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Abstract
Synapse remodeling is an important mechanism for activity-dependent changes in neuronal transmission. However, the link between activity and resulting synaptic changes is not well understood. In this dissertation, I have studied the roles of Polo-like kinase 2 (Plk2) in this process. Plk2 is an activity-inducible kinase important for homeostatic downregulation of excitatory synapse number and strength following chronic overactivity. The central discovery of this thesis was the identification of a direct interaction between Plk2 and NSF, an ATPase involved in the stabilization of AMPARs at the plasma membrane surface.
In this thesis, using molecular biology and biochemistry techniques, I show that Plk2 expression was upregulated with chronic overexcitation, and became targeted to NSF. Plk2 engagement of NSF profoundly disrupted the interaction of NSF with GluR2, promoting extensive loss of surface GluR2 expression via increased endocytosis and slowed recycling without accompanying synapse loss. Furthermore, no dramatic effect was observed on surface GluR1 receptors, suggesting a major shift in subunit composition to GluR2-lacking AMPARs.
Molecular dissection of the NSF-Plk2 interaction revealed that the NSF-Plk2 interaction occurred through a novel binding site on Plk2 independent of the conserved “polo box” motif required for all known polo kinase interactions. This finding extends the repertoire of possible regulatory functions for polo family members.
Further, this work involved examining the functional effects of Plk2 binding NSF using electrophysiology and I report that the NSF-interaction domain of Plk2 was sufficient to cause reduced AMPA receptor signaling. Thus, Plk2 binding NSF alone results in significantly decreased synaptic strength.
Finally, I also studied the function of Plk3, a highly related Plk2-family member. Although Plk3 was capable of binding NSF and causing dissociation of NSF and GluR2 when expressed exogenously, Plk3 did not play a physiological role through the mechanism defined here in response to overexcitation.
These data support a model in which heightened synaptic activity leads to decreased cell surface AMPAR expression via the direct dissociation of NSF from GluR2 by Plk2. Thus, my dissertation work supports a model whereby Plk2 binding NSF serves to directly link synaptic activity and subsequent changes in synaptic strength and subunit composition.
