Skeletal muscle contractions are initiated by an increase in Ca 2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca 2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca 2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca 2+ in internal stores, which is consistent with increased SR luminal Ca 2+ . These findings define critical roles for Stac3 in EC coupling and human disease.zebrafish | skeletal muscle | excitation-contraction coupling | dihydropyridine receptor | Native American myopathy

The genetic, molecular and neuronal mechanism underlying circadian activity rhythms is well characterized in the brain of Drosophila. The small ventrolateral neurons (s-LNs) and pigment dispersing factor (PDF) expressed by them are especially important for regulating circadian locomotion. Here we describe a novel gene, Dstac, which is similar to the stac genes found in vertebrates that encode adaptor proteins, which bind and regulate L-type voltage-gated Ca channels (CaChs). We show that Dstac is coexpressed with PDF by the s-LNs and regulates circadian activity. Furthermore, the L-type CaCh, Dmca1D, appears to be expressed by the s-LNs. Since vertebrate Stac3 regulates an L-type CaCh we hypothesize that Dstac regulates Dmca1D in s-LNs and circadian activity.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder where recent evidence suggests pathogenesis may be mediated by inflammatory processes. The molecular architecture of the disease remains to be fully elucidated. We performed single-nucleus transcriptomics and unbiased proteomics using postmortem tissue obtained from the prefrontal cortex of 12 individuals with late-stage PD and age-matched controls. We analyzed ~80,000 nuclei and identified eight major cell types, including brain-resident T cells, each with distinct transcriptional changes in line with the known genetics of PD. By analyzing Lewy body pathology in the same postmortem tissue, we found that a-synuclein pathology is inversely correlated with chaperone expression in excitatory neurons. Examining cell-cell interactions, we found a selective abatement of neuron-astrocyte interactions and enhanced neuroinflammation. Proteomic analyses of the same brains identified synaptic proteins in prefrontal cortex that were preferentially downregulated in PD. Strikingly, comparing this dataset to a regionally similar published analysis for Alzheimer's disease (AD), we found no common differentially expressed genes in neurons, but identified many shared differentially expressed genes in glial cells, suggesting that disease etiology in PD and AD are likely distinct. These data are presented as a resource for interrogating the molecular and cellular basis of PD and other neurodegenerative diseases.

Contraction of skeletal muscle is initiated by excitation-contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca2+ release. EC coupling requires dihydropyridine receptors (DHPR) located at triads, which are junctions between the transverse (T) tubule and SR membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers we find that DHPR is transported along the longitudinal SR in a microtubule independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRβ, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads.

Stac3 regulates excitation-contraction coupling (EC coupling) in vertebrate skeletal muscles by regulating the L-type voltage-gated calcium channel (Ca v channel). Recently a stac -like gene, Dstac , was identified in Drosophila and found to be expressed by both a subset of neurons and muscles. Here, we show that Dstac and Dmca1D, the Drosophila L-type Ca v channel, are necessary for normal locomotion by larvae. Immunolabeling with specific antibodies against Dstac and Dmca1D found that Dstac and Dmca1D are expressed by larval body-wall muscles. Furthermore, Ca 2+ imaging of muscles of Dstac and Dmca1D deficient larvae found that Dstac and Dmca1D are required for excitation-contraction coupling. Finally, Dstac appears to be required for normal expression levels of Dmca1D in body-wall muscles. These results suggest that Dstac regulates Dmca1D during EC coupling and thus muscle contraction.