This R21 application is prepared in response to PA-10-138, Development of Animal Models and Related Biological Materials For Research. In this application, we propose to establish both cellular and animal models that will allow us to characterize the mechanism of FUS/TLS mutations. The FUS/TLS locus on human chromosome 16 is originally identified to encode an oncogene implicated in malignant liposarcoma and myeloid leukemia. More recent evidence shows that mutations in FUS/TLS are found in familial cases of amyotrophic lateral sclerosis (ALS). These results indicate that further investigations to the mechanisms of FUS/TLS gene will have profound impacts on many disciplines in biomedical research, including cancer biology, neurodegeneration, aging, and mental health. Although most mutations are found in the highly conserved C-terminal region of the FUS protein, the precise pathogenic mechanisms for these mutations have not been fully elucidated. Among the FUS mutations, the most common form in adult ALS patients is the R521C mutation. In addition, our recent study indicates that the P525L mutation in FUS leads to a more aggressive and rapidly progressive form of ALS in young patients. Currently, it is unclear how FUS-R521C and FUS-P525L cause neuronal degeneration, or why FUS-P525L tends to affect younger patients. Our recent data, however, indicated that the spinal motor neurons in patients with P525L mutation show mislocalization of mutant FUS proteins in neuronal cytoplasm with abnormal FUS protein aggregates that are associated with markedly disorganized intracellular organelles, including endoplasmic reticulum (ER) and mitochondria. These results lead us to hypothesize that mutant FUS proteins dominantly inhibit normal RNA and protein synthesis and thereby suppressing neuronal functions and survival. Our objectives are to further investigate the mechanisms of mutant FUS proteins in neuronal degeneration using both primary neurons and transgenic mice. Since synaptic degeneration plays a central role in neurodegenerative diseases, we believe that the establishment of these models will contribute to understanding of the mutant FUS proteins in the development and maintenance of synaptic connectivity of motor neurons within the spinal cord and at the neuromuscular junction (NMJ). We propose two specific aims to achieve these goals. In Aim 1, we will characterize the mechanisms by which FUS-R521C and FUS-P525L mutant proteins affect synaptic degeneration and cell death in primary neurons. In Aim 2, we propose to characterize if mice expressing FUS-R521C or FUS-P525L develop motor behavioral phenotype and motor neuron pathology similar to patients with corresponding mutations. We believe that the establishment of these models will have important contributions to the missions of many institutions at NIH, and fulfills the criteria of high rewards and high impacts for the R21 funding mechanism. Once available, these models will serve as novel platforms to identify new therapeutic targets for diseases caused by FUS/TLS mutations. PUBLIC HEALTH RELEVANCE: Statement of Relevance to Public Health Amyotrophic lateral sclerosis, also known as ALS or Lou Gehrig's Disease, is a neurodegenerative condition that involves motor neurons in the brain and spinal cord. Each year, approximately 6,000 new patients are diagnosed with ALS in the US, and more than 30,000 Americans are living with ALS at any given time. Recent findings show that patients with mutations in the FUS/TLS gene show prominent accumulation of mutant FUS proteins in the cytoplasm of motor neurons, raising the hypothesis that these mutant proteins may dominantly inhibit normal RNA and protein synthesis and thereby suppressing neuronal functions and survival. This application proposes to establish both cellular and transgenic mouse models, which will allow us to investigate the mechanism of neuronal cell death in familial ALS with FUS mutations and identify new therapeutic targets for this devastating disease.
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