BMAL1 regulation of CaMKII⍺: Biochemical Mechanisms for Synaptic Timekeeping

Abstract

Biological timing is essential to health. Every 24 hours, animals undergo massive, predictable behavioral plasticity (sleep/wake, fast/feed) in synchrony with light cycles. In mammals, circadian behavioral rhythms are regulated by an endogenous molecular clock made of a transcription-translation feedback loop. However, the synaptic basis for behavioral rhythms remains unclear. Considering the spatial complexity of neurons, synapses are situated far from the source of rhythmic transcript yet manage to synchronize across circuits to give rise to rhythmic behaviors and states. The idea that mechanisms which occur “locally” at the synapse has been put forth to explain this spatial-temporal enigma for rhythmic synaptic function. In support of the hypothesis that there are local synaptic mechanisms for circadian behaviors, the Lipton lab previously found that the clock protein, BMAL1, rhythmically localizes to hippocampal synapses, dependent on phosphorylation at S42. A phospho-incompetent mouse model, Bmal1-S42A, loses a rhythmic reduction of synaptic BMAL1 and displays a loss of circadian variation of long-term potentiation (LTP) at the CA3-Schaffer collateral-CA1 synapse. The synaptic BMAL1 interactome is enriched with pre-synaptic vesicle proteins as well as with CaMKII⍺, a neuronal, Ca2+-dependent kinase with roles in pre-synaptic vesicle release and post-synaptic LTP. In vitro assays with recombinant protein show that BMAL1 potentiates the autophosphorylation of CaMKII⍺ at T286. This site endows CaMKII⍺ with Ca2+-autonomy, allowing it to act as a molecular ‘memory’ of historical Ca2+ transients, potentially on circadian timescales. This suggests that the basis for aberrant synaptic rhythms in Bmal1-S42A is mis-regulation of CaMKII⍺ by BMAL1. These data formed the hypothesis that synaptic BMAL1 regulates circadian synaptic functions through direct modulation of CaMKII⍺. The aims of this thesis work have thus been to (1) identify the domains required for BMAL1-CaMKII⍺ binding and (2) characterize BMAL1 regulation of CaMKII⍺ activity as it relates to synaptic function. To probe the binding interface of BMAL1-CaMKII⍺, I performed co-immunoprecipitations of protein variants in HEK293T cells and tested for the requirement of phosphorylation sites and functional domains. With respect to BMAL1, its central structured region is sufficient and required to bind CaMKII⍺, while flanking disordered regions are modulatory but insufficient. Concerning CaMKII⍺, we find that BMAL1 binds its open conformation and that the kinase domain is sufficient. However, BMAL1 does not bind the substrate-pocket – a conserved binding site for several CaMKII⍺ substrate-activators. BMAL1 thus binds a novel CaMKII⍺ regulatory site, leaving the substrate-pocket subject to regulation. Synapsin1 (SYN1) is a pre-synaptic CaMKII⍺ substrate which interacts with synaptic vesicle (SV) membranes to stabilize the vesicle pool. SYN1 phosphorylation by CaMKII⍺ promotes SV dispersion. In Bmal1-WT synapses, we found a rhythm in SV number. In Bmal1-S42A, where rhythmic synaptic BMAL1 is lost, the SV pool is larger, arrhythmic and insensitive to evoked release. To explain these findings, in a heterologous system I tested the effect of BMAL1 on binding and phosphorylation of SYN1 by CaMKII⍺ and found that BMAL1 potentiates SYN1-CaMKII⍺ binding but inhibits SYN1 phosphorylation. These data suggest that rhythmic synaptic BMAL1 confers circadian inhibition to CaMKII⍺. If BMAL1-mediated regulation of CaMKII⍺ forms the basis of a synaptic oscillator, what comprises an opposing arm of this loop? In parallel investigations, we found evidence that BMAL1 promotes the ubiquitination of CaMKII⍺. Collaborators who study CaMKII⍺ degradation confirmed that the open state of CaMKII⍺, which BMAL1 stabilizes, is more vulnerable to ubiquitination and degradation. Taking these data together with our previously published work suggests that the rhythmic localization of BMAL1 to the synapse is required for circadian synaptic functions because it tunes CaMKII⍺ activity and protein stability in a time-of-day dependent manner. These findings support an emerging model for synaptic timekeeping whereby circadian protein-kinase regulation and degradation form a biochemical basis for rhythmic synaptic function. This model expands the scope of contributory mechanisms to cellular rhythmicity beyond rhythmic gene expression alone. Thus, in settings of co-morbid circadian disruption, neurological disease and synaptic dysfunction, targeting the synaptic BMAL1-CaMKII⍺ interaction, or other yet undiscovered molecular nodes of the synaptic clock, could be an untapped preventative and therapeutic frontier.