Changhe Wang

Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte

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 Ca2+-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 Ca2+-dependent vesicular exocytosis, and small vesicles support glutamate release from astrocytes.

Modulation of dopamine release in the striatum by physiologically relevant levels of nicotine

Striatal dopamine (DA) release can be independently triggered not only by action potentials (APs) in dopaminergic axons but also APs in cholinergic interneurons (ChIs). Nicotine causes addiction by modulating DA release, but with paradoxical findings. Here, we investigate how physiologically relevant levels of nicotine modulate striatal DA release. The optogenetic stimulation of ChIs elicits DA release, which is potently inhibited by nicotine with an IC50 of 28 nM in the dorsal striatum slice. This ChI-driven DA release is predominantly mediated by α6β2* nAChRs. Local electrical stimulus (Estim) activates both dopaminergic axons and ChIs. Nicotine does not affect the AP(DA)-dependent DA release (AP(DA), AP of dopaminergic axon). During burst Estim, nicotine permits the facilitation of DA release by prevention of DA depletion. Our work indicates that cholinergic stimulation-induced DA release is profoundly modulated by physiologically relevant levels of nicotine and resolves the paradoxical observation of nicotines effects on striatal DA release.

Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells

Aims/hypothesisInsulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca2+ and cAMP: exocytosis is triggered by Ca2+ and mediated by the cAMP/protein kinase A (PKA) signalling pathway. However, the causal link between these two processes in primary beta cells remains undefined.MethodsTime-resolved confocal imaging of fluorescence resonance energy transfer signals was performed to visualise PKA activity, and combined membrane capacitance recordings were used to monitor insulin secretion from patch-clamped rat beta cells.ResultsMembrane depolarisation-induced Ca2+ influx caused an increase in cytosolic PKA activity via activating a Ca2+-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca2+ oscillations and PKA activation. ADCY8 knockdown significantly reduced the level of depolarisation-evoked PKA activation and impaired replenishment of the readily releasable vesicle pool. Pharmacological inhibition of PKA by two inhibitors reduced depolarisation-induced PKA activation to a similar extent and reduced the capacity for sustained vesicle exocytosis and insulin release.Conclusions/interpretationOur findings suggest that depolarisation-induced Ca2+ influx plays dual roles in regulating exocytosis in rat pancreatic beta cells by triggering vesicle fusion and replenishing the vesicle pool to support sustained insulin release. Therefore, Ca2+ influx may be important for glucose-stimulated insulin secretion.

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Significance With a combination of HPLC and carbon fiber electrodes, we demonstrate that grafted neural stem cells directly release dopamine in the damaged striatum in vivo and partially rescue a Parkinson’s disease (PD) model. (i) Primitive neural stem cell–dopamine-like neuron (pNSC–DAn) retained tyrosine hydroxylase expression and reduced the PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in striatum in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical micro-carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (not from the injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation. Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.

Temporal components of cholinergic terminal to dopaminergic terminal transmission in dorsal striatum slices of mice

The timing of synaptic transmission is critical to synaptic plasticity in the striatum. However, the timing of striatal dopamine (DA) release induced by cholinergic interneurons (ChIs) in the striatum is unclear. In this study, we focused on the temporal components of DA release and replenishment triggered by different pathways. We show that stimulation of ChIs induces DA release with a total delay of 20.6 ms, including 2.8 ms for action potential firing of ChIs, 7.0 ms for cholinergic transmission between acetylcholine terminals and DA terminals, and 10.8 ms for downstream DA release. The delay of DA release via this ChI pathway is 1.9 times that via the nigrostriatal pathways. We describe the time course of recovery of DA release via the two pathways and that of vesicle replenishment in DA terminals. Our work provides an example of unravelling the temporal building blocks during fundamental synaptic terminal–terminal transmission.

Intracellular TRPA1 mediates Ca2+ release from lysosomes in dorsal root ganglion neurons

The temperature-sensitive TRP channel, TRPA1, is known to mediate Na+ and Ca2+ influx at the plasma membrane of sensory neurons. In this study, the authors show that TRPA1 is also present on the lysosomal membrane and mediates lysosome Ca2+ release in dorsal root ganglion neurons.

Synaptotagmin‐11 inhibits clathrin‐mediated and bulk endocytosis

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 Parkinsons 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.

Two distinct vesicle pools for depolarization-induced exocytosis in somata of dorsal root ganglion neurons

Non‐technical summary  Ca2+‐regulated exocytosis is essential for neurotransmitter and hormone release. As well as this type of exocytosis, the somata of dorsal root ganglion (DRG) neurons also show Ca2+‐independent but voltage‐dependent exocytosis. It is unclear whether these two types of exocytosis use the same or different vesicle pools in DRG neurons. Here, we found that they were separable in response to the same stimulation in low external Ca2+ solution. Depletion of the Ca2+‐dependent vesicle pool did not affect the Ca2+‐independent but voltage‐dependent exocytosis. These results show that DRG neurons exhibit two distinct types of exocytosis that use different vesicle pools.

Molecular Mechanisms for the Coupling of Endocytosis to Exocytosis in Neurons

Neuronal communication and brain function mainly depend on the fundamental biological events of neurotransmission, including the exocytosis of presynaptic vesicles (SVs) for neurotransmitter release and the subsequent endocytosis for SV retrieval. Neurotransmitters are released through the Ca2+- and SNARE-dependent fusion of SVs with the presynaptic plasma membrane. Following exocytosis, endocytosis occurs immediately to retrieve SV membrane and fusion machinery for local recycling and thus maintain the homeostasis of synaptic structure and sustained neurotransmission. Apart from the general endocytic machinery, recent studies have also revealed the involvement of SNARE proteins (synaptobrevin, SNAP25 and syntaxin), synaptophysin, Ca2+/calmodulin, and members of the synaptotagmin protein family (Syt1, Syt4, Syt7 and Syt11) in the balance and tight coupling of exo-endocytosis in neurons. Here, we provide an overview of recent progress in understanding how these neuron-specific adaptors coordinate to ensure precise and efficient endocytosis during neurotransmission.

Extracellular Ca2+ per se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca(2+), in this calcium hypothesis, serves as a reservoir of Ca(2+) source. Recently we find that extracellular Ca(2+)per se inhibits the [Ca(2+)]i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca(2+) or both. In this work we showed that, in physiological condition, extracellular Ca(2+) per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca(2+) in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca(2+) directly triggered vesicle exocytosis without eliciting intracellular Ca(2+). We propose that intracellular Ca(2+) and extracellular Ca(2+)per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca(2+), while the vesicle quantal size was mainly determined by extracellular Ca(2+) in chromaffin cells physiologically.