A Novel Inhibitory Pathway Modulates the Fraction of Release-Competent Synaptic Vesicles

Abstract

Homeostatic synaptic plasticity (HSP) maintains circuits within a dynamic range suitable for encoding information. HSP-induced changes in neurotransmission result from chronic increases in activity and lead to compensatory decreases in vesicle release (and vice versa). One mechanism for modulating release in response to a homeostatic challenge is to shift the fractional size of resting vs. recycling vesicle pools. In addition, strong evidence indicates that cyclin-dependent kinase 5 (CDK5) is critical for determining the fraction of vesicles in the resting, non-releasable state. Although CDK5 activity strongly affects synaptic release, the specific mechanism(s) exerting this function remains unknown. Current investigations aim to identify and characterize the CDK5 effector pathway that is required for its maintenance of the resting pool of vesicles. Here we test the hypothesis that tomosyn, a soluble R-SNARE protein is this CDK5 effector. Indeed our data indicate that tomosyn can be phosphorylated by CDK5 in vitro. We next determined the functional reliance of CDK5 on the presence of tomosyn by knocking-down (KD) or overexpressing (OE) tomosyn. These experiments utilized cultured rat hippocampal neurons (15-21 DIV) expressing vGLUT1-pHluorin, an optical reporter of vesicle release. Tomosyn KD increased mobilization of vesicles into recycling (active) pools (68% vs. 51% of totally releasable pool (TRP) in control); and, conversely, tomosyn OE resulted in a decrease in the recycling pool (36% of TRP). Pharmacological inhibition of CDK5 via roscovitine (100μM, 30m) mirrored the tomosyn KD results wherein a significantly greater fraction of vesicles appeared in a releasable state (70% of TRP). Importantly, combination of the two perturbations (tomosyn KD + roscovitine) did not result in an additive increase in the releasable pool (72% of TRP) further demonstrating that CDK5 and tomosyn likely exert control over vesicle release through a common signaling pathway.