Phosphorylation of Synapsin I by Cyclin-Dependent Kinase-5 Sets the Ratio between the Resting and Recycling Pools of Synaptic Vesicles at Hippocampal Synapses

Abstract

Cyclin-dependentkinase-5(Cdk5)wasreportedtodownscaleneurotransmissionbysequesteringsynapticvesicles(SVs)intherelease-reluctant resting pool, but the molecular targets mediating this activity remain unknown. Synapsin I (SynI), a major SV phosphoprotein involved in the regulation of SV trafficking and neurotransmitter release, is one of the presynaptic substrates of Cdk5, which phosphorylates it in its C-terminal region at Ser 549 (site 6) and Ser (site 7). Here we demonstrate that Cdk5 phosphorylation of SynI fine tunes the recruitment of SVs to the active recycling pool and contributes to the Cdk5-mediated homeostatic responses. Phosphorylation of SynI by Cdk5 is physiologically regulated and enhances its binding to F-actin. The effects of Cdk5 inhibition on the size and depletion kinetics of the recycling pool, as well as on SV distribution within the nerve terminal, are virtually abolished in mouse SynI knock-out (KO) neurons or in KO neurons expressing the dephosphomimeticSynI mutants at sites 6,7 or site 7 only. The observation that the single site-7 mutant phenocopies the effects of the deletion of SynI identifies this site as the central switch in mediating the synaptic effects of Cdk5 and demonstrates that SynI is necessary and sufficient for achieving the effects of the kinase on SV trafficking. The phosphorylation state of SynI by Cdk5 at site 7 is regulated during chronic modification of neuronal activity and is an essential downstream effector for the Cdk5-mediated homeostatic scaling.

TARANIS Functions with Cyclin A and Cdk1 in a Novel Arousal Center to Control Sleep in Drosophila

“…Interestingly, large-scale screens for short-sleeping mutants in fruit flies and zebrafish have identified several channel proteins such as SHAKER, REDEYE, and ETHER-A-GO-GO [6, 49, 50], and channel modulators such as SLEEPLESS and WIDE AWAKE [51, 52]. Thus it is plausible that Cdk1 regulates sleep by phosphorylating substrates that modulate the function of synaptic ion channels or proteins involved in synaptic vesicle fusion, as has previously been demonstrated for Cdk5 at mammalian synapses [53]. …”

Synaptic Vesicle-Recycling Machinery Components as Potential Therapeutic Targets

“…Small-molecule screens for modulators of synapsin I phosphorylation in primary neurons have already identified more potential compounds (Chan et al, 2014). Cyclin-dependent kinase-5 phosphorylation of synapsin I regulates the resting and recycling pools of synaptic vesicles in an activity-dependent manner and may be another pathway for pharmacological intervention (Verstegen et al, 2014). Four protein kinase inhibitors (staurosporine, quercetagetin, roscovitin, 70159800251) were found to compete against ATP for binding to the ATP binding site of synapsin I, highlighting one more potential mechanism to alter synapsin-mediated activity for therapeutic advantage (Defranchi et al, 2010).…”

Neuronal hyperactivity causes Na+/H+ exchanger-induced extracellular acidification at active synapses

“…Primary neurons were infected with ex.E²GFP alone or co-infected with mCherry-syn-I (Verstegen et al, 2014) at 10 DIV at 10 multiplicity of infection (MOI), and the experiments were performed 7-8 days post-infection (17-18 DIV). For multi-electrode array experiments, neurons were infected at 10 DIV, and recordings performed at 17-18 DIV.…”