CK2 Phosphorylation Is Required for Regulation of Syntaxin 1A Activity in Ca2+-Triggered Release in Neuroendocrine Cells

The polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin1A (Syx), was previously shown by us to act as a fusion clamp in PC12 cells, as charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release. Using a Syx-based FRET probe (CSYS), we demonstrated that 5RK is required for a depolarization-induced Ca+2-dependent opening (close-to-open transition; CDO) of Syx, which involves the vesicular SNARE synaptobrevin2 and occurs concomitantly with Ca2+-triggered release. Here, we investigated the mechanism underlying the CDO requirement for 5RK and identified phosphorylation of Syx at Ser-14 (S14) by casein kinase 2 (CK2) as a crucial molecular determinant. Thus, following biochemical verification that both endogenous Syx and CSYS are constitutively S14 phosphorylated in PC12 cells, dynamic FRET analysis of phospho-null and phospho-mimetic mutants of CSYS and the use of a CK2 inhibitor revealed that the S14 phosphorylation confers the CDO requirement for 5RK. In accord, amperometric analysis of catecholamine release revealed that the phospho-null mutant does not support Ca2+-triggered release. These results identify a functionally important CK2 phosphorylation of Syx that is required for the 5RK-regulation of CDO and for concomitant Ca2+-triggered release. Further, also spontaneous release, conferred by charge neutralization of 5RK, was abolished in the phospho-null mutant.

Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release, however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release. In non-neuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of non-photobleached probes within 10-seconds. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

Nasal epithelium: new insights and differences of the cytokine profile between normal subjects and subjects with allergic rhinitis

Background: The role of the nasal epithelium in the induction of a proper cytokine response in normal subjects and subjects with allergic rhinitis is still not completely elucidated. Methodology: We aimed to compare nasal epithelial immune responses in allergic rhinitis patients of different ages compared to healthy volunteers. Primary nasal epithelial cells from 47 subjects (33 normal and 17 with allergic rhinitis) were collected and cultured. Their unstimulated supernatants were analysed for 21 cytokines and chemokines. Statistical analysis was performed with the R statistical software and the RStudio interface. Results: Differences of the spontaneous release of epithelial cytokines and chemokines were noticed between the two study groups. The levels of GMCSF, MIP1A, MIP1B, IL28A, TNFA, CCL5 were significantly lower in the allergic rhinitis group compared to healthy volunteers’ group, independent of age. Most differences were noticed in the younger allergic rhinitis group (0-12 years old). Conclusions: Despite the cross-sectional nature of the study and the limited number of subjects, allergic rhinitis appears to be associated with dysfunction of cytokine and chemokine spontaneous release from nasal epithelial cells which may represent an abnormal innate immunity maturation pattern.

Swept-Source Optical Coherence Tomography Angiography in Vitreomacular Traction Syndrome

Purpose: A swept-source optical coherence tomography angiography (SS-OCTA) analysis of vasculature in vitreomacular traction (VMT) before and after surgery as well as 15 months’ “watchful waiting” follow-up data. Methods: A retrospective analysis of 38 eyes. Patients were divided into group 1: untreated (20 eyes); group 2: untreated, spontaneous release of traction (4 eyes); and group 3: vitrectomy (14 eyes). Results: In all cases, SS-OCTA of the choriocapillaris revealed a hyporeflective area, which disappeared after traction release. In group 1, none of the analyzed factors significantly changed. In group 2, visual acuity (VA) improved from 0.3 logMAR to 0.1 logMAR. None of the following parameters significantly changed: central choroidal thickness, superficial fovea avascular zone (sFAZ), deep fovea avascular zone (dFAZ), and vessel densities. In 1 eye a lamellar macular hole formed. Factors increasing the chances of spontaneous release of traction were width of traction and central retinal thickness ( P < .05). In group 3, VA improved from 0.27 Snellen (0.6 logMAR) to 0.44 Snellen (0.4 logMAR) ( P < .05). Postoperative OCTA revealed significant decreases in central retinal thickness ( P < .001), the parameters sFAZ, and dFAZ ( P < .05). Conclusions: sFAZ and dFAZ decreased after vitrectomy but not after spontaneous release of traction. VA was better in eyes with spontaneous release of traction. The degree of improvement in VA was greater in the vitrectomy group. In all cases a hyporeflective area is visible in the choriocapillaris layer in SS-OCTA. It disappears when traction is released. Early treatment, at least in patients with lower VA, might be beneficial.

DYNAMICS OF NEUROMUSCULAR TRANSMISSION REPRODUCED BY CALCIUM-DEPENDENT SERIAL TRANSITIONS IN THE VESICLE FUSION COMPLEX

Neuromuscular transmission, from spontaneous release to facilitation and depression was accurately reproduced by a mechanistic kinetic model of sequential maturation transitions in the molecular fusion complex. The model incorporates three predictions. First, sequential calcium-dependent forward transitions take vesicles from docked to pre-primed to primed states, followed by fusion. Second, pre-priming and priming are reversible. Third, fusion and recycling are unidirectional. The model was fed with experimental data from previous studies while the backward (β) and recycling (ρ) rate constant values were fitted. Classical experiments were successfully reproduced when every forward (α) rate constant had the same value, and both backward rate constants were 50-100 times larger. Such disproportion originated an abruptly decreasing gradient of resting vesicles from docked to primed states. Simulations also predict that: i. Spontaneous release reflects primed to fusion spontaneous transitions. ii. Calcium elevations synchronize the series of forward transitions that lead to fusion. iii Facilitation reflects a transient increase of priming following calcium-dependent transitions. iv. Backward transitions and recycling restore the resting state. v. Depression reflects backward transitions and slow recycling after intense release. Such finely-tuned kinetics offers a mechanism for collective non-linear transitional adaptations of a homogeneous vesicle pool to an ever-changing pattern of electrical activity.

Recurrent and Spontaneous Release of Epiretinal Membrane in a Toddler

We report a case of an epiretinal membrane (ERM) in a 3-year-old girl, which was accidently discovered after a strabismus surgery. The ERM occurred twice in 2 years and spontaneously released within 3 months, which has not been previously reported.

Synaptotagmin-7–mediated activation of spontaneous NMDAR currents is disrupted in bipolar disorder susceptibility variants

Synaptotagmin-7 (Syt7) plays direct or redundant Ca2+ sensor roles in multiple forms of vesicle exocytosis in synapses. Here, we show that Syt7 is a redundant Ca2+ sensor with Syt1/Doc2 to drive spontaneous glutamate release, which functions uniquely to activate the postsynaptic GluN2B-containing NMDARs that significantly contribute to mental illness. In mouse hippocampal neurons lacking Syt1/Doc2, Syt7 inactivation largely diminishes spontaneous release. Using 2 approaches, including measuring Ca2+ dose response and substituting extracellular Ca2+ with Sr2+, we detect that Syt7 directly triggers spontaneous release via its Ca2+ binding motif to activate GluN2B-NMDARs. Furthermore, modifying the localization of Syt7 in the active zone still allows Syt7 to drive spontaneous release, but the GluN2B-NMDAR activity is abolished. Finally, Syt7 SNPs identified in bipolar disorder patients destroy the function of Syt7 in spontaneous release in patient iPSC-derived and mouse hippocampal neurons. Therefore, Syt7 could contribute to neuropsychiatric disorders through driving spontaneous glutamate release.

Air-stable 18-electron adducts of Schrock catalysts with tuned stability constants for spontaneous release of the active species

Schrock alkylidenes are highly versatile, very active olefin metathesis catalysts, but their pronounced sensitivity to air still hinders their applications. Converting them into more robust but inactive 18-electron adducts was suggested previously to facilitate their handling. Generating the active species from the inactive adducts, however, required a high-temperature Lewis acid treatment and resulted in an insoluble by-product, limiting the practicality of the methodology. Herein, we introduce an approach to circumvent the inconvenient, costly, and environmentally taxing activation process. We show that 18-electron adducts of W- and Mo-based Schrock catalysts with finite stability constants (typically K = 200–15,000 M−1) can readily be prepared and isolated in excellent yields. The adducts display enhanced air-stability in the solid state, and in solution they dissociate spontaneously, hence liberating the active alkylidenes without chemical assistance.