Biochemical and Biophysical Tuning of Presynaptic Function by the Clock Protein BMAL1

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Project Summary The circadian system is an ancient mechanism which evolved in organisms to adaptively align internal state with environmental cues. It is comprised of a molecular oscillator in every cell which, through rhythmic gene expression, regulates predictable behavioral plasticity that is engendered by rhythmic synaptic function. The spatiotemporal segregation of the synapse from the soma precludes the molecular oscillator from being the mechanistic provenance for rhythmic synaptic processes and has warranted investigation for a local synaptic clock. The Lipton lab identified that BMAL1 – a core component of the circadian mechanism – is rhythmically localized to synapses where it interacts with the synaptic kinase Ca2+/calmodulin-dependent kinase (CaMKII⍺) and organizes the circadian assembly of synaptic vesicle pools. The diurnal localization of BMAL1 to the synapse is lost in a phosphoincompetent mouse model (Bmal-S42A) which also loses circadian dynamics of synaptic vesicle clusters paired with impaired learning and memory. These findings link the circadian system to the regulation of synaptic plasticity and synapse generated behavior, in a manner which is independent of the core transcriptional clock. It remains unknown how BMAL1, either through biochemical and/or biophysical mechanisms, rhythmically assembles synaptic vesicle pools in phase with circadian time. The overarching goal of this proposed work is to gain insight into how the circadian clock biochemically and biophysically assembles synaptic vesicles in a manner which regulates their circadian compartmentalization and dynamics. With two related but independent aims, this proposal investigates the biochemical interactions that BMAL1 makes with the synaptic kinase CaMKII⍺, the biomolecular condensation of that interaction and the presynaptic signals that are BMAL1-dependent for their plasticity. Aim 1 proposes to define the structural elements in the BMAL1 and CaMKII⍺ protein sequences which are required for their interactions. This will be accomplished by conducting a series of complementary in vitro assays in cells and with recombinant protein that assess interaction of these proteins and condensation of these proteins into phase separated liquid-like droplets. The effect of these biochemical and biophysical interactions on synaptic vesicles will then be evaluated using a non-neuronal system which reconstitutes synaptic vesicle-like structures in both form and function. Aim 2 will then identify the presynaptic, neuromodulator systems which require and recruit pBMAL1(S42) for their acute presynaptic plasticity control on synaptic vesicles. A screen for neuromodulators which depend on pBMAL1(S42) for synaptic vesicle control will be conducted. Signaling experiments will then be performed in cultured neurons and in vivo to determine if the neuromodulator serotonin, a key regulator of rhythms and sleep/wake, potentiates pBMAL1 (S42). Such collective knowledge gained from this proposal has the potential to uncover druggable targets for modifying the temporal organization of presynaptic plasticity in neurological syndromes which share circadian and synaptic function.