Functional analysis of pathogenic mutations in Neurofibromatosis Type-1 (NF1)

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Neurofibromatosis type-1 (NF1) is an inherited neurological disorder affecting about 1 in 3000 people. Mutations in the NF1 gene underlie this disease, with patients’ symptoms including pigmentation defects, cognitive deficits, skeletal and vascular abnormalities, and neurofibromas - benign tumors associated with peripheral nerves. NF1 patients also frequently develop malignant tumors, including peripheral nerve sheath tumors. Further, the NF1 gene has been identified among the most frequently mutated tumor suppressor genes in a number of other cancers including glioblastoma and lung adenocarcinoma. The NF1 gene encodes a large protein, called neurofibromin, the central portion of which (termed the GAP domain) negatively regulates Ras, an important cell signaling protein. NF1 mutations often result in loss of neurofibromin or impair its GAP domain, resulting in elevated Ras signaling. However, many NF1 pathogenic missense mutations have been identified that are predicted to affect regions of neurofibromin distinct from the GAP domain. This suggests that other parts of the highly conserved protein are also essential for its function. We hypothesize that amino acid substitutions outside of the GAP domain may disrupt important protein interactions or subcellular localization of neurofibromin, which could perturb its activity and potentially contribute to the varied clinical symptoms of NF1. This hypothesis will be addressed using a fruit fly (Drosophila) model of NF1 to investigate the molecular and cellular consequences of NF1 missense mutations. Specific Aim 1: We will use Drosophila to rapidly assess the residual function of fly NF1 containing the corresponding mutations from NF1 patients. We will determine if NF1 mutations negatively affect cell signaling and localization within neurons. In addition, mutants will be tested in functional assays including their ability to rescue the growth and behavioral defects of NF1 mutant flies. This will enable us to correlate cellular and molecular phenotypes to specific mutations that disrupt different regions of neurofibromin. Specific Aim 2: We have recently conducted proteomic studies in Drosophila to identify proteins that associate with neurofibromin in neurons. These studies give possible new clues as to the function of neurofibromin in neurons. We will confirm that these putative interactors exist in protein complexes with neurofibromin and explore the functional significance using genetics studies, as well as biochemical and cell fractionation experiments. We anticipate that these studies involving rapid functional testing in an in vivo Drosophila model of NF1 will allow us to establish genotype-phenotype relationships for a number of patient-derived NF1 mutations, as well as further define the functional role of neurofibromin in neurons by investigating novel protein interactors. This knowledge will help prioritize NF1 mutations for further analysis in human cells in the discovery of new biomarkers and therapeutic targets for NF1.