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Membrane trafficking pathways mediate key microglial activities such as cell migration, cytokine secretion, and phagocytosis. However, the underlying molecular mechanism remains poorly understood. Previously, we found that synaptotagmin‐11 (Syt11), a non‐Ca2+‐binding Syt associated with Parkinson's disease (PD) and schizophrenia, inhibits cytokine release and phagocytosis in primary microglia. Here we reported the in vivo function of Syt11 in microglial immune responses using an inducible microglia‐specific Syt11‐conditional‐knockout (cKO) mouse strain. Syt11‐cKO resulted in activation of microglia and elevated mRNA levels of IL‐6, TNF‐α, IL‐1β, and iNOS in various brain regions under both resting state and LPS‐induced acute inflammation state in adult mice. In a PD mouse model generated by microinjection of preformed α‐synuclein fibrils into the striatum, a reduced number of microglia migrated toward the injection sites and an enhanced phagocytosis of α‐synuclein fibrils by microglia were found in Syt11‐cKO mice. To understand the molecular mechanism of Syt11 function, we identified its direct binding proteins vps10p‐tail‐interactor‐1a (vti1a) and vti1b. The linker domain of Syt11 interacted with both proteins and a peptide derived from it competitively inhibited the interaction of Syt11 with vti1a/vti1b in vitro and in cells. Importantly, application of this peptide induced more cytokine secretion in wild‐type microglia upon LPS treatment, phenocopying defects in Syt11 knockdown cells. Altogether, we propose that Syt11 inhibits microglial activation in vivo and regulates cytokine secretion through interactions with vti1a and vti1b. [ABSTRACT FROM AUTHOR]

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac ([I.sub.f])/neuronal ([I.sub.h]) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and [I.sub.h] channels in neurons. This raises the possibility of [Ca.sup.2+] permeation in [I.sub.f], the [I.sub.h] counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 [Ca.sup.2+] signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by [I.sub.f] in rat ventricular myocytes. We observed [Ca.sup.2+] influx when HCN/[I.sub.f] channels were activated. [Ca.sup.2+] influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an [I.sub.f] channel blocker, inhibited [I.sub.f] and [Ca.sup.2+] influx at the same time. Quantitative analysis revealed that [Ca.sup.2+] flux contributed to ~0.5% of current produced by the HCN2 channel or [I.sub.f]. The associated increase in [Ca.sup.2+] influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which [I.sub.f] current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a [Ca.sup.2+] chelator), preactivation of [I.sub.f] channels significantly reduced the action potential duration, and theeffect was blocked by another selective [I.sub.f] channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of [I.sub.f] channels had no effects on action potential duration. Our data extend our previous discovery of [Ca.sup.2+] influx in [I.sub.h] channels in neurons to [I.sub.f] channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels

α‐Synuclein is a central player in Parkinson's disease (PD) pathology. Various point mutations in α‐synuclein have been identified to alter the protein‐phospholipid binding behavior and cause PD. Therefore, exploration of α‐synuclein‐phospholipid interaction is important for understanding the PD pathogenesis and helping the early diagnosis of PD. Herein, a phospholipid‐decorated liquid crystal (LC)‐aqueous interface is constructed to investigate the binding between α‐synucleins (wild‐type and six familial mutant A30P, E46K, H50Q, G51D, A53E and A53T) and phospholipid. The application of deep learning analyzes and reveals distinct LC signatures generated by the binding of α‐synuclein and phospholipid. This system allows for the identification of single point mutant α‐synucleins with an average accuracy of 98.3±1.3% in a fast and efficient manner. We propose that this analytical methodology provides a new platform to understand α‐synuclein‐lipid interactions, and can be potentially developed for easy identification of α‐synuclein mutations in common clinic. [ABSTRACT FROM AUTHOR]

Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca2+-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural- functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis. [ABSTRACT FROM AUTHOR]

α-Synuclein is a central player in Parkinson's disease (PD) pathology. Various point mutations in α-synuclein have been identified to alter the protein-phospholipid binding behavior and cause PD. Therefore, exploration of α-synuclein-phospholipid interaction is important for understanding the PD pathogenesis and helping the early diagnosis of PD. Herein, a phospholipid-decorated liquid crystal (LC)-aqueous interface is constructed to investigate the binding between α-synucleins (wild-type and six familial mutant A30P, E46K, H50Q, G51D, A53E and A53T) and phospholipid. The application of deep learning analyzes and reveals distinct LC signatures generated by the binding of α-synuclein and phospholipid. This system allows for the identification of single point mutant α-synucleins with an average accuracy of 98.3±1.3% in a fast and efficient manner. We propose that this analytical methodology provides a new platform to understand α-synuclein-lipid interactions, and can be potentially developed for easy identification of α-synuclein mutations in common clinic., (© 2021 Wiley-VCH GmbH.)

Microglia are professional phagocytes in the brain and deficiency in their phagocytic activity plays an important role in Parkinson's disease. This protocol mainly describes the phagocytosis assay for uptake of α-synuclein preformed fibrils, a pathologic form of α-synuclein, by primary microglia., Competing Interests: Competing interestsAuthors declare no conflicts of interest., (Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.)

Astrocytes release a variety of signaling molecules including glutamate, D-serine, and ATP in a regulated manner. Although the functions of these molecules, from regulating synaptic transmission to controlling specific behavior, are well documented, the identity of their cellular compartment(s) is still unclear. Here we set out to study vesicular exocytosis and glutamate release in mouse hippocampal astrocytes. We found that small vesicles and lysosomes coexisted in the same freshly isolated or cultured astrocytes. Both small vesicles and lysosome fused with the plasma membrane in the same astrocytes in a Ca(2+)-regulated manner, although small vesicles were exocytosed more efficiently than lysosomes. Blockade of the vesicle glutamate transporter or cleavage of synaptobrevin 2 and cellubrevin (both are vesicle-associated membrane proteins) with a clostridial toxin greatly inhibited glutamate release from astrocytes, while lysosome exocytosis remained intact. Thus, both small vesicles and lysosomes contribute to Ca(2+)-dependent vesicular exocytosis, and small vesicles support glutamate release from astrocytes.

Our previous observations showed that several stimuli, including high-K(+) solution, glutamate, and voltage pulses, induce somatic noradrenaline (NA) secretion from locus ceruleus (LC) neurons. Hypocretin (orexin), a hypothalamic peptide critical for normal wakefulness, has been shown to evoke NA release from the axon terminals of LC neurons. Here, we used amperometry to test the effect of hypocretin-1 (HCRT) on NMDA receptor-mediated somatodendritic release in LC neurons. Either HCRT or NMDA applied alone dose-dependently induced somatodendritic secretion. Bath application of HCRT notably potentiated NMDA receptor-mediated somatodendritic NA release. This potentiation was blocked by SB 334867, a selective HCRT receptor (Hcrtr 1) antagonist, or bisindolylmaleimide, a specific protein kinase C (PKC) inhibitor, indicating the involvement of Hcrtr 1 and PKC. Consistent with this, phorbol 12-myristate 13-acetate, a PKC activator, mimicked the HCRT-induced potentiation. Furthermore, HCRT enhanced NMDA-induced intracellular Ca(2+) elevation via activation of Hcrtr 1 and PKC, which may contribute to HCRT-potentiated somatodendritic secretion. These results suggest that HCRT modulates LC activity not only by regulating noradrenergic input to its targets, but also by affecting noradrenergic communication in the soma and dendrites.

In order to investigate the central nervous mechanism and the diseases involved in catecholamine transmitter secretion, the dynamics of catecholamine release is studied in single cell, brain slice or in vivo. Noradrenaline is an important neurotransmitter and modulator in the central nervous system (CNS) and the peripheral nervous system (PNS). In the present paper, we first compared three real-time methods used to measure noradrenaline secretion in single cells (membrane capacitance, amperometry and confocal fluorescence microscopy imaging). Compared to the electrophysiological method and fluorescence microscopy, the basic usage of the carbon fiber electrode (CFE) in neuroscience research was presented as an example. Then, we presented a primary description of ion channels, including voltage-gated Na(+), K(+) and Ca(2+) channels in locus coeruleus (LC) neurons in rat brain slices. Finally, we presented example recordings of combined patch-clamp and amperometry measurements in LC neurons, indicating Ca(2+)-dependent quantal noradrenaline release following Ca(2+) influx through Ca(2+) channels.