and ATXN2 knockout motor neurons in the context of TDP-43 perturbation to explore global transcriptomic changes that arise from these changes in cellular state that are associated with progression of ALS (ATXN2 intact) and what pathways could be implicated in protection from ALS-associated neurodegeneration (ATXN2 Knockout).
Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism.
Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism.