G.O.20: AAV-mediated overexpression of Ubiquilin2 mimics ALS and ALS with dementia in naive mice

Abstract

Mutations in Ubiquilin2 (UBQLN2) gene have been identified in familial forms of Amyotrophic lateral sclerosis (fALS) and ALS/dementia. Ubiquilin2 pathology was also described in both non-UBQLN2 fALS and in sporadic ALS cases. Ubiquilin2 is involved in degradation of ubiquitinated proteins, and functional studies have shown the formation of abnormal protein aggregation in Ubiquilin2 overexpressing cells. According to the evidence suggesting a toxic gain of function of the mutated Ubiquilin2, we set up a new animal model of UBQLN2-ALS by AAV vector expression of mutant or wild type UBQLN2 in the central nervous system of naive mice. Recombinant AAV10 carrying the human mutant (Pro497His) or wild-type Ubiquilin2 cDNA under control of the phosphoglycerate-kinase (PGK) promoter, were delivered through intracerebroventricular injection in newborn FVB mice. An AAV10 encoding the green fluorescent protein (GFP), injected under the same conditions, was used as control. The expression of human Ubiquilin2 in brain and spinal cord extracts of infected animals was evidenced by western blot, one month after injection. Immunofluorescence analyses demonstrated the presence of Ubiquilin2-positive inclusions in the brain and the spinal cord of mutant Ubiquilin2 overexpressing animals, similarly to ALS/dementia patients. The injected animals displayed a reduced brain size compared to controls, a severe astrogliosis in both the spinal cord and the brain, and a reduced number of ChAT + motor neurons in the whole spinal cord. Ubiquilin2-injected mice had a shortened life span and a body weight loss phenotype; they developed muscle weakness, with loss of muscle mass and strength. Finally, to monitor anxiety, one of the first sign of dementia, mice were subjected to the light/dark box test, which revealed a strong anxious phenotype in Ubiquilin2-injected mice. This model will be useful to further dissect the molecular mechanisms of this complex pathology and to find new therapeutic targets.