Rapid excitatory transmission at glutamatergic synapses occurs through AMPA-type glutamate receptors (AMPAR), whose abundance within the postsynaptic density (PSD) is a critical determinant of synaptic strength. Synaptic AMPAR levels are regulated by membrane trafficking events, which are critical for proper development and maintenance of neuronal circuits. Internalization and downregulation of postsynaptic receptors is thought to occur at the endocytic zone (EZ), a stably positioned clathrin-coated pit located adjacent to the PSD;however, the functional significance of this spatial positioning remains unclear. Close positioning of the EZ to the PSD could allow for rapid control of endocytosis in response to synaptic activity and enable continuous capture and recycling of AMPARs lost from the synapse by lateral diffusion. In addition, coupling the EZ to the PSD could be important for various forms of synaptic plasticity. Our preliminary data indicate that the EZ is coupled to the PSD by dynamin-3, which exists in a complex with the endocytic GTPase dynamin-2 and the PSD protein homer. Importantly, we found that expression of dynamin-3-P800L, which lacks the homer binding site, or RNAi knockdown of dynamin-3, leads to uncoupling of the EZ from the PSD, but has no effect on clathrin assembly in the dendritic shaft. Thus, cells expressing the dynamin-3 P800L mutant or dynamin-3 shRNA contain many spines lacking an endocytic zone, thereby allowing us to analyze the functions of local spine endocytosis and spatial positioning between the EZ and PSD. The objectives of this research proposal are to (1) determine whether endocytic cycling within spines maintains control over AMPA receptor abundance and activity, (2) test if spine maturation and synapse maintenance require the continued presence of a local endocytic zone, and (3) determine if activity-induced upregulation of homer-1a leads to loss of spine EZs and whether local spine endocytosis contributes to homeostatic scaling. These experiments will provide a greater understanding of how spatial coupling between the EZ and PSD controls synaptic activity and plasticity and will also uncover how protein complexes and scaffolds within dendritic spines are spatially positioned and cooperate to establish functional neuronal circuits.Proper targeting and delivery of neurotransmitter receptors, ion channels, and adhesion molecules is critical for neuronal development and plasticity. Deficiencies in trafficking of these cargoes have been implicated in conditions such as epilepsy, Alzheimer's, Huntington's, and Parkinson's disease. These experiments will provide important insight into how perturbation of protein trafficking leads to neurological disease.
|Newpher, Thomas M; Ehlers, Michael D (2009) Spine microdomains for postsynaptic signaling and plasticity. Trends Cell Biol 19:218-27|
|Newpher, Thomas M; Ehlers, Michael D (2008) Glutamate receptor dynamics in dendritic microdomains. Neuron 58:472-97|