We will characterize mechanisms through which TrkB regulates inhibitory synapse formation and maintenance in the cerebellum with the expectation that the insights obtained will prove important for understanding the role of TrkB in many regions of the brain. Extending our prior demonstrations that TrkB controls inhibitory synapse formation throughout the cerebellum and has important pre- and postsynaptic cell- autonomous roles, we will use high resolution stochastic optical reconstruction microscopy (STORM) imaging to characterize the localizations of inhibitory synapse-associated cell surface and synaptic scaffold proteins and determine the effects of TrkB activation and inhibition on their presence at the synapse. Extending our recent observation that adult TrkB activity is required to maintain inhibitory synapses and that reactivation of TrkB signaling after earlier inhibition results in reappearance at the synapse of many synaptic proteins, we will examine the effects of adult TrkB inhibition and reactivation on the molecular composition of these synapses. Using cell culture we will examine in more detail the appearance and disappearance of proteins associated with the synapse following TrkB activation and inactivation. We shall determine whether TrkB functions in part through control of protein synthesis or turnover. We shall also examine the effects of TrkB activity on the kinetics of gephyrin stability, insertion and removal a synaptic sites in cell culture. Finally, we more critically examine our model that TrkB acts in par through control molecular assembly of the proteins that form the synaptic scaffold. We will determine the effects of TrkB activation and inactivation in vivo and in vitro on the distribution f gephyrin and other postsynaptic scaffold proteins in detergent soluble and resistant fractions, the interactions of these proteins with binding partners and on phosphorylation and other post-translational modifications.
This application will characterize mechanisms through which a neurotrophic factor implicated in neuronal survival named brain-derived neurotrophic factor (BDNF) signaling through a receptor tyrosine kinase named (TrkB) regulate synapse formation and maintenance in the cerebellum, a brain region that controls motor performance and coordination, such as walking and running. While the proposed studies focus on the cerebellum, the mechanistic insights are likely to be useful in understanding the function of this protein and its receptor in many regions of the brain, including those involved in memory, mood, and addictive behaviors. Human genetic studies have implicated BDNF in schizophrenia and other mental disorders. Rare human mutations in TrkB result in severe mental retardation, obesity, and cardiac disorders. The major aim of this proposal is to understand the mechanisms through which these genes control synapse formation and neuronal circuit function within the brain with the expectation that discoveries will identify novel targets for drugs to alleviate these devastatig disorders as well as increasing our knowledge of how the brain works in health and disease.