Characteristics of FUS-Mediated Toxicity in Amyotrophic Lateral Sclerosis
Abstract
Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous disease with many etiologies; both environmental and genetic factors are thought to play a significant role. Previous ALS research has focused heavily on the potential role of mutations in superoxide dismutase 1 (SOD1). Studies primarily done in models of SOD1-ALS have identified several pathogenic mechanisms as potential therapeutic targets. However, preclinical and clinical trials targeting these mechanisms have by and large failed. The complexity of the disease may play a big factor in these failures; therefore, the premise for this work is to investigate alternative therapeutic targets for ALS based on patient-specific genotypes and/or phenotypes. This thesis characterizes a form of ALS caused by mutations in FUSed in Liposarcoma (FUS) in order to identify potentially converging and/or unique pathogenic mechanisms among ALS subtypes. Initial studies characterizing mutant FUS in astrocytes revealed that expression of this mutant protein recapitulated key pathology observed in ALS patients, and it did not overtly kill astrocytes. Like mutant SOD1, mutant FUS exerted non-cell autonomous toxicity on wild type motor neurons. Mutant FUS toxicity was mediated through the inflammatory factor TNF&agr;, and neutralization of this soluble factor rescued toxicity. Although astrocytes did not appear to die as a direct result of mutant FUS expression, significant transcriptome changes were observed using microarray analysis. These findings prompted analysis of neural/glial antigen 2 (NG2) positive cells and revealed that in purified populations of astrocytes, counts of glial fibrillary acidic protein (GFAP) positive cells decreased while NG2+ cells increased. This was accompanied by an increase in NG2 levels identified by western blot. In order to then examine cell-intrinsic mechanisms of FUS toxicity, an in vitro model of the mammalian neuromuscular junction was established by co-culturing motor neurons expressing mutant FUS with differentiated myotubes. These studies identified two key targets of mutant FUS at the presynaptic neuronal terminal, SV2 and synaptophysin. Knock down of wild type FUS led to reductions of mRNA and protein of these synaptic vesicle proteins, while expression of mutant FUS led to reductions of protein levels only. These changes decreased the functional ability of mutant FUS neurons to exocytose, revealing a novel role for wild type FUS, and uncovering potential motor neuron–specific therapeutic targets in FUS-ALS. Intrinsic cellular toxicity was also probed by analysis of mitochondrial bioenergetics, which showed a significant decrease in the mitochondrial membrane potential in mutant FUS–expressing neuron-like cells. Lastly, studies targeting netrin-1, a soluble factor shown to induce toxicity in SOD1-mediated ALS, showed that rescue of FUS-astrocytic toxicity could not be rescued through netrin-1 neutralization. Collectively, these data shed light on potential new pathogenic pathways underlying FUS-ALS and contribute to a better understanding of FUS-mediated toxicity in ALS. Future studies will be necessary to further explore these pathways with the ultimate goal of developing improved treatment strategies aimed at these potential new therapeutic targets.
Subject Area
Molecular biology|Neurosciences|Toxicology|Surgery
Recommended Citation
Kia, Azadeh Tehrani, "Characteristics of FUS-Mediated Toxicity in Amyotrophic Lateral Sclerosis" (2014). ProQuest ETD Collection - Thomas Jefferson University. AAI3705077.
https://jdc.jefferson.edu/dissertations/AAI3705077
