Neurotoxic mechanisms by which the USP14 inhibitor IU1 depletes ubiquitinated proteins and Tau in rat cerebral cortical neurons: Relevance to Alzheimer's disease

In Alzheimer's disease Proteasome activity is reportedly downregulated, thus increasing it could be therapeutically beneficial. The proteasome-associated Deubiquitinase USP14 disassembles polyubiquitin-chains, potentially delaying proteasome-dependent protein degradation. We assessed the protective efficacy of inhibiting or downregulating USP14 in rat and mouse (Usp14^axJ) neuronal cultures treated with prostaglandin J2 (PGJ2). IU1 concentrations (_HIU1>25μM) reported by Others to inhibit USP14 and be protective in non-neuronal cells, reduced PGJ2-induced Ub-protein accumulation in neurons. However, _HIU1 alone or with PGJ2 is neurotoxic, induces calpain-dependent Tau cleavage, and decreases E1~Ub thioester levels and 26S Proteasome assembly, which are energy-dependent processes. We attribute the two latter _HIU1 effects to ATP-deficits and mitochondrial Complex I inhibition, as shown herein. These _HIU1 effects mimic those of mitochondrial inhibitors in general, thus supporting that ATP-depletion is a major mediator of _HIU1-actions. In contrast, low IU1 concentrations (_LIU1≤25μM) or USP14 knockdown by siRNA in rat cortical cultures or loss of USP14 in cortical cultures from ataxia (Usp14^axJ) mice, failed to prevent PGJ2-induced Ub-protein accumulation. PGJ2 alone induces Ub-protein accumulation and decreases E1~Ub thioester levels. This seemingly paradoxical result may be attributed to PGJ2 inhibiting some deubiquitinases (such as UCH-L1 but not USP14), thus triggering Ub-protein stabilization. Overall, IU1-concentrations that reduce PGJ2-induced accumulation of Ub-proteins are neurotoxic, trigger calpain-mediated Tau cleavage, lower ATP, E1~Ub thioester and E1 protein levels, and reduce Proteasome activity. In conclusion, pharmacologically inhibiting (with low or high IU1 concentrations) or genetically down-regulating USP14 fail to enhance proteasomal degradation of Ub-proteins or Tau in neurons.

Alzheimer's; Calpain; Deubiquitinase; Mitochondria; Tau; USP14; Ubiquitin-activating enzyme.