CMT AND NEUROGENIC DISEASE

Dominant mutations in the gene encoding the nucleic acid binding protein FUS cause ∼5% of familial amyotrophic lateral sclerosis (ALS). While some FUS mutants mislocalize to the cytoplasm due to disruption of a nuclear localization sequence, many remain predominantly nuclear and may exert a gain of toxicity within the nucleus. We seek to discern the mechanism(s) by which FUS mutants ultimately injure aging motor neurons and to develop effective therapeutic approaches to defend against these insults. We identified a novel and robust nuclear phenotype caused by FUS mutants: impaired stress-responsive processing of sub-nuclear assemblies known as promyelocytic leukemia (PML) nuclear bodies. PML nuclear bodies occur in many cell types (including CNS and muscle) and mediate stress-responsive regulation of nuclear protein homeostasis, transcription, DNA-damage pathways, and cellular senescence, yet their potential role in ALS has not been explored. We observed that PML bodies were abnormally enlarged both in cell lines and in human ALS fibroblasts expressing mutant FUS. Exposure to mild oxidative stress or proteasome inhibition aggravated this phenotype in FUS mutant cells. We developed a high-content imaging-based assay to screen for small molecule compounds that exacerbate or ameliorate the PML body abnormality. Using the LOPAC library of 1,280 pharmacologically active compounds, we identified a set of hits that implicate impaired cellular redox homeostasis as an important determinant of the phenotype and confirmed this using human ALS fibroblasts. We have established transgenic mice harboring ALS-linked FUS variants and observed age-dependent loss of the connection between motor nerves and muscle in mice that express the hFUS-R495X mutant. We are using a lentiviral reporter to test whether a subset of the prioritized hit compounds modulate defects of nuclear protein homeostasis in cultured primary neurons, glia, or muscle cells obtained from FUS mutant mice.