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Therapeutic Effect of Rapamycin on TDP-43-Related Pathogenesis in Ischemic Stroke.

ABSTRACT: Stroke is a major cause of death and disability across the world, and its detrimental impact should not be underestimated. Therapies are available and effective for ischemic stroke (e.g., thrombolytic recanalization and mechanical thrombectomy); however, there are limitations to therapeutic interventions. Recanalization therapy has developed dramatically, while the use of adjunct neuroprotective agents as complementary therapies remains deficient. Pathological TAR DNA-binding protein (TDP-43) has been identified as a major component of insoluble aggregates in numerous neurodegenerative pathologies, including ALS, FTLD and Alzheimer's disease. Here, we show that increased pathological TDP-43 fractions accompanied by impaired mitochondrial function and increased gliosis were observed in an ischemic stroke rat model, suggesting a pathological role of TDP-43 in ischemic stroke. In ischemic rats administered rapamycin, the insoluble TDP-43 fraction was significantly decreased in the ischemic cortex region, accompanied by a recovery of mitochondrial function, the attenuation of cellular apoptosis, a reduction in infarct areas and improvements in motor defects. Accordingly, our results suggest that rapamycin provides neuroprotective benefits not only by ameliorating pathological TDP-43 levels, but also by reversing mitochondrial function and attenuating cell apoptosis in ischemic stroke.

SUBMITTER: Tsou YS

PROVIDER: S-EPMC9820757 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Therapeutic Effect of Rapamycin on TDP-43-Related Pathogenesis in Ischemic Stroke.

Tsou Yi-Syue YS Lai Jing-Huei JH Chen Kai-Yun KY Chang Cheng-Fu CF Huang Chi-Chen CC

International journal of molecular sciences 20221230 1

Stroke is a major cause of death and disability across the world, and its detrimental impact should not be underestimated. Therapies are available and effective for ischemic stroke (e.g., thrombolytic recanalization and mechanical thrombectomy); however, there are limitations to therapeutic interventions. Recanalization therapy has developed dramatically, while the use of adjunct neuroprotective agents as complementary therapies remains deficient. Pathological TAR DNA-binding protein (TDP-43) ha ...[more]

PMID: 36614118

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Project description:BackgroundTDP-43 has been identified as a disease-associated protein in several chronic neurodegenerative disorders and increasing evidence suggests its potentially pathogenic role following brain injuries. Normally expressed in nucleus, under pathological conditions TDP-43 forms cytoplasmic ubiquitinated inclusions in which it is abnormally phosphorylated and cleaved to generate a 35 and a 25 kDa C-terminal fragments. In the present study, we investigated age-related expression patterns of TDP-43 in neurons and glia and its role as modulator of inflammation following ischemic injury.MethodsWild-type and TDP-43 transgenic mice of different age groups were subjected to transient middle cerebral artery occlusion. The role of TDP-43 in modulation of inflammation was assessed using immunofluorescence, Western blot analysis, and in vivo bioluminescence imaging. Finally, post-mortem stroke human brain sections were analyzed for TDP-43 protein by immunohistochemistry.ResultsWe report here an age-related increase and formation of ubiquitinated TDP-43 cytoplasmic inclusions after stroke. The observed deregulation in TDP-43 expression patterns was associated with an increase in microglial activation and innate immune signaling as revealed by in vivo bioluminescence imaging and immunofluorescence analysis. The presence of ubiquitinated TDP-43 aggregates and its cleaved TDP-35 and TDP-25 fragments was markedly increased in older, 12-month-old mice leading to larger infarctions and a significant increase in in neuronal death. Importantly, unlike the hallmark neuropathological features associated with chronic neurodegenerative disorders, the TDP-43-positive cytoplasmic inclusions detected after stroke were not phosphorylated. Next, we showed that an increase and/or overexpression of the cytoplasmic TDP-43 drives the pathogenic NF-κB response and further increases levels of pro-inflammatory markers and ischemic injury after stroke in age-dependent manner. Finally, analyses of the post-mortem stroke brain tissues revealed the presence of the cytoplasmic TDP-43 immunoreactive structures after human stroke.ConclusionTogether, our findings suggest that the level of cytoplasmic TDP-43 increases with aging and may act as an age-related mediator of inflammation and neuronal injury after stroke. Thus, targeting cytoplasmic TDP-43 may have a therapeutic potential after stroke.

Project description:BackgroundThe brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy.MethodsWe used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model.ResultsHere we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions.ConclusionsHere we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.

Project description:BackgroundAmyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative condition that is characterized by progressive loss of motor neurons and the accumulation of aggregated TAR DNA Binding Protein-43 (TDP-43, gene: TARDBP). Increasing evidence indicates that environmental factors contribute to the risk of ALS. Dioxins, related planar polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that activate the aryl hydrocarbon receptor (AHR), a ligand-activated, PAS family transcription factor. Recently, exposure to these toxicants was identified as a risk factor for ALS.MethodsWe examined levels of TDP-43 reporter activity, transcript and protein. Quantification was done using cell lines, induced pluripotent stem cells (iPSCs) and mouse brain. The target samples were treated with AHR agonists, including 6-Formylindolo[3,2-b]carbazole (FICZ, a potential endogenous ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, and benzo(a)pyrene, an abundant carcinogen in cigarette smoke). The action of the agonists was inhibited by concomitant addition of AHR antagonists or by AHR-specific shRNA.ResultsWe now report that AHR agonists induce up to a 3-fold increase in TDP-43 protein in human neuronal cell lines (BE-M17 cells), motor neuron differentiated iPSCs, and in murine brain. Chronic treatment with AHR agonists elicits over 2-fold accumulation of soluble and insoluble TDP-43, primarily because of reduced TDP-43 catabolism. AHR antagonists or AHR knockdown inhibits agonist-induced increases in TDP-43 protein and TARDBP transcription demonstrating that the ligands act through the AHR.ConclusionsThese results provide the first evidence that environmental AHR ligands increase TDP-43, which is the principle pathological protein associated with ALS. These results suggest novel molecular mechanisms through which a variety of prevalent environmental factors might directly contribute to ALS. The widespread distribution of dioxins, PCBs and PAHs is considered to be a risk factor for cancer and autoimmune diseases, but could also be a significant public health concern for ALS.

Dataset Information

Therapeutic Effect of Rapamycin on TDP-43-Related Pathogenesis in Ischemic Stroke.

Publications

Therapeutic Effect of Rapamycin on TDP-43-Related Pathogenesis in Ischemic Stroke.

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