Mutant TDP-43 in Astrocytes Kills Motor Neurons in Rats through Neurotoxic Gain and Neuroprotective Loss in Astrocytes
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ABSTRACT: Mutation in TDP-43 is causative to amyotrophic lateral sclerosis (ALS). TDP-43 is a multifunctional ribonucleoprotein and is reproted to regulate thousands of genes in neurons, but how astrocytes contribute to TDP-43 pathogenesis is not known. This study examined how mutant TDP-43 in astrocytes kills motor neurons and causes ALS phenotypes. Primary astrocytes were isolated from transgenic rats expressing mutant TDP-43 or from control rats without mutant TDP-43 expression. Cultured astrocytes were induced to express mutant human TDP-43 and their gene expression profiles were determined by microarray assays. Microarray analysis revealed that hundreds of genes were altered in astrocytes in response to mutant TDP-43 expression. As mutant TDP-43 transgene is under the control of tetracycline-regulated pomoter elements (TRE), mutant TDP-43 expression is subjected to Doxycline regulation. Astrocytes isolated from GFAP-tTA/TRE-TDP43M337V rats were desiginated as M337V groups and astrocytes isolated from GFAP-tTA single transgenic rats were desiginated as tTA control groups. Total RNA was isolated from cultured astrocytes at varying times (3, 4, or 6 days after Dox withdrawal) after mutant TDP-43 was induced in astrocytes. Upon mutant TDP-43 induction in astroyctes, gene expression profiles in astroyctes were determined by Illumina Direct Hybridization Assay and compared between tTA and M337V groups at the varying time points of mutant TDP-43 induction.
Project description:Changes in gene expression profile of CLL cells in response to an antigen-specific Th cell clone that is specific for a Ckappa peptide of mouse Abs. Mouse anti human BCR mAbs were used to ligate BCR and deliver antigen to CLL cells. 32-45 x10e6 PBMC were incubated overnight with or without 2 μg/ml of Ms κ+IgG anti-kappa and anti-lambda mAbs. The PBMC were then cultured in presence or absence of 12.5 x10e6 T18 cells. On day 3, CLL cells were purified by negative selection using CD3 and CD14 Dynabeads (Invitrogen). RNA from CLL cells was controlled and quantified on a NanoDrop1000 (Thermo Scientific, Wilmington, DE, USA). 290ng per sample was amplified and labeled with the TotalPrep™-96 RNA Amplification Kit (Illumina, San Diego, CA, USA). Sample quality was further tested by Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). 1500ng of biotin labeled cRNA was used to hybridize onto Illumina HumanWG-6 v3 Expression BeadChips. After scanning, the results were imported into Illumina GenomeStudio v. 2010.1, Gene Expression module v. 1.6.0 for quality control and data export. Analysis was performed utilizing GeneSpring GX v11 software (Agilent, Santa Clara, CA, USA). In GeneSpring GX, Logbase 2-transformed data were normalized to the 75 percentile with baseline transformation to median of all samples. Flag Information: Lower cutoff for 'Present' call: 0.8, Upper cutoff for 'Absent' call: 0.6 Testing antigen dependent Th cell - CLL cell collaboration, effects on gene expression of CLL cells
Project description:Mutation in TDP-43 is causative to amyotrophic lateral sclerosis (ALS). TDP-43 is a multifunctional ribonucleoprotein and is reproted to regulate thousands of genes in neurons, but how astrocytes contribute to TDP-43 pathogenesis is not known. This study examined how mutant TDP-43 in astrocytes kills motor neurons and causes ALS phenotypes. Primary astrocytes were isolated from transgenic rats expressing mutant TDP-43 or from control rats without mutant TDP-43 expression. Cultured astrocytes were induced to express mutant human TDP-43 and their gene expression profiles were determined by microarray assays. Microarray analysis revealed that hundreds of genes were altered in astrocytes in response to mutant TDP-43 expression.
Project description:The majority of patients with amyotrophic lateral sclerosis (ALS) have abnormal TDP-43 aggregates in the nucleus and/or cytosol of their surviving neurons and glia. Although accumulating evidence indicates that astroglial dysfunctions contribute to motor neuron degeneration in ALS, the normal physiological functions of TDP-43 in astrocytes are largely unknown and whether the loss of astroglial TDP-43 contributes to ALS remains to be clarified. Here, we showed that TDP-43 deleted astrocytes showed cell-autonomously enhanced GFAP immunoreactivity without affecting astrocyte or microglia proliferation. At the transcriptomic level, TDP-43 deleted astrocytes resemble the A1-reactive astrocytes and induce microglia to increase C1q expression. These astrocytic changes do not cause the loss of motor neurons in spinal cords or denervation at the neuromuscular junctions. In contrast, there was a selective reduction of mature oligodendrocytes, but not oligodendrocyte precursor cells, suggesting a tri-glial dysfunction mediated by TDP-43-deleted astrocytes. Mice with astroglial TDP-43 deletion developed motor, but not sensory, deficits. Taken together, our results demonstrate that TDP-43 is required to maintain the protective functions of astrocytes relevant to the development of motor deficits in mice.
Project description:Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.
Project description:TDP-43 is an RNA binding protein involved in amyotrophic lateral sclerosis and other neurodegenerative diseases. The purpose of this study was to determine if loss of TDP-43 function leads to accumulation of repetitive element transcripts, double-stranded RNA (dsRNA) and innate immune activation that may be involved in disease pathology. TDP-43 was knocked down in primary rat astrocytes via siRNA, cells were treated with/without ATP (an immune modulator), and polyA RNA-seq was performed to profile gene expression. Immunoprecipitation/RNA-seq was also performed using a dsRNA-specific antibody to identify potential dsRNAs resulting from TDP-43 knockdown.
Project description:TDP-43 is a DNA/RNA-binding protein that regulates gene expression and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its role in endothelial cells (ECs), angiogenesis and vascular function. Using inducible EC-specific TDP-43 knockout mice, we show that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity and blood vessel stability. Postnatal EC-specific deletion of TDP-43 leads to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupts the blood-brain barrier and triggers vascular degeneration. These vascular defects are associated with an inflammatory response in the central-nervous system with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupts fibronectin matrix around sprouting vessels and reduces -catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.
Project description:Transactive response DNA-binding protein 43 (TDP-43) is a predominantly nuclear, ubiquitously expressed RNA and DNA-binding protein. It recognises and binds to UG repeats and is involved in pre-mRNA splicing, mRNA stability and microRNA metabolism. TDP-43 is essential in early embryonic development but accumulates in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar degeneration (FTLD). It is not known yet whether cytoplasmic aggregates of TDP-43 are toxic or protective but they are often associated with a loss of TDP-43 from the nucleus and neurodegeneration may be caused by a loss of normal TDP-43 function or a gain of toxic function. Here we present a proteomic study to analyse the effect of loss of TDP-43 on the proteome. Our results indicate that TDP-43 is an important regulator of RNA metabolism and intracellular transport. We show that Ran-binding protein 1 (RanBP1), DNA methyltransferase 3 alpha (Dnmt3a) and chromogranin B (CgB) are downregulated upon TDP-43 knockdown. Subsequently, transportin 1 level is increased as a result of RanBP1 depletion. Improper regulation of these proteins and the subsequent disruption of cellular processes may play a role in the pathogenesis of the TDP-43 proteinopathies ALS and FTLD.
Project description:To investigate how translation changed as a function of TDP-43 CR loss, mass spectrometry (MS) based approach was performed to monitor the dynamics of TDP-43-associated protein complexes in response to CR deletion using TDP-43 knockout HEK293 cells expressing strep tagged wild type TDP-43 (TDP-43WT) or TDP-43ΔCR mutant.
Project description:Cytoplasmic aggregation TAR DNA binding protein 43 (TDP-43) is a hallmark pathology of motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). The disease-relevant protein-protein interactome of TDP-43 remains incompletely defined, elucidation of which will increase understanding of the molecular mechanisms responsible for disease. In this study, we aimed to identify true TDP-43 protein partners within the nucleus and cytoplasm and interactions at specific pathological stages of disease by correlating identified TDP-43 interaction partners in in human embryonic kidney HEK293, mouse neuroblastoma Neuro2A and mouse primary neurons. Using this approach combined with APEX2 proximity labelling and immunoprecipitation, and coupled with mass spectrometry analysis of protein interactors from nuclear and cytoplasmic fractions, we identified 58 putative wild-type TDP-43 interactors, including novel binding partners responsible for RNA metabolism and splicing. We verified the level of interaction of these protein partners in two other models: (1) a model presenting early pathological change showing TDP-43WT condensates in the nucleus through arsenite treatment and (2) a model stimulating another early-disease stage with TDP-43 containing ALS missense mutations (G294V and A315T) in the nucleus. We found that most interactors that presenting had a weaker affinity to mutant TDP-43 in the cytoplasm are involved in the translational machinery which could, over time, contribute to neurodegeneration. Understanding early pathological changes to TDP-43 in the nucleus and its specific interactions partners at different disease stages is critical to better understand ALS and FTLD mechanisms and provide potential therapeutic targets and novel biomarkers.