TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways

Nuclear factor TDP-43 is known to play an important role in several neurodegenerative pathologies. In general, TDP-43 is an abundant protein within the eukaryotic nucleus that binds to many coding and non-coding RNAs and influence their processing. Using Drosophila, we have performed a functional screening to establish the ability of major hnRNP proteins to affect TDP-43 overexpression/depletion phenotypes. Interestingly, we observed that lowering hnRNP and TDP-43 expression has a generally harmful effect on flies locomotor abilities. In parallel, our study has also identified a distinct set of hnRNPs that is capable of powerfully rescuing TDP-43 toxicity in the fly eye (Hrb27c, CG42458, Glo and Syp). Most importantly, removing the human orthologs of Hrb27c (DAZAP1) in human neuronal cell lines can correct several pre-mRNA splicing events altered by TDP-43 depletion. Moreover, using RNA sequencing analysis we show that DAZAP1 and TDP-43 can co-regulate an extensive number of biological processes and molecular functions potentially important for the neuron/motor neuron pathophysiology. Our results suggest that changes in hnRNP expression levels can significantly modulate TDP-43 functions and affect pathological outcomes.

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“…Thus, the analyzed events were the splicing of SKAR/POLDIP3 exon 3 (48); TNIK exon 15 (14); STAG2 exon 30b (49); and MADD exon 31 (49). …”

Section: Resultsmentioning

confidence: 99%

“…To this aim, we examined the levels of MADD and BRD8 expression levels as these were previously reported to be reduced following the TDP-43 RNAi (49). TNIK was also added as a control, since its splicing levels were affected by most of these hnRNPs.…”

Section: Resultsmentioning

confidence: 99%

Major hnRNP proteins act as general TDP-43 functional modifiers both in Drosophila and human neuronal cells

Appocher

Mohagheghi

Cappelli

et al. 2017

Nucleic Acids Research

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“…3c). We also evaluated splicing in TDP-43 target genes POLDIP3/SKAR, STAG2, and FNIP1 [11, 14, 55], but found no changes in their respective splicing profiles (Supplemental Fig. 3c).…”

Section: Resultsmentioning

confidence: 99%

The phosphatase calcineurin regulates pathological TDP-43 phosphorylation

et al. 2016

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Detergent insoluble inclusions of TDP-43 protein are hallmarks of the neuropathology in over 90% of amyotrophic lateral sclerosis (ALS) cases and approximately half of frontotemporal dementia (FTLD-TDP) cases. In TDP-43 proteinopathy disorders, lesions containing aggregated TDP-43 protein are extensively post-translationally modified, with phosphorylated TDP-43 (pTDP) being the most consistent and robust marker of pathological TDP-43 deposition. Abnormally phosphorylated TDP-43 has been hypothesized to mediate TDP-43 toxicity in many neurodegenerative disease models. To date several different kinases have been implicated in the genesis of pTDP, but no phosphatases have been shown to reverse pathological TDP-43 phosphorylation. We have identified the phosphatase calcineurin as an enzyme binding to and catalyzing the removal of pathological C-terminal phosphorylation of TDP-43 in vitro. In C. elegans models of TDP-43 proteinopathy, genetic elimination of calcineurin results in accumulation of excess pTDP, exacerbated motor dysfunction, and accelerated neurodegenerative changes. In cultured human cells, treatment with FK506 (tacrolimus), a calcineurin inhibitor, results in accumulation of pTDP species. Lastly, calcineurin co-localizes with pTDP in degenerating areas of the central nervous system in subjects with FTLD-TDP and ALS. Taken together these findings suggest calcineurin acts on pTDP as a phosphatase in neurons. Furthermore, patient treatment with calcineurin inhibitors may have unappreciated adverse neuropathological consequences.

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“…To test this hypothesis, we examined the exon 31 skipping in MADD gene. MADD gene is one of the previously identified, direct targets of TDP-43, and TDP-43 knockdown in HEK293 cells has been reported to increase the level of exon-skipped MADD gene (28). We transfected EGFP, wt, C71G or M114T mutant PFN1 into HEK293 cells and observed that C71G or M114T mutant PFN1 significantly increased the levels of the exon 31-skipped MADD gene, whereas wt PFN1 did not change the splicing profiles of the MADD gene (Fig.…”

Section: Overexpression Of Fals Mutant Pfn1 Increased the Cytoplasmicmentioning

confidence: 87%

“…6, A and B, 1.55 Ϯ 0.22% in EGFP, 0.96 Ϯ 0.23% in wt PFN1 (p ϭ 0.83), 6.39 Ϯ 0.81% in C71G PFN1 (p ϭ 0.0001), 3.92 Ϯ 0.47% in M114T PFN1 (p ϭ 0.0086)). We also examined exon 7 skipping in the FNIP1 gene, exon 20 skipping in the BRD8 gene, or exon 3 skipping in the SKAR gene (28,29), although no changes in the splicing profiles of these genes were observed among HEK293 cells transfected with EGFP, wt, C71G, or M114T mutant PFN1 (data not shown). Human HDAC6 mRNA has been reported to be down-regulated in TDP-43-silenced HEK293 cells (30).…”

Section: Overexpression Of Fals Mutant Pfn1 Increased the Cytoplasmicmentioning

confidence: 99%

Familial Amyotrophic Lateral Sclerosis-linked Mutations in Profilin 1 Exacerbate TDP-43-induced Degeneration in the Retina of Drosophila melanogaster through an Increase in the Cytoplasmic Localization of TDP-43

Matsukawa

Hashimoto

Matsumoto

et al. 2016

Journal of Biological Chemistry

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons. Causative genes for familial ALS (fALS), e.g. TARDBP or FUS/TLS, have been found, among which mutations within the profilin 1 (PFN1) gene have recently been identified in ALS18. To elucidate the mechanism whereby PFN1 mutations lead to neuronal death, we generated transgenic Drosophila melanogaster overexpressing human PFN1 in the retinal photoreceptor neurons. Overexpression of wild-type or fALS mutant PFN1 caused no degenerative phenotypes in the retina. Double overexpression of fALS mutant PFN1 and human TDP-43 markedly exacerbated the TDP-43-induced retinal degeneration, i.e. vacuolation and thinning of the retina, whereas co-expression of wild-type PFN1 did not aggravate the degenerative phenotype. Notably, co-expression of TDP-43 with fALS mutant PFN1 increased the cytoplasmic localization of TDP-43, the latter remaining in nuclei upon co-expression with wild-type PFN1, whereas co-expression of TDP-43 lacking the nuclear localization signal with the fALS mutant PFN1 did not aggravate the retinal degeneration. Knockdown of endogenous Drosophila PFN1 did not alter the degenerative phenotypes of the retina in flies overexpressing wild-type TDP-43. These data suggest that ALS-linked PFN1 mutations exacerbate TDP-43-induced neurodegeneration in a gain-of-function manner, possibly by shifting the localization of TDP-43 from nuclei to cytoplasm. Amyotrophic lateral sclerosis (ALS)2 is a progressive neurodegenerative disorder characterized by selective loss of upper and lower motor neurons. Approximately 10% of ALS cases are inherited as an autosomal dominant trait (familial ALS; fALS), and mutations in a number of causative genes, e.g. Cu/Zn superoxide dismutase (1) and TARDBP (2-5), have been identified in fALS cases. Recently mutations within the PFN1 gene were identified in pedigrees of ALS 18 (OMIM614808) (6, 7). Moreover, the E117G variant in PFN1 is also considered a genetic risk factor for ALS (8, 9). However, the mechanisms whereby PFN1 mutations induce degeneration and death of motor neurons remain elusive. PFN1 is a protein implicated in the regulation of actin assembly by binding to monomeric actin (10). Homozygous PFN1 knock-out mice die shortly after fertilization (11), and brain-specific conditional knock-out of the PFN1 gene during development led to cerebellar hypoplasia and abnormal neuronal migration (12), indicating the roles of PFN1 in development. However, there have been few reports on the pathological functions of fALS mutant PFN1; the level of PFN1-bound actin was shown to be reduced upon overexpression of the fALS mutant PFN1 compared with wild-type (wt) PFN1 in HEK293 cells (6). A recent report (13) showed that the fALS mutant PFN1 failed to restore growth of PFN1 mutant yeast. These data suggested a partial loss-of-function mechanism in neurodegeneration caused by fALS mutations in PFN1.TAR DNA-binding protein of 43 kDa (TDP-43) is a member of hetero...

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TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways

Cited by 65 publications

References 72 publications

Major hnRNP proteins act as general TDP-43 functional modifiers both in Drosophila and human neuronal cells

Major hnRNP proteins act as general TDP-43 functional modifiers both in Drosophila and human neuronal cells

The phosphatase calcineurin regulates pathological TDP-43 phosphorylation

Familial Amyotrophic Lateral Sclerosis-linked Mutations in Profilin 1 Exacerbate TDP-43-induced Degeneration in the Retina of Drosophila melanogaster through an Increase in the Cytoplasmic Localization of TDP-43

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