The Role Of TDP-43 In The Pathogeneis Of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis(ALS) is a fatal neurodegenerative disorder that is characterized by the loss of motor neurons of the brainstem, cortical regions and spinal cord, leading to progressive paralysis and eventually death from respiratory failure. Ubiquitinated inclusions and neuroinflammation are the defining features of ALS. Landmark discoveries of ALS pathogenesis were the identification of mutations in TDP-43, but the effects of TDP-43 on disease pathogenesis is largely unknown. Here, we show the roles of TDP-43 in the regulations of autophagy and microglial activation, the cellular processes that are tightly associated in the pathogenesis of amyotrophic lateral sclerosis, which is of help to explain molecular mechanism of disease etiology. RESULT I Abnormality in autophagy and lysosomal pathway contributes to TDP-43-linked amyotrophic lateral sclerosisAim: To study the effects of TDP-43 on autophagy–lysosome pathway and explore its potential molecular mechanism.Methods: Knockdown of TDP-43 in HEK293, HeLa, SH-SY5 Y, ATG5-WT MEF and ATG5-KO MEF cells. Overexpression of EGFP-TFEB in TDP-43-depleted cells, TFEB nuclear translocation was examined using fluorescence microscope. Subcellular fractionation and Western blotting analyses were used to check TFEB nuclear translocation in TDP-43-depleted cells. Immunofluorescence analysis was applied to examine the lysosomal localization of mTOR, and Western blotting analysis was used to detect the phosphorylation of mTORC1 substrate p70S6 K in TDP-43-depleted cells. Western blotting and quantitative RT-PCR(qRT-PCR) experiments were performed to examine the protein and mRNA levels of the members of Ragulator-Rag-mTORC1 complex in TDP-43 knockdown cells. RNA immunoprecipitation(RIP) was performed to detect whether TDP-43 could bind to raptor mRNA. Immunofluorescence analysis was performed to detect the localization of mTOR and TFEB using fluorescence microscope in TDP-43-depleted cells re-transfected with siRNA against raptor or HA-raptor. Flow cytometry analysis was applied to detecte the fluorescent intensity and immunofluorescence analysis was performed to check the location of LC3 and p62 in TDP-43 knockdown cells. Western blotting and qRT-PCR analyses were applied to detect the protein and mRNA level of lysosomal and autophagic genes in TDP-43-depleted cells. Western blotting and qRT-PCR analyses were performed to examine lysosomal and autophagic genes in both TFEB and TDP-43-deficient cells; and electron microscope(EM) analysis was used to examine autophagosome and lysosome numbers. Western blotting was applied to analyze the protein level of lysosomal and autophagic genes in TDP-43-depleted cells that were starved or treated with Bafilomycin A1. Fluorescence microscope was used to observe the number of autophagosomes and autolysosomes in TDP-43-depleted cells transfected with mCherry-EGFP-LC3. MTT assays were performed to detect the cell viability in TDP-43-depleted ATG5-WT MEF and ATG5-KO MEF cells transfected with EGFP-Nhtt-150 Q. Western blotting analysis was used to detect the genes involved in autophagosome-lysosome fusion, and immunofluorescence was applied to examine LC3/LAMP1-RFP co-localization in TDP-43-depleted cells. Western blotting analysis was used to examine the phosphorylation of mTORC1 substrates p70S6 K and 4E-BP1 in TBPH KO flies. qRT-PCR was used to detect the mRNA level of raptor, lysosomal and autophagic genes in TBPH KO flies. TBPH knockout flies that were treated with PA(to inhibit autophagy) or Rapamycin(to activate autophagy) were subjected to larval locomotion and fly developmental viability assays.Results: A dramatic TFEB nuclear translocation was observed in TDP-43-depleted cells using fluorescence microscope and subcellular fractionation assays. Depletion of TDP-43 prevented the lysosomal localization of mTOR and caused a reduction of the phosphorylation of mTORC1 substrate p70S6 K. A decerased protein level of raptor in the absence of TDP-43, but not p18 and RagB, was observed. qRT-PCR analysis further confirmed a dramatic decrease mRNA level of raptor upon treatment with siRNA against TDP-43, but not p18, mTOR and RagB. In addition, RNA immunoprecipitation(RIP) showed a direct intertion of TDP-43 with raptor mRNA. RNAi-mediated suppression of TDP-43 and raptor prevented mTOR to localize to lysosomes, leading to a significant TFEB nuclear translocation. However, mTOR was redistributed to lysosomes and TFEB shuttled back to the cytoplasm in TDP-43-depleted cells upon re-transfection of raptor. In TDP-43-depleted cells, a markedly increased punctuate LC3 was presented. The protein and mRNA levels of lysosomal and autophagic genes were increased in TDP-43-depleted cells, whereas the increased expression levels of relative genes were no longer increased in TDP-43 deficient cells upon treatment with siRNA against TFEB. In addition, electron microscope(EM) analysis also demonstrated that depletion of TFEB blocked the increased autophagosome and lysosome numbers in TDP-43-depleted cells. Depletion of TDP-43 increased the number of autophagosomes in cells expressing mCherry-EGFP-LC3 even under starvation or Bafilomycin A1 treatment. Loss of MTT assays showed that knockdown of TDP-43 caused an obvious decrease of cell viability in ATG5-WT MEF cells transfected with EGFP-Nhtt-150 Q, but not in ATG5-KO MEF cells. Decrease of Dynactin 1 level with a significant decreased LC3/LAMP1–RFP co-localization was observed in TDP-43-deficient cells. Thus data suggest that a decrease of Dynactin 1 causes an impairment of the fusion of autophagosome and lysosome in in TDP-43-deficient cells, leading to accumulations of autophagosome and lysosome, which induces a susceptibility of cells to stress. A dramatic decrease of the phosphorylation of S6 K and 4E-BP1 in TBPH KO flies was also observed, and qRT-PCR showed a decrease mRNA level of raptor and an increase mRNA level of lysosomal and autophagic genes in TBPH KO flies. Treated TBPH KO larval with rapamycin greatly aggravated larval locomotion defects, but treated with PA ameliorated locomotion defects. In addition, TBPH KO flies treatment with rapamycin also impaired the developmental viability, but treatment with PA improved the developmental viability.Conclusion: Depletion of TDP-43 greatly caused a dramatic TFEB nuclear translocation and impaired the activity of mTORC1 by targeting raptor, one of the components of mTORC1, resulting in increased autophagosomal and lysosomal biogenesis. However, depletion of TDP-43 also impaired autophagosome-lysosome fusion by targeting dynactin 1. Both of these caused overwhelmed accumulation of autophagosomes and lysosomes, contributing to TDP-43-mediated neurodegeneration.RESULT II Neuroinflammation contributes to TDP-43-linked amyotrophic lateral sclerosisTDP-43-depleted models. Aim: To explore the contribution of microglia and astrocytes to neurotoxicity inMethods: Knockdown of TDP-43 in primary cultured microglia, primary cultured astrocytes and BV2 cells. Western blotting and quantitative RT-PCR(q RT-PCR) were performed to examine the expression level of COX-2 and i NOS, two markers of neuroinflammation, in TDP-43-depleted microglia and astrocytes that were treated with LPS or not. Western blotting analysis was used to check cell signaling pathways in TDP-43-depleted microglia. Western blotting and q RT-PCR were applied to analyze the protein and m RNA levels of COX-2 in TDP-43-depleted microglia treated with a set of inhibitors. Luciferase reporter gene assay was applied to detecte the transcriptional activity of AP1 and ELISA kit was used to examine the production of PGE2 in TDP-43-depleted microglia with or without U0126 or celecoxib treatment. Conditioned medium assays were performed to check both cortical neuron and motor neuron viability.Results: A dramatic increased protein level of COX-2, but not i NOS, in TDP-43-depleted microglia either treated with or without LPS, whereas either COX-2 or i NOS protein level was unchanged in TDP-43-depleted astrocytes treated with LPS or not. q RT-PCR analysis further confirmed a significant increase of COX-2 m RNA level in TDP-43-depleted microglia, whereas i NOS m RNA level was not changed. Loss of TDP-43 in microglia greatly upregulated phosphorylated MEK and ERK, whereas phosphorylated JNK or p38 was unchanged. In addition, total JNK, p38, MEK and ERK protein levels were unchanged. COX-2 protein and m RNA levels were not upregulated in TDP-43-depleted microglia that was treated with U0126(MEK1 inhibitor). However, TDP-43-dificient microglia that were incubated with SP600125(JNK inhibitor), SB216763(GSK3β inhibitor), BAY(NF-κB inhibitor) and SB203580(p38 inhibitor) kept unchanged for COX-2. Luciferase reporter gene assay demonstrated that loss of TDP-43 in microglia remarkably upregulated AP1 transcriptional activity. However, AP1 transcriptional activity was no longer increased under U0126 treatment, suggesting microglial activation induced by TDP-43-depletion is mediated by MEK-ERK pathway. Moreover, the production of PGE2 was significantly upregulated in TDP-43-depleted microglia, but not in TDP-43-depleted astrocytes. In TDP-43-depleted microglia treated with U0126 or celecoxib(COX-2 inhibitor), the production of PGE2 was no longer increased. Condition medium collected from TDP-43-depleted microglia greatly induced both cortical and motor neuron death, however, this effect was blocked by celecoxib.Conclusion: TDP-43-deficiency selectively activates microglia, but not astrocytes. The COX-2-PGE2 production by TDP-43-depletion is meadted by MAPK/ERK pathway. Thus, our data suggest that the selective regulation of microglia by TDP-43 may contribute to TDP-43-related neuroinflammation and neurotoxicity.