Abstract: In the nervous system, protein ubiquitination subserves multiple cellular processes, utilizing an enzymatic cascade that consists of ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3). The main function of the E3 in this pathway is to covalently link a small 76 residue protein called ubiquitin on to selected protein targets known as substrates. It is estimated that E3s represent 500 – 1000 genes in the human genome. We found 83 different E3s that are mutated in 70 different types of neurological disease providing supporting evidence that disruptions in ubiquitin signaling is a precipitating factor in human neurological conditions. Although a multitude of E3s are mutated in numerous neurological disorders, there is still a paucity of knowledge regarding the function and substrates of disease- relevant E3s in the brain. In this proposal, we will elucidate key functions for two E3s, TRIAD3A and CHIP, which are mutated in a heterogenous neurological disorder called Gordon Holmes syndrome (GHS). Major pathophysiologies of GHS include hypothalamic dysfunction, dementia and neurodegeneration. Individuals with RNF216/TRIAD3 and CHIP/STUB1 mutations also exhibit hypogonadotropic hypogonadism, which is thought to be caused by a dysfunctional hypothalamic-pitiutary-gonadal (HPG) axis due to deficiencies in the release of gonadotropin- releasing hormone (GnRH) within GnRH positive neurons in the hypothalamus. Here, we hypothesize that the brain-expressed isoform of RNF216/TRIAD3, TRIAD3A and CHIP regulate a suite of substrates, whose ubiquitination gives rise to the diversity of phenotypes observed in GHS. In Aim 1, we will develop a ubiquitin substrate capture platform known as “Orthogonal Ubiquitin Transfer” to profile the substrate specificities of TRIAD3A and CHIP in hippocampal neurons. In Aim 2, we will verify the synergy between TRIAD3A and CHIP in neuron development. Taken together, our study will provide the first comprehensive overview of brain-specific TRIAD3A and CHIP substrates that alter physiological functions in select brain regions, and will identify new molecular targets to compensate for the deficiency of these two enzymes in GHS patients. More broadly, our work will provide a mechanistic perspective of how malfunctions of E3s cause neurological diseases. By identifying common substrate profiles for TRIAD3A and CHIP, we will be able to reveal key mechanistic deficiencies associated with GHS, which may ultimately be effective in guiding the treatment of GHS and similar neurodegenerative diseases.