To evaluate senescence mechanisms, including senescence-associated secretory phenotype (SASP), in the motor-neuron disease model hSOD1-G93A, we quantified the expression of p16 and p21 and the senescence-associated β galactosidase (SA-β-gal) in nervous tissue. As SASP markers, we measured the mRNA levels of Il1a, Il6, Ifna, and Ifnb. Furthermore, we explored if an alteration of alternative splicing is associated with senescence by measuring the Adipor2 cryptic exon inclusion levels, a specific splicing variant repressed by TAR-DNA binding of 43 kDa (Tdp-43). Transgenic mice show an atypical senescence profile with high p16 and p21 mRNA and protein in glia, without the canonical increase in SA-β-gal activity. Consistent with SASP, there is an increase in Il1a and Il6 expression, associated with increased TNFR and M-CSF protein levels, with females being partially protected. TDP-43 splicing activity is compromised in this model. Senolytic drug Navitoclax does not alter the present 'model's disease progression. This lack of effect is reproduced in vitro, in contrast with Dasatinib and quercetin, which diminish p16 and p21. Our findings show a non-canonical profile of senescence biomarkers in the model hSOD1-G93A.
Previous evidence links the formation of extranuclear inclusions of transcription factors, such as ERK, Jun, TDP-43, and REST, with oxidative, endoplasmic-reticulum, proteasomal, and osmotic stress. To further characterize its extranuclear location, we performed a high-content screening based on confocal microscopy and automatized image analyses of an epithelial cell culture treated with hydrogen peroxide, thapsigargin, epoxomicin, or sorbitol at different concentrations and times to recreate the stresses mentioned above. We also performed a subcellular fractionation of the brain from transgenic mice overexpressing the Q331K-mutated TARDBP, and we analyzed the REST-regulated mRNAs. The results show that these nuclear proteins exhibit a mitochondrial location, together with significant nuclear/extranuclear ratio changes, in a protein and stress-specific manner. The presence of these proteins in enriched mitochondrial fractions in vivo confirmed the results of the image analyses. TDP-43 aggregation was associated with alterations in the mRNA levels of the REST target genes involved in calcium homeostasis, apoptosis, and metabolism. In conclusion, cell stress increased the mitochondrial translocation of nuclear proteins, increasing the chance of proteostasis alterations. Furthermore, TDP-43 aggregation impacts REST target genes, disclosing an exciting interaction between these two transcription factors in neurodegenerative processes.
Previous evidence links the formation of extranuclear inclusions of transcription factors, such as ERK, Jun, TDP-43, and REST with oxidative, endoplasmic-reticulum, proteasomal, and osmotic stress. To further characterize its extranuclear location, we performed a high-content screening based on confocal microscopy and automatized image analyses of an epithelial cell culture treated with hydrogen peroxide, thapsigargin, epoxomicin, or sorbitol at different concentrations and times to recreate the stresses mentioned above. We also performed subcellular fractionation of the brain from transgenic mice overexpressing the Q331K mutated TARDBP, and we analyzed REST-regulated mRNAs. The results show that these nuclear proteins exhibit a mitochondrial location, together with significant nuclear/extranuclear ratio changes, in a protein and stress-specific manner. The presence of these proteins in enriched mitochondrial fractions in vivo confirmed the results of image analyses. TDP-43 aggregation was associated with alteration in mRNA levels of REST target genes involved in calcium homeostasis, apoptosis, and metabolism. In conclusion, cell stress increased mitochondrial translocation of nuclear proteins, increasing the chance of proteostasis alterations. Further, TDP-43 aggregation impacts REST target genes, disclosing an exciting interaction between these two transcription factors in neurodegenerative processes.
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