Failure of Autolysosome Fusion Results in Impaired Autophagy in UBQLN2-Linked ALS-FTD (P02.167)

OBJECTIVE: To further explore the pathogenic mechanism of UBQLN2-mediated ALS and ALS-FTD, and the effect of mutant UBQLN2 on autophagy. BACKGROUND: Mutations in UBQLN2 cause ALS and ALS-FTD. Pathological inclusions containing UBQLN2 are a common pathological feature in ALS and ALS-FTD. UBQLN2 sits at the crossroads of protein degradation through the ubiquitin-proteasome system (UPS), and bulk lysosomal degradation via autophagy. Alterations in autophagy have been proposed to contribute to pathogenesis in several neurodegenerative diseases including ALS. However, the precise mechanism behind autophagy malfunction in ALS is poorly understood. DESIGN/METHODS: To study the effect of UBQLN2 mutations on autophagy, neuro-2a cells were transiently transfected with expression vectors containing wildtype (wt) UBQLN2, P497H-UBQLN2 or P506T-UBQLN2. For flow cytometry and imaging studies, cells were co-transfected with an autophagosome marker (LC3-GFP). Fourty-eight hours post-transfection cells were collected using a BD LSRFortessa flow cytometer and analyzed using BD FACSDiva software. For imaging studies, cells were fixed 24 hours post-transfection, immunolabeled with anti-UBQLN2, anti-p62 or anti-LAMP1 antibodies and analyzed using a Zeiss LSM 510 Meta laser scanning confocal microscope. Endogenous LC3 and p62 turnover assay was performed using Western blotting according to standard protocols. RESULTS: We found that cells expressing mutant UBQLN2 accumulate autophagosomes and autophagosome precursors. Expression of mutant UBQLN2 leads to an accumulation of autophagosome-associated proteins, LC3 and p62. After autophagic induction, autophagosomes in mutant UBQLN2 expressing cells fail to mature into autolysosomes and degrade LC3 and p62. CONCLUSIONS: Our data implicate UBQLN2 in autophagy, and suggest that impaired autophagy due to the failure of autolysosome fusion is central to the pathogenesis of UBQLN2-linked ALS and ALS-FTD and may explain the pathology seen in ALS and FTD patients. Hence, autophagy represents an attractive target for designing rational therapeutics in ALS and FTD. Supported by: NIH Grants (NS050641, NS078504, T32 AG20506), Les Turner ALS Foundation, Vena E. Schaff ALS Research Fund, Harold Post Research Professorship, Herbert and Florence C. Wenske Foundation. Disclosure: Dr. Fecto has nothing to disclose. Dr. Esengul has nothing to disclose. Dr. Deng has nothing to disclose. Dr. Siddique has nothing to disclose.