Munc13 proteins control regulated exocytosis in mast cells.

Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knock-out (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation but not with changes in MC development, distribution, numbers, or morphology. In vitro assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events (i.e. granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that although Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response.

The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity.

Natural anion channelrhodopsins (ACRs) discovered in the cryptophyte alga Guillardia theta generate large hyperpolarizing currents at membrane potentials above the Nernst equilibrium potential for Cl- and thus can be used as efficient inhibitory tools for optogenetics. We have identified and characterized new ACR homologs in different cryptophyte species, showing that all of them are anion-selective, and thus expanded this protein family to 20 functionally confirmed members. Sequence comparison of natural ACRs and engineered Cl--conducting mutants of cation channelrhodopsins (CCRs) showed radical differences in their anion selectivity filters. In particular, the Glu90 residue in channelrhodopsin 2, which needed to be mutated to a neutral or alkaline residue to confer anion selectivity to CCRs, is nevertheless conserved in all of the ACRs identified. The new ACRs showed a large variation of the amplitude, kinetics, and spectral sensitivity of their photocurrents. A notable variant, designated "ZipACR", is particularly promising for inhibitory optogenetics because of its combination of larger current amplitudes than those of previously reported ACRs and an unprecedentedly fast conductance cycle (current half-decay time 2-4 ms depending on voltage). ZipACR expressed in cultured mouse hippocampal neurons enabled precise photoinhibition of individual spikes in trains of up to 50 Hz frequency.

Neuro-Ophthalmology Cases for the Neurologist.

Neurologists should be aware of specific urgent and emergent neuro-ophthalmic conditions, including giant cell arteritis, arterial dissection, intracranial aneurysm, pituitary apoplexy, and invasive sino-orbital fungal infection (eg, mucormycosis). Early recognition and treatment can greatly impact patient morbidity and mortality, including the preservation of vision and life. Neurologists should be cognizant of the key and differentiating clinical and radiographic features for these presentations.

Ischemic stroke in patients with gliomas at The University of Texas-M.D. Anderson Cancer Center.

Patients with gliomas are at risk of cerebrovascular accidents (CVA) with potential consequences on survival, function, and local tumor control. Our objective was to provide information about CVA in patients with gliomas and to estimate survival in this group. We reviewed all adult glioma patients with ischemic CVA at the University of Texas-M.D. Anderson Cancer Center from 2003 through 2014. We extracted demographic, clinical, imaging, treatment and outcome data. We used descriptive summary data and estimated or compared survival rates where appropriate. 60 of 6500 patients (0.1%) with high-grade (HGG, n = 47) or low-grade glioma (LGG, n = 13) had ischemic CVA Thirty-two (53%) patients had postoperative strokes, and 20 (33%) had CVA after 2 weeks of surgery. Forty-one patients (68%) had gross total resection. For HGG and CVA, the poststroke median overall survival was 17 months versus 61 months in LGG and CVA (P = 0.03; hazard ratio (HR): 2.8; 95% CI 1.07-4.60). Survival stratified by modified Rankin Scale grade was significant (X(2) = 9.8, P = 0.007). Five patients received bevacizumab before stroke onset; none responded to antiangiogenic therapy. There was no stroke-related death. At our institution for 10 years, ischemic CVA in glioma patients was a rare complication, clearly associated in half of cases to surgery, and with a variable negative impact on performance status and neurologic function. In this group, patients with more neurological deficits lived less. The survival difference between and within subgroups was most likely due to tumor grade. More research is necessary to improve prevention of postoperative stroke in glioma patients.

Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells.

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.