Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are progressive and fatal neurodegenerative disorders showing mislocalization and cytosolic accumulation of TDP-43 inclusions in the central nervous system. The decrease in the efficiency of the clearance systems in aging, as well as the presence of genetic mutations of proteins associated with cellular proteostasis in the familial forms of TDP-43 proteinopathies, suggest that a failure of these protein degradation systems is a key factor in the aetiology of TDP-43 associated disorders. Here we show that the internalization of human pre-formed TDP-43 aggregates in the murine neuroblastoma N2a cells promptly resulted in their ubiquitination and hyperphosphorylation by endogenous machineries, mimicking the post-translational modifications observed in patients. Moreover, our data identify mitochondria as the main responsible sites for the alteration of calcium homeostasis induced by TDP-43 aggregates, which, in turn, stimulates an increase in reactive oxygen species and, finally, caspase activation. The inhibition of TDP-43 proteostasis in the presence of selective inhibitors against the proteasome and macroautophagy systems revealed that these two systems are both severely involved in TDP-43 accumulation and have a strong influence on each other in neurodegenerative disorders associated with TDP-43.