Abstract
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.
Highlights
IntroductionThe presence of protein aggregates is a pathological hallmark for several neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), to name a few [1]
Oxidative stress induced by H2 O2 in HMEC cells led to changes in the nuclear and non-nuclear distribution of p-TDP-43, while the nuclear intensity of p-TDP-43 was decreased in the milder oxidative conditions tested (Figure 1A and Supplemental Figure S1)
Complex V coimmunostaining with these factors (Figure 1B) indicated that the degree of colocalization increased significantly in all the cases after oxidative stress, exceeding z’-values of 0.5, except for p-ERK
Summary
The presence of protein aggregates is a pathological hallmark for several neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), to name a few [1]. Beta-amyloid aggregates characterize the AD neurofibrillary tangles and intracellular aggregates. Aggregates of highly phosphorylated TDP-43 are present in the cytosol of the remaining motor neurons in ALS. Whether (and how) these aggregates are mere bystanders of ongoing pathogenic processes or whether they constitute bona fide cellular noxa is still under debate
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