IDENTIFICATION AND CHARACTERIZATION OF NOVEL MOLECULAR PLAYERS FROM THE UBIQUITINOME IMPLICATED IN THE REGULATION OF AMYLOID PRECURSOR PROTEIN

Abstract

Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder worldwide and has been associated for decades to the Amyloid Precursor Protein (APP), one hallmark in the pathogenesis of AD. However, it is still unclear how APP protein levels are maintained. Increasing evidence implicates the dysfunction of the two main degradative pathways: Ubiquitin-Proteasome System (UPS) and Autophagic/Lysosomal pathway (ALP) in the pathogenesis of AD. Moreover, it has been described that APP is ubiquitylated within its cytosolic domain, modification that acts as a unifying signal between both pathways. To gain insights into the role of UPS and ALP on APP regulation, we performed High Content siRNA Screening (HCS) including 1.187 genes of the human “ubiquitinome” in H4 neuroglioma cells stably expressing APP-EGFP as reporter. We identified the deubiquitinating (DUB) enzyme PSMD14, as the major hit among all the analyzed targets. PSMD14 corresponds to a structural subunit of the 19S proteasome with DUB activity that removes ubiquitin (Ub) chains of substrates during proteasomal degradation. We found that specific PSMD14 KD or its pharmacological inhibition by CZM caused a robust increase in APP levels, confirming its role on APP regulation. Additionally, our studies revealed an unexplored function of PSMD14 in Golgi apparatus homeostasis. We found that inhibition of PSMD14 activity by CZM caused a decrease in Golgi to ER retrograde transport. Moreover, because previous studies revealed the involvement PSMD14 activity in the clearance of protein aggregates by ALP, a pathway that have been recently linked with Golgi to ER retrograde transport, we explored the effect of PSMD14 inhibition on autophagosomes biogenesis. We found that PSMD14 activity is crucial for LC3-dependent autophagosome formation. Our results revealed an unexpected mechanism of interplay between UPS and ALP pathways, showing that active PSMD14 contributes with the biogenesis of autophagosomes. Importantly, inhibition of PSMD14 caused the increase in APP full-length due to the impairment in this interplay with important implication in cellular proteostasis and AD pathogenesis. Keywords: Alzheimer's disease, APP, UPS, ALP, HCS