THE ROLE OF NUB1 IN THE CLEARANCE OF TAU AGGREGATES IN ALZHEIMER'S DISEASE

Abstract

BACKGROUND: Intraneuronal aggregations of abnormal hyperphosphorylated tau are a hallmark of Alzheimer’s disease. Previous data revealed that NEDD8 ultimate buster 1 (NUB1) reduces the aggregation and phosphorylation of tau in an in vitro model. NUB1 is a ubiquitin-like (UBL)/ ubiquitin-associated (UBA) protein that targets the down regulation of ubiquitin-like modifiers (NEDD8, FAT10), and aggregation-prone proteins leading to neurodegenerative disease (synphilin-1, huntingtin). The aim of the study is to understand how NUB1 affects tau aggregation and phosphorylation. METHODS: A GFP-tau inducible neuroblastoma cell line was generated and treated with proteasome inhibitor to induce the formation of aggresomes. GFP-tau cells were transduced with NUB1 lentiviral vectors both in the absence and presence of proteasome inhibitor, followed by western blotting, filter trap assays, immunoprecipitation and immunofluorescence. RESULTS: Inhibition of the proteasome induced the formation of GFP-tau aggresomes that were positive for phospho-tau (p-tau) markers (AT8, pS396). Filter trap assays revealed that NUB1 significantly decreased the ratio of p-tau to tau in the total cellular fraction (both soluble and insoluble), as well as in only the detergent insoluble fraction, both with and without proteasome inhibition. However, the ratio of tau and p-tau in the insoluble fraction to that in the total fraction revealed that the NUB1-mediated decrease in the levels of detergent insoluble tau and p-tau exceeded that of the overall decline in total levels, suggesting NUB1 can target aggregation-prone tau and p-tau. This effect of NUB1 was more pronounced for p-tau following proteasome inhibition. Blocking the proteasome induced the upregulation of p62 and increased the ratio of LC3B-II to LC3B-I. NUB1 induced further upregulation of autophagy following proteasome inhibition as seen by an increase in the LC3B-II/LC3B-I ratio and changes in LC3B positive puncta in cells. Finally, GFP-tau aggresomes were positive for p62 and NUB1 overexpression promoted the interaction between GFP-tau and p62. CONCLUSIONS: These data suggest that NUB1 may play a role in the clearance of aggregation-prone tau and p-tau following inhibition of the proteasome via autophagy.