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[
MicroPubl Biol,
2023]
Cytoplasmic inclusions consisting of transactive response DNA-binding protein 43 (TDP-43) are a key hallmark of TDP-43 proteinopathies like amyotrophic lateral sclerosis (ALS). Caenorhabditis elegans is considered a useful model for studying the molecular mechanisms underlying TDP-43 toxicity in vivo . Here, we assessed different neuronal systems through established behavioral assays and extended the phenotypic characterisation of a C. elegans model expressing wildtype human TDP-43 ( hTDP-43 ) pan-neuronally. Our data show that neuronal expression of hTDP-43 in C. elegans disrupts chemotaxis and decreases fecundity. The basal slowing response, on the other hand, appears to be preserved in the presence of hTDP-43.
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[
International Worm Meeting,
2009]
Aging related neurodegenerative disorders are frequently characterized by lesions containing deposits of insoluble protein aggregates. Recently, the lesions seen in the degenerating neurons of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) were discovered to consist primarily of the TDP-43 protein. Likewise TDP-43 inclusions are also seen in many cases of other diseases leading to dementia including Alzheimer''s disease (AD), Parkinson''s disease (PD), dementia with Lewy bodies (DLB), and Guam amyotrophic lateral sclerosis/Parkinson''s dementia complex (ALS/PDC). Disorders with deposits of TDP-43 are now referred to as TDP-43 proteinopathy disorders. Of these, ALS is of particular interest because there is a causal link between TDP-43 and ALS. TDP-43 mutations cause some forms of familial ALS proving that abnormal TDP-43 can be a cause of neurodegeneration, although the mechanism by which TDP-43 abnormalities lead to neurodegeneration is poorly understood. To model TDP-43 neurotoxicity, we have transgenically expressed human TDP-43 protein in the neurons of C. elegans. Neuronal expression of normal human TDP-43 causes a relatively mild Unc phenotype, while expression of ALS causing mutant forms of TDP-43 leads to a robust Unc phenotype. Consistent with the mild Unc phenotype, we see little if any neurodegeneration caused by the wild type TDP-43 transgene. However, ALS mutant TDP-43 expressing transgenic animals exhibit dramatic degeneration of motor neurons. As in authentic human TDP-43 proteinopathy disorders, we observe the accumulation of insoluble TDP-43 protein in our model, both for wild type and mutant TDP-43 transgenes. This is in keeping with the observation that wild type TDP-43 forms inclusions in most sporadic ALS cases, while mutant TDP-43 is known to form aggregates in familial ALS cases. Detailed characterization of TDP-43 associated phenotypes and analysis of the genes and pathways contributing to TDP-43 mediated neurodegeneration are underway. The long term goal of this work is to develop neuroprotective strategies for neurodegenerative disorders with TPD-43 protein deposits.
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[
Ann Neurol,
2013]
OBJECTIVE: Kinase hyperactivity occurs in both neurodegenerative disease and cancer. Lesions containing hyperphosphorylated aggregated TDP-43 characterize amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions. Dual phosphorylation of TDP-43 at serines 409/410 (S409/410) drives neurotoxicity in disease models; therefore, TDP-43-specific kinases are candidate targets for intervention. METHODS: To find therapeutic targets for the prevention of TDP-43 phosphorylation, we assembled and screened a comprehensive RNA interference library targeting kinases in TDP-43 transgenic Caenorhabditis elegans. RESULTS: We show CDC7 robustly phosphorylates TDP-43 at pathological residues S409/410 in C. elegans, in vitro, and in human cell culture. In frontotemporal lobar degeneration (FTLD)-TDP cases, CDC7 immunostaining overlaps with the phospho-TDP-43 pathology found in frontal cortex. Furthermore, PHA767491, a small molecule inhibitor of CDC7, reduces TDP-43 phosphorylation and prevents TDP-43-dependent neurodegeneration in TDP-43-transgenic animals. INTERPRETATION: Taken together, these data support CDC7 as a novel therapeutic target for TDP-43 proteinopathies, including FTLD-TDP and amyotrophic lateral sclerosis.
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[
MicroPubl Biol,
2023]
Inclusions consisting of transactive response DNA-binding protein 43 (TDP-43) are a characteristic feature of amyotrophic lateral sclerosis (ALS). <i>Caenorhabditis elegans</i> has been instrumental in studying the underlying mechanisms of TDP-43 pathology. Here, we extend the possibilities of previous studies by examining a <i>C. elegans</i> model expressing human wild-type <i>TDP-43</i> ( <i>hTDP-43</i> ) pan-neuronally. We show that disease-related (hyper)phosphorylation and cytosolic localisation of hTDP-43 are present in hTDP-43 worms and that these features can be enhanced by adjusting the environmental temperature.
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Bird, Thomas, Guthrie, Chris, Kraemer, Brian, Leverenz, James, Liachko, Nicole, McMillan, Pamela
[
International Worm Meeting,
2013]
Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) are severe progressive neurodegenerative diseases characterized by lesions containing aggregated, hyperphosphorylated TDP-43. In addition, mutations in TDP-43 have been shown to cause some cases of ALS and FTLD-TDP. To study the cellular, molecular, and genetic underpinnings of TDP-43 mediated neurotoxicity in a tractable model system, we have developed a C. elegans model of TDP-43 proteinopathy. Expression of familial ALS-mutant TDP-43 in all C. elegans neurons causes severe motor dysfunction, and recapitulates some characteristic features of ALS and FTLD-TDP including decreased lifespan, neuronal degeneration, hyperphosphorylation and ubiquitination of TDP-43, and accumulation of detergent insoluble aggregates. We have shown that in C. elegans, phosphorylation of TDP-43 at serine residues 409/410 drives mutant TDP-43 toxicity. To identify kinases involved in the pathological phosphorylation of TDP-43, we have assembled and screened an RNA interference (RNAi) library targeting the majority of C. elegans kinases. 456 predicted kinases were individually inactivated by RNAi and tested for modification of TDP-43 dependent behavioral phenotypes. 12 kinase-inactivating RNAi treatments suppressed TDP-43 driven motor phenotypes. Null mutant analysis of these candidates in C. elegans identified 2 kinases whose loss of function suppresses TDP-43 movement defects and reduces TDP-43 phosphorylation in vivo. We have demonstrated that the human homolog of one candidate TDP-43 kinase,
cdc-7, directly phosphorylates TDP-43 in vitro and in mammalian cells. Further, we have demonstrated that treatment with a CDC-7 inhibitor decreases TDP-43 phosphorylation in vitro and prevents neurodegeration in vivo. The C. elegans model of TDP-43 proteinopathy serves as a powerful system to explore the biology of human disease and rapidly test interventions. TDP-43 specific kinases identified using this system may represent effective therapeutic targets for TDP-43 proteinopathies such as FTLD-TDP and ALS.
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[
International Worm Meeting,
2021]
Accumulation of hyperphosphorylated TDP-43 protein in neuronal aggregates is the major pathological feature of two devastating neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD-TDP). To study the cellular, molecular, and genetic underpinnings of TDP-43 mediated neurotoxicity in a tractable model system, we have developed a C. elegans model of TDP-43 proteinopathy expressing human disease-causing mutant TDP-43 pan-neuronally (TDP-43 tg). These transgenic animals display early, progressive uncoordinated movement (Unc), decreased lifespan, and age-dependent neurodegeneration. Using this model, we have found that phosphorylation of TDP-43 increases mutant TDP-43 toxicity. To identify phosphatases controlling TDP-43 phosphorylation, we have screened an RNA interference (RNAi) library targeting most known or predicted C. elegans phosphatases. 167 candidate genes were individually tested for modification of TDP-43 dependent behavioral phenotypes and for changes in the phosphorylation status of TDP-43 by immunoblot. From this primary screen we have identified 15 phosphatase genes that modify TDP-43 phenotypes. Additional work on these genes will provide mechanistic insight into the environmental and cellular triggers of TDP-43 phosphorylation, and provide potential novel avenues for therapeutic interventions into TDP-43 proteinopathies such as ALS and FTLD-TDP.
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[
J Neurosci,
2010]
Neurodegenerative disorders characterized by neuronal and glial lesions containing aggregated pathological TDP-43 protein in the cytoplasm, nucleus, or neurites are collectively referred to as TDP-43 proteinopathies. Lesions containing aggregated TDP-43 protein are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). In addition, mutations in human TDP-43 cause ALS. We have developed a Caenorhabditis elegans model of TDP-43 proteinopathies to study the cellular, molecular, and genetic underpinnings of TDP-43-mediated neurotoxicity. Expression of normal human TDP-43 in all C. elegans neurons causes moderate motor defects, whereas ALS-mutant G290A, A315T, or M337V TDP-43 transgenes cause severe motor dysfunction. The model recapitulates some characteristic features of ALS and FTLD-U including age-induced decline in motor function, decreased life span, and degeneration of motor neurons accompanied by hyperphosphorylation, truncation, and ubiquitination of TDP-43 protein that accumulates in detergent-insoluble protein deposits. In C. elegans, TDP-43 neurotoxicity is independent of activity of the cell death caspase CED-3. Furthermore, phosphorylation of TDP-43 at serine residues 409/410 drives mutant TDP-43 toxicity. This model provides a tractable system for additional dissection of the cellular and molecular mechanisms underlying TDP-43 neuropathology.
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[
J Biomed Sci,
2023]
BACKGROUND: Amyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43. METHODS: By applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43. RESULTS: Four PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system. CONCLUSIONS: Our study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.
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Petrucelli, Leonard, Roberts, Christine M., Link, Christopher D., Hutter, Harald, Ash, Peter E.A., Saldi, Tassa, Fonte, Virginia
[
International Worm Meeting,
2009]
TDP-43 is a conserved RNA binding protein with known roles in mRNA splicing and stability. Cytoplasmic deposition of TDP-43 has been linked to multiple neurodegenerative diseases, including ALS and frontotemporal lobar dementia (FTLD). We have engineered pan-neuronal expression of human TDP-43 protein in C. elegans, with the goal of generating a convenient in vivo model of TDP-43 neurotoxicity. Full-length (wild type) human TDP-43 expressed in C. elegans is nuclear as is observed in human cells. Transgenic worms with neuronal human TDP-43 expression exhibit an uncoordinated phenotype and have abnormal motorneuron synapses. By using this uncoordinated phenotype as a read-out of TDP-43 neurotoxicty, we have investigated the contribution of specific TDP-43 domains as well as TDP-43 sub-cellular localization to toxicity. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. In contrast, N-terminal deletions result in the formation of toxic cytoplasmic aggregates. Mutation of the TDP-43 nuclear localization signal (NLS) results in cytoplasmic deposition of full-length TDP-43, which is not toxic. Mutations that alter two TDP-43 caspase cleavage sites (D89/219E), however, do not reverse TDP-43 toxicity. Our results demonstrate that TDP-43 neurotoxicty can result from either nuclear activity of the full-length protein or accumulation of cytoplasmic aggregates composed of C-terminal fragments. These results suggest that there may be (at least) two different mechanisms of TDP-43 neurotoxicity.
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[
International Worm Meeting,
2015]
Accumulation of the protein TDP-43 in neuronal aggregates is the major pathological feature of two devastating neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD-TDP). TDP-43 is an essential protein and is involved in many aspects of RNA metabolism from transcription to translation, although its most critical contribution may be regulation of the majority of cellular mRNA splicing events. Disruption of the normal activity of TDP-43, either by mutation of TDP-43 or alterations in TDP-43 regulatory pathways, promotes neuronal dysfuction and degeneration in a variety of model systems including C. elegans, Drosophila, zebrafish, mammalian cell culture, and mice. To study the cellular, molecular, and genetic underpinnings of TDP-43 mediated neurotoxicity in a tractable model system, we have developed C. elegans models of TDP-43 proteinopathy expressing either wild type or disease-causing mutant TDP-43 pan-neuronally (TDP-43 tg). These transgenic animals display early, progressive motor dysfunction, decreased lifespan, and age-dependent degeneration of specific types of neurons, including GABA-ergic and dopaminergic neurons (1). However, not all TDP-43 expressing neurons undergo apparent neurodegeneration, indicating differences in sensitivities of specific populations of neurons to the presence of TDP-43. Surveying the range of responses to TDP-43 may provide a set of shared characteristics for populations of neurons susceptible or resistant to aberrant TDP-43. To investigate neuronal function in TDP-43 tg animals, we are utilizing a panel of behavioral, stress response, and stress survival assays. Results from these assays have identified individual sensory neurons with functional impairments in the absence of early neuronal cell body degeneration. These TDP-43 sensitive neurons will allow dissection of the effects of neurotoxic TDP-43 on neuronal function, and provide insight into the upstream processes leading to TDP-43 dependent neurodegeneration. 1. N. F. Liachko, C. R. Guthrie, B. C. Kraemer, Phosphorylation Promotes Neurotoxicity in a Caenorhabditis elegans Model of TDP-43 Proteinopathy. J Neurosci 30, 16208-16219 (2010).