2017 — 2018 |
Donnelly, Christopher James Pandey, Udai B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Molecular Mechanisms of C9orf72 and Matrin 3 Interaction in Als @ University of Pittsburgh At Pittsburgh
Abstract GGGGCC hexanucleotide repeat expansions (G4C2), in the non-coding region of the C9orf72 gene on chromosome 9p21, are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. G4C2 expansions in C9orf72 cause a loss of nuclear membrane integrity which in turn leads to mislocalization of nuclear TDP-43 (another ALS causing protein) in the spinal cords of C9 ALS patients. Recently, expanded hexanucleotide repeat binding proteins have been identified in neurons differentiated from induced pluripotent stem cells and motor cortex tissue from C9-ALS patients. Matrin-3 (MATR3) interacts with hexanucleotide repeats in two independent studies. Mutations in MATR3, an RNA binding nuclear matrix protein, are a known genetic cause of ALS. MATR3 interacts with nuclear matrix proteins such as Lamin A suggesting potential role of MATR3 in regulating nuclear matrix architecture and function. MATR3 positive cytoplasmic inclusions have been identified in an ALS patient known to carry the C9orf72 repeat expansion suggesting a potential link between these two forms of ALS. MATR3 binds to the G4C2 in C9orf72 and the binding motif recognized by MATR3 has been found to be enriched in sporadic ALS patients? alternative splicing cassette exons. However, the physiological consequences of the interaction between MATR3 and expanded G4C2 repeats, as well as the relevance of this interaction to ALS pathogenesis, are not yet known. We found that ectopic expression of wild type (WT) MATR3 strongly suppresses neurodegenerative phenotype in a Drosophila model of mutant C9ORF72 hexanucleotide repeat expansions (HNE). We observed that ablating the RNA binding domain in WT-MATR3 blocks its ability to suppress C9-HNE toxicity in vivo, suggesting that the rescue phenotype depends on HNE RNA transcripts binding to WT-MATR3. Here, we propose to investigate this novel interaction between WT-MATR3 and C9ORF72-HNE and its impact on C9ORF72-HNE neurodegenerative phenotype. In this R21 proposal, we will thoroughly examine the relationship between MATR3 and C9 using biochemical, genetic and molecular approaches in mammalian cell cultures, induced pluripotent stem cells from C9 patients and Drosophila models of C9orf72-mediated neurodegeneration.
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0.956 |
2017 — 2018 |
Donnelly, Christopher James Pandey, Udai B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Nuclear Export Inhibition as a Novel Therapeutic Target For C9orf72 Als/Ftd @ University of Pittsburgh At Pittsburgh
Project Abstract The C9ORF72 repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This G4C2 repeat expansion has been shown to generate toxic RNA molecules that sequester proteins which disrupts their function. These RNAs can also be translated into dipeptide repeats (DPRs) via a non-canonical pathway and accumulate. Both G4C2 RNAs and DPRs have been shown to be toxic when they accumulate in neurons and glia. We and others have recently shown that nucleocytoplasmic trafficking is disrupted in C9ORF72 fly models and induced pluripotent stem cell (iPSC) neurons due to the presence of these molecules. Importantly, genetically enhancing nuclear protein import or reducing nuclear export is neuroprotective in G4C2-expressing flies. In this R21 research proposal, we will employ various fly models of G4C2 RNA and DPR based toxicity and test multiple compounds to pharmacologically modulate nucleocytoplasmic trafficking using inhibitors of the nuclear exporter, exportin-1. Compounds that rescue neural degeneration and motor phenotypes in fly models of C9ORF72 ALS/FTD will then be tested for efficacy in C9ORF72 iPSC motor neurons. We will first investigate C9ORF72 ALS/FTD related pathology including G4C2 RNA foci and DPR accumulation. Next we will assess if these compounds protect against known susceptibility to extracellular stressors and survival employing longitudinal imaging analysis. Finally, we will determine how these compounds affect nucleocytoplasmic trafficking deficits in C9ORF72 ALS motor neurons. Taken together, these studies will determine if pharmacologically modulating the nucleocytoplasmic trafficking pathway is a viable therapeutic strategy to treat neurodegenerative diseases related to the C9ORF72 repeat expansion. Furthermore, we will have identified the neuroprotective mechanism of action for these novel compounds.
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0.956 |
2018 — 2021 |
Donnelly, Christopher James |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
An Optogenetic Approach to Establish the Interplay Between Stress Granules and Tdp-43 Proteinopathy in Als/Ftd @ University of Pittsburgh At Pittsburgh
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder marked by loss of motor neurons of the spinal cord and cortex. ALS is characterized by significant heterogeneity in disease onset and patient presentation. Over the last twenty years, mutations in over 35 different genes have been uncovered as causal in the development of familial forms of ALS (fALS); however, fALS only accounts for roughly 10% of all ALS cases. The remaining 90% of patients suffer from sporadic ALS (sALS), with no family history of disease and unknown causes of pathogenesis. Regardless of all this genetic and pathogenic complexity, remarkably nearly every single ALS patient (both fALS and sALS) share a common neuropathology in the form of aberrant cytoplasmic inclusions of a protein called TAR DNA-binding protein of 43 kDa (TDP-43) found in degenerating regions of the nervous system. Insight drawn from studies of fALS-linked mutations have implicated stress granule dysfunction in disease development, but the exact mechanisms by which the stress granules may directly regulate TDP-43 aggregation remains unclear. By utilizing a novel optogenetic approach to induce either TDP-43 inclusions or functional stress granules, we can manipulate these processes at a previously unattainable level of control. These experiments will explore the contribution of stress granule dysfunction to TDP-43 inclusion neurotoxicity. We hypothesize that perturbations in stress granule dynamics contribute to neurotoxic TDP-43 aggregation in ALS. We will first investigate the contribution of stress granules to light-induced TDP-43 inclusions. We will then determine if chronic or persistent stress granule formation initiates TDP-43 proteinopathy. Finally, we will perform a genome-wide RNAi screen to identify novel pathways that protect against TDP-43 inclusion toxicity and assess whether this protection is dependent on stress granule regulation.
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0.956 |
2019 — 2020 |
Donnelly, Christopher James Pandey, Udai B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Oligonucleotide Approach to Antagonize Neurotoxic Tdp-43 Assemblies in Dementia @ University of Pittsburgh At Pittsburgh
Cytoplasmic inclusions of the TAR DNA-binding protein of 43 kDa (TDP-43) is frequently found in degenerating neurons of patients with Amyotrophic lateral sclerosis, Frontotemporal Dementia, and Alzheimer?s Disease. TDP-43 is a ubiquitously-expressed, tightly-regulated, and predominantly nuclear DNA/RNA-binding protein and the cause and mechanism underlying the formation of TDP-43 protein inclusions remains unclear. Furthermore, modeling these inclusions has proven challenging and it is unclear if these are neurotoxic or neuroprotective. To address these issues, we developed an optogenetic approach to induce TDP-43 inclusions in live cells. We show that these inclusions mimic the properties of TDP-43 inclusions found in patient neurons that are neurotoxic. We further show that RNA binding status dictates the propensity for TDP-43 for form inclusions in live cells. In this grant, we propose to identify the characteristics of neurotoxic TDP-43 assemblies employing our light-induced system. Specifically, we will describe the post- translational modifications, localization, and material state of neurotoxic TDP-43 assemblies. Since our preliminary data indicates that RNA binding state determines whether TDP-43 proteins can oligomerize into a pathogenic conformation, we propose to study whether bait oligonucleotides designed to bind RNA deficient TDP-43 proteins confer neuroprotection in cortical neuron cultures, and in vivo using a TDP-43 Drosophila model system developed in our labs. These studies have the potential to address pervasive questions regarding the neurotoxic state of TDP-43 assemblies and the development of a new use for oligonucleotide therapeutics.
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0.956 |
2021 |
Belzil, Veronique Donnelly, Christopher James Heiman, Myriam Kellis, Manolis (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Identification of Tdp-43 Modifiers Through Single-Cell Transcriptional and Epigenomic Dissection of Als and Ftld-Mnd @ University of Pittsburgh At Pittsburgh
Abstract Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two fatal neurodegenerative conditions with no current treatment to prevent, decelerate or stop neuronal death in patients. ALS and FTLD are clinically distinct but show an overlap in postmortem brain pathology and genetic factors: nuclear clearance and cytoplasmic accumulation of TDP-43 in affected central nervous system (CNS) regions is observed in 98% of ALS and 50% of FTLD patients. While initial symptoms lead to the diagnosis of either ALS or FTLD, up to 50% of ALS patients eventually develop symptoms of FTLD, with ~15% of patients ultimately receiving both diagnoses (FTLD with motor neuron disease, FTLD/MND). Mutations in the gene encoding TDP-43 (TARDBP) lead to rare cases of ALS, while TDP-43 pathology is observed in patients carrying more prevalent mutations, such as a pathological C9orf72 hexanucleotide repeat expansion (C9orf72+)?the most common genetic cause of ALS and FTLD identified thus far. TDP-43 therefore appears to be a pivotal and convergent factor in the pathogenesis of both ALS and FTLD. Despite this, however, the reasons for selective vulnerability of motor neurons, the mechanisms responsible for TDP-43 mislocalization, and the impact on neuronal health of nuclear TDP-43 exclusion and aberrant liquid-liquid phase separation underlying cytoplasmic demixing remain unknown. To address this challenge, in Aim 1, we systematically profile the transcriptional and epigenomic alterations of ALS and FTLD/MND patients at single-cell resolution using post-mortem CNS samples. In Aim 2, we integrate the resulting datasets to study the link between genetic, epigenomic, transcriptional, and cellular signatures of ALS and FTLD/MND. We associate these links with available clinical information, elucidate the genes and biological pathways altered in each, and predict new therapeutic targets. In Aim 3, we validate the molecular and cellular effects of these targets by assessing their impact on neuronal viability and TDP-43 functions/aggregation using high-throughput directed perturbation experiments. We study both cell-autonomous and non-cell-autonomous effects of these perturbations in human dura fibroblast-derived iPSC neurons and astroglia. In Aim 4, we perform neuropathological analyses of TDP-43 modifiers in ALS and FTLD/MND postmortem tissues, and endeavor to rescue in vivo pathology and phenotypes in a mouse model. The resulting datasets, analyses, and dura-derived iPSCs will provide an invaluable resource to understand the mechanisms of TDP-43 pathology in ALS and FTLD/MND, and may reveal putative therapeutic targets able to mitigate TDP-43 pathology through genetic manipulation.
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0.956 |