ROLE OF THE PRESYNAPTIC SCAFFOLDING PROTEINS BASSOON AND PICCOLO IN REGULATION OF PRESYNAPTIC CALCIUM HOMEOSTASIS AND NEUROTRANSMITTER RELEASE

Synaptic transmission involves neurotransmitter release following action potential arrival at the synaptic terminal. This process is critically dependent on the regulation of presynaptic calcium homeostasis and the precise localization of voltage-gated calcium channels. Bassoon (Bsn) and Piccolo (Pclo) are large multidomain presynaptic scaffolding proteins. They are localized at the active zone (AZ) and mediate the organization of multiple AZ components that are involved in synaptic vesicle (SV) priming and fusion. We investigated the effects of Bsn and Pclo deletion on the regulation of calcium channel localization and lateral mobility, presynaptic evoked calcium influx and homeostasis, together with glutamate and GABA release in mature primary neurons from conditional Bsn and Pclo knockout (KO) mice. Utilizing live-cell imaging of cultured neurons expressing genetically-encoded sensors of vesicle recycling, calcium, glutamate and GABA we observed specific effects on calcium channel mobility, SV recycling, calcium influx and evoked neurotransmitter release upon loss of Bsn or Pclo. Quantitative immunostainings revealed changes in components of the presynaptic release machinery in Pclo and Bsn synapses. To futher assess the functional consequences of altered calcium influx, Seahorse assay was performed to examine mitochondrial activity in Pclo and Bsn KO neurons, given the role of calcium homeostasis in mitochondrial function at the synapse. Together, our findings indicate that Bsn and Pclo have distinct roles in the regulation of presynaptic calcium homeostasis and neurotransmitter release.