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Sleep circuitry evolved to have both dedicated and context-dependent modulatory elements. Identifying modulatory subcircuits and understanding their molecular machinery is a major challenge for the sleep field. Previously, we identified 25 sleep-regulating microRNAs in

A null mutation of theDrosophila calcium/calmodulin-dependent protein kinase IIgene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution ofCaMKIImRNA, which consists of a short 3′-untranslated region (UTR) form lacking regulatory elements that guide local translation. The selective loss of the long 3′UTR mRNA inCaMKII-null larvae allows us to test its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower inCaMKIImutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non–sex-specific and rescued by a fosmid containing the entire wild-typeCaMKIIlocus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3′UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3′UTR form ofCaMKIImRNA and a process that requires zygotic transcription ofCaMKIImRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release, which cannot be fulfilled by synaptic CaMKII from the other pool.SIGNIFICANCE STATEMENTAs a regulator of synaptic development and plasticity, CaMKII has important roles in both normal and pathological function of the nervous system.CaMKIIshows high conservation betweenDrosophilaand humans, underscoring the usefulness ofDrosophilain modeling its function.Drosophila CaMKII-null mutants remain viable throughout development, enabling morphological and electrophysiological characterization. Although the structure of the synapse is normal, maternally contributed CaMKII does not localize to synapses. Zygotic production ofCaMKIImRNA with a long 3′-untranslated region is necessary for modulating spontaneous neurotransmission in an activity-dependent manner, but not for viability. These data argue that regulation of CaMKII localization and levels by local transcriptional processes is conserved. This is the first demonstration of distinct functions forDrosophila CaMKIImRNA variants.

Drosophila ether-à-go-go (eag) is the founding member of a large family of voltage-gated K+channels, the KCNH family, which includes Kv10, 11 and 12, (Ganetzky et al. 1999). Concurrent binding of calcium/calmodulin (Ca2+/CaM) to N-and C-terminal sites inhibits mammalian EAG1 channels at sub-micromolar Ca2+concentrations (Schonherr et al. 2000), likely by causing pore constriction (Whicher and MacKinnon 2016). Although theDrosophilaEAG channel was believed to be Ca2+-insensitive (Schonherr et al. 2000), both the N-and C-terminal sites are conserved. Here we show thatDrosophilaEAG is inhibited by high Ca2+concentrations that are only present at plasma membrane Ca2+channel microdomains. To test the role of this regulationin vivo, we engineered mutations that block CaM-binding to the major C-terminal site of the endogenouseaglocus, disrupting Ca2+-dependent inhibition.eagCaMBD mutants have reduced evoked release from larval motor neuron presynaptic terminals and show decreased Ca2+influx in stimulated adult projection neuron presynaptic terminals, consistent with an increase in K+conductance. These results are predicted by a conductance-based multi-compartment model of the presynaptic terminal in which some fraction of EAG is localized to the Ca2+channel microdomains that control neurotransmitter release. The reduction of release in the larval neuromuscular junction drives a compensatory increase in motor neuron somatic excitability. This misregulation of synaptic and somatic excitability has consequences for systems-level processes and leads to defects in associative memory formation in adults.New and NoteworthyRegulation of excitability is critical to tuning the nervous system for complex behaviors. We demonstrate here that the EAG family of voltage-gated K+channels exhibit conserved gating by Ca2+/CaM. Disruption of this inhibition inDrosophilaresults in decreased evoked neurotransmitter release due to truncated Ca2+influx in presynaptic terminals. In adults, disrupted Ca2+dynamics cripples memory formation. These data demonstrate that the biophysical details of channels have important implications for cell function and behavior.

A null mutation of the Drosophila calcium/calmodulin-dependent protein kinase II gene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution of CaMKII mRNA, which consists of a short 3′-untranslated region (UTR) form lacking regulatory elements that guide local translation. The selective loss of the long 3′UTR mRNA in CaMKII-null larvae allows us to test its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower in CaMKII mutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non–sex-specific and rescued by a fosmid containing the entire wild-type CaMKII locus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3′UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3′UTR form of CaMKII mRNA and a process that requires zygotic transcription of CaMKII mRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release, which cannot be fulfilled by synaptic CaMKII from the other pool.

Genetic causes for disturbances of locomotor behavior can be due to muscle, peripheral neuron, or central nervous system pathologies. The Drosophila melanogaster homolog of human CASK (also known as caki or camguk) is a molecular scaffold that has been postulated to have roles in both locomotion and plasticity. These conclusions are based on studies using overlapping deficiencies that largely eliminate the entire CASK locus, but contain additional chromosomal aberrations as well. More importantly, analysis of the sequenced Drosophila genome suggests the existence of multiple protein variants from the CASK locus, further complicating the interpretation of experiments using deficiency strains. In this study, we generated small deletions within the CASK gene that eliminate gene products containing the CaMK-like and L27 domains (CASK-β), but do not affect transcripts encoding the smaller forms (CASK-α), which are structurally homologous to vertebrate MPP1. These mutants have normal olfactory habituation, but exhibit a striking array of locomotor problems that includes both initiation and motor maintenance defects. Previous studies had suggested that presynaptic release defects at the neuromuscular junction in the multigene deficiency strain were the likely basis of its locomotor phenotype. The locomotor phenotype of the CASK-β mutant, however, cannot be rescued by expression of a CASK-β transgene in motor neurons. Expression in a subset of central neurons that does not include the ellipsoid body, a well-known pre-motor neuropil, provides complete rescue. Full-length CASK-β, while widely expressed in the nervous system, appears to have a unique role within central circuits that control motor output.

CASK is an evolutionarily conserved scaffolding protein that has roles in many cell types. In Drosophila, loss of the entire CASK gene or just the CASK- transcript causes a complex set of adult locomotor defects. In this study, we show that the motor initiation component of this phenotype is due to loss of CASK- in dopaminergic neurons and can be specifically rescued by expression of CASK- within this subset of neurons. Functional imaging demonstrates that mutation of CASK- disrupts coupling of neuronal activity to vesicle fusion. Consistent with this, locomotor initiation can be rescued by artificially driving activity in dopaminergic neurons. The molecular mechanism underlying this role of CASK- in dopaminergic neurons involves interaction with Hsc70-4, a molecular chaperone previously shown to regulate calcium-dependent vesicle fusion. These data suggest that there is a novel CASK- -dependent regulatory complex in dopaminergic neurons that serves to link activity and neurotransmitter release. Fil: Slawson, Justin B. Brandeis University; Estados Unidos Fil: Kuklin, Elena A. Brandeis University; Estados Unidos Fil: Mukherjee, Konark. Brandeis University; Estados Unidos Fil: Pírez, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Brandeis University; Estados Unidos Fil: Donelson, Nathan C. Brandeis University; Estados Unidos Fil: Griffith, Leslie C. Brandeis University; Estados Unidos