sirolimus and Frontotemporal-Lobar-Degeneration

Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes.

Topics: Adaptor Proteins, Signal Transducing; Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Frontotemporal Lobar Degeneration; Gene Knockdown Techniques; Locomotion; Phenotype; Sequestosome-1 Protein; Sirolimus; TOR Serine-Threonine Kinases; Zebrafish; Zebrafish Proteins

Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, have been associated with familial amyotrophic lateral sclerosis (fALS), which is a fatal neurodegenerative disease that causes progressive muscular weakness and has overlapping clinical and pathologic characteristics with frontotemporal lobar degeneration. However, the role of autophagy in regulation of FUS-positive stress granules (SGs) and aggregates remains unclear. We found that the ALS-linked FUS(R521C) mutation causes accumulation of FUS-positive SGs under oxidative stress, leading to a disruption in the release of FUS from SGs in cultured neurons. Autophagy controls the quality of proteins or organelles; therefore, we checked whether autophagy regulates FUS(R521C)-positive SGs. Interestingly, FUS(R521C)-positive SGs were colocalized to RFP-LC3-positive autophagosomes. Furthermore, FUS-positive SGs accumulated in atg5(-/-) mouse embryonic fibroblasts (MEFs) and in autophagy-deficient neurons. However, FUS(R521C) expression did not significantly impair autophagic degradation. Moreover, autophagy activation with rapamycin reduced the accumulation of FUS-positive SGs in an autophagy-dependent manner. Rapamycin further reduced neurite fragmentation and cell death in neurons expressing mutant FUS under oxidative stress. Overall, we provide a novel pathogenic mechanism of ALS associated with a FUS mutation under oxidative stress, as well as therapeutic insight regarding FUS pathology associated with excessive SGs.

Topics: Amyotrophic Lateral Sclerosis; Animals; Autophagy; Cells, Cultured; Cytoplasmic Granules; Female; Frontotemporal Lobar Degeneration; Gene Expression Regulation; Genetic Association Studies; Humans; Male; Mice; Mutation; Neurons; Oxidative Stress; RNA-Binding Protein FUS; Sirolimus