Impaired Activity of the Ubiquitin–Proteasome System in Transgenic Mice Expressing ALS/Dementia-Linked Mutant UBQLN2 (P02.170)

OBJECTIVE: To further explore the pathogenic mechanism of UBQLN2-mediated ALS and ALS/dementia and the effect of mutant UBQLN2 on the ubiquitin-proteasome system (UPS). BACKGROUND: The UPS may be responsible for the accumulation of potentially harmful ubiquitinated proteins in neurodegenerative disorders. In ALS, UPS dysfunction may be central to motor neuron death. Recently, we showed that mutations in UBQLN2, which encodes the ubiquitin-like protein ubiquilin 2, cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Ubiquilin 2 is a member of the ubiquilin family, which regulates the degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Therefore, our findings linked abnormalities in ubiquilin 2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, indicating a common pathogenic mechanism. DESIGN/METHODS: We first developed transgenic mice expressing an ALS/dementia-linked UBQLN2 mutation (P497H) using the human genomic DNA transgene. These mice recapitulate the behavioral and pathological characteristics of human ubiquilinopathy. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed the UBQLN2-P497H transgenic mice with transgenic mice expressing a fluorescently tagged UPS reporter substrate (Ub-G76V-GFP). In Ub-G76V-GFP/UBQLN2-P497H double-transgenic mice, we stained brain sections and performed immunohistochemisrty using antibodies generated against ubiquilin2 and ubiquitin. Western blotting was also performed to confirm our results. RESULTS: Prominent ubiquilin2-positive inclusions were observed in the hippocampus of Ub-G76V-GFP/UBQLN2-P497H double-transgenic mice. Confocal microscopy shows co-localization of ubiquilin2 with ubiquitin and Ub-G76V-GFP in these hippocampal inclusions, consistent with UPS impairment and accumulation of Ub-G76V-GFP due to inefficient reporter degradation. Accumulation of ubiquitinated proteins and Ub-G76V-GFP in these mice was also confirmed using Western blotting. CONCLUSIONS: Our data suggest that UPS impairment occurs in hippocampal neurons in UBQLN2-P497H transgenic mice and may be central to neurodegeneration observed in UBQLN2-linked ALS and ALS/dementia. 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. Gorrie has nothing to disclose. Dr. Zhai has nothing to disclose. Dr. Liu has nothing to disclose. Dr. Deng has nothing to disclose. Dr. Siddique has nothing to disclose.