Determination of the mechanism underlying Thorase modulation of AMPARs mediated neurotransmission

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Project Summary/Abstract My scientific training includes protein biochemistry during my doctorate and post-doctoral training in neuroscience. My long-term career goal is to become an independent investigator focusing on mechanisms of neurologic diseases related to the AAA+ ATPase Thorase. My goal over the next three years is to acquire additional knowledge and expertise in structural biology in order to elucidate how Thorase is structured and organized in oligomeric complexes to interact with α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors (AMPARs). With the help of a team of outstanding mentors, we have developed a structured training program that includes extensive hands-on research training in protein crystallization, negative staining, and single particle cryo-electron microscopy, as well as taking formal courses, presenting at seminars, attending scientific meetings, and training in grant writing. This training program will help me achieve the goals proposed in this K01 and ultimately prepare me to be in a position to compete for R01 funding. Thorase plays a critical role in controlling synaptic plasticity, learning, and memory by modulating the expression of surface AMPARs. Mutations in Thorase have been associated with schizophrenia and in some cases, neonates expressing loss-of-function Thorase demonstrate extreme hypertonia, encephalopathy, seizures, and early death. Mice with Thorase mutations have defects in glutamatergic signaling and deficits in behavior. The abnormalities linked to defects in Thorase function can be normalized by the AMPAR antagonist, perampanel, both in patients and mice. These findings suggest that Thorase could be an important mediator of neurologic diseases, such as schizophrenia, that are linked to compromised AMPAR-mediated glutamatergic neurotransmission. How Thorase modulates AMPAR- mediated glutamatergic neurotransmission remains to be elucidated. AAA+ ATPases are known to form oligomeric complexes that are critical for their functions. The structure of Thorase and the exact number of protomers present in its oligomeric complex are currently unknown. To probe these questions, my mentors and I have designed a series of studies that will provide vital information underlying the mechanism of Thorase-mediated AMPAR trafficking/recycling, as well as other Thorase functions in neurons. To attain these goals, the research aims of the proposal are to (i) determine the different oligomeric states of Thorase using single particle cryo-EM; (ii) identify specific residues of Thorase that are critical for AMPAR trafficking; (iii) determine the crystal structure of Thorase bound to the C-terminus of the AMPAR subunit GluA2 using x-ray crystallography; and (iv) identify new interacting protein partners of Thorase oligomeric complexes. Knowing the structure of Thorase and how its oligomeric complex interacts with AMPARs will advance our understanding of how Thorase mediates changes in synaptic plasticity via AMPAR signaling. This study may also identify new potential therapeutic targets for Thorase and AMPAR-mediated neurologic disease.