Shyue An Chan

Dysregulation of brain-derived neurotrophic factor expression and neurosecretory function in Mecp2 null mice.

Disruptions in brain-derived neurotrophic factor (BDNF) expression are proposed to contribute to the molecular pathogenesis of Rett syndrome (RTT), a severe neurological disorder caused by loss-of-function mutations in methyl-CpG-binding protein-2 (MeCP2). Although MeCP2 is a transcriptional regulator of BDNF, it is unknown how MeCP2 mutations affect transynaptic BDNF signaling. Our findings demonstrate an early, abnormal neurosecretory phenotype in MeCP2-deficient neurons characterized by significant increases in the percentage of cellular BDNF content available for release. However, loss of MeCP2 also results in deficits in total cell BDNF content that are developmentally regulated in a cell-type-specific manner. Thus, the net effect of MeCP2 loss on absolute BDNF secretion changes with age and is determined by both the amount of BDNF available for release and progressive declines in total cellular BDNF. We propose, therefore, that loss of MeCP2 function disrupts transynaptic BDNF signaling by perturbing the normal balance between BDNF protein levels and secretion. However, mutant neurons are capable of secreting wild-type levels of BDNF in response to high-frequency electrical stimulation. In addition, we found elevated exocytic function in Mecp2 −/y adrenal chromaffin cells, indicating that the Mecp2 null mutation is associated with alterations of neurosecretion that are not restricted to BDNF. These findings are the first examples of abnormal neuropeptide and catecholamine secretion in a mouse model of RTT.

Physiological stimuli evoke two forms of endocytosis in bovine chromaffin cells

1 Exocytosis and endocytosis were measured following single, or trains of, simulated action potentials (sAP) in bovine adrenal chromaffin cells. Catecholamine secretion was measured by oxidative amperometry and cell membrane turnover was measured by voltage clamp cell capacitance measurements. 2 The sAPs evoked inward Na+ and Ca2+ currents that were statistically identical to those evoked by native action potential waveforms. On average, a single secretory granule underwent fusion following sAP stimulation. An equivalent amount of membrane was then quickly internalised (τ= 560 ms). 3 Stimulation with sAP trains revealed a biphasic relationship between cell firing rate and endocytic activity. At basal stimulus frequencies (single to 0.5 Hz) cells exhibited a robust membrane internalisation that then diminished as firing increased to intermediate levels (1.9 and 6 Hz). However at the higher stimulation rates (10 and 16 Hz) endocytic activity rebounded and was again able to effectively maintain cell surface near pre‐stimulus levels. 4 Treatment with cyclosporin A and FK506, inhibitors of the phosphatase calcineurin, left endocytosis characteristics unaltered at the lower basal stimulus levels, but blocked the resurgence in endocytosis seen in control cells at higher sAP frequencies. 5 Based on these findings we propose that, under physiological electrical stimulation, chromaffin cells internalise membrane via two distinct pathways that are separable. One is prevalent at basal stimulus frequencies, is lessened with increased firing, and is insensitive to cyclosporin A and FK506. A second endocytic form is activated by increased firing frequencies, and is selectively blocked by cyclosporin A and FK506.

PACAP regulates immediate catecholamine release from adrenal chromaffin cells in an activity‐dependent manner through a protein kinase C‐dependent pathway

Adrenal medullary chromaffin cells are a major peripheral output of the sympathetic nervous system. Catecholamine release from these cells is driven by synaptic excitation from the innervating splanchnic nerve. Acetylcholine has long been shown to be the primary transmitter at the splanchnic‐chromaffin synapse, acting through ionotropic nicotinic acetylcholine receptors to elicit action potential‐dependent secretion from the chromaffin cells. This cholinergic stimulation has been shown to desensitize under sustained stimulation, yet catecholamine release persists under this same condition. Recent evidence supports synaptic chromaffin cell stimulation through alternate transmitters. One candidate is pituitary adenylate cyclase activating peptide (PACAP), a peptide transmitter present in the adrenal medulla shown to have an excitatory effect on chromaffin cell secretion. In this study we utilize native neuronal stimulation of adrenal chromaffin cells in situ and amperometric catecholamine detection to demonstrate that PACAP specifically elicits catecholamine release under elevated splanchnic firing. Further data reveal that the immediate PACAP‐evoked stimulation involves a phospholipase C and protein kinase C‐dependant pathway to facilitate calcium influx through a Ni2+ and mibefradil‐sensitive calcium conductance that results in catecholamine release. These data demonstrate that PACAP acts as a primary secretagogue at the sympatho‐adrenal synapse under the stress response.

Low Frequency Stimulation of Mouse Adrenal Slices Reveals a Clathrin‐Independent, Protein Kinase C‐Mediated Endocytic Mechanism

Evidence suggests that chromaffin cells employ separate mechanisms for evoked endocytosis and granule recycling when stimulated at basal (∼0.5 Hz) and stress‐activated (∼15 Hz) rates. Previous studies have focused mainly on elucidating the cellular mechanisms responsible for membrane recycling under conditions similar to the stress‐activated state and indicate a clathrin/dephosphin‐mediated retrieval via coated pits. However, the mechanism for membrane internalisation at basal stimulus intensity remains largely unexplored. We electrically stimulated chromaffin cells in adrenal tissue slices at the sympathetic basal firing rate and measured cell capacitance in the perforated voltage clamp configuration. A new method for the separation of non‐secretory from secretory cell capacitance signals is presented. Simultaneous catecholamine release was measured electrochemically to isolate the exocytic from endocytic components of the capacitance responses. Using this approach we demonstrate that firing patterns that mimic basal sympathetic input results in rapid and graded membrane retrieval. We show that block of the calcium‐mediated protein phosphatase 2B, a common step in clathrin‐mediated processes, did not alter endocytosis elicited at basal firing levels. We further blocked clathrin‐mediated retrieval with a clathrin/dephosphin‐disrupting peptide (PP‐19) and found endocytosis to be blocked at 15 Hz stimulation but complete and indistinguishable from control cells at 0.5 Hz stimulation. Lastly, pharmacological treatments show that conventional isoforms of protein kinase C (cPKC) are required for the 0.5 Hz‐evoked retrieval mechanism. From these data we conclude that unlike endocytosis evoked under stress conditions, basal firing activity results in a clathrin‐independent rapid membrane retrieval mediated through conventional isoforms of PKC.

Increased secretory capacity of mouse adrenal chromaffin cells by chronic intermittent hypoxia: involvement of protein kinase C

Previous studies have shown that catecholamine secretion from the adrenal medulla plays a critical role in chronic intermittent hypoxia (CIH)‐induced alterations in cardiovascular function. In the present study we examined the cellular mechanisms associated with the effects of CIH on adrenal chromaffin cell catecholamine secretion. Experiments were performed on adult male mice (C57/BL6) that were exposed to 1–4 days of CIH or to normoxia. Perforated patch electrical capacitance recordings were performed on freshly prepared adrenal medullary slices that permit separating the chromaffin cell secretion from sympathetic input. CIH resulted in a significant increase in the readily releasable pool (RRP) of secretory granules, and decreased stimulus‐evoked Ca2+ influx. Continuous hypoxia (CH) either for 2.5 h (equivalent to hypoxic duration accumulated over 4 days of CIH) or for 4 days were ineffective in evoking changes in the RRP and Ca2+ influx. CIH activated PKC in adrenal medullae as evidenced by increased phosphorylation of PKC at Thr514 and PKC inhibitors prevented CIH‐induced increases in the RRP and restored stimulus‐evoked attenuation of Ca2+ influx. CIH resulted in elevated thio‐barbituric acid reactive substances (TBARSs, an index of oxidized proteins) and an antioxidant prevented CIH‐induced changes in the RRP, suggesting the involvement of reactive oxygen species (ROS). These results demonstrate that CIH increases the RRP in adrenal chromaffin cells via ROS‐mediated activation of PKC and suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to elevated catecholamine levels.

Pituitary Adenylate Cyclase-activating Peptide (PACAP) Recruits Low Voltage-activated T-type Calcium Influx under Acute Sympathetic Stimulation in Mouse Adrenal Chromaffin Cells

Background: The acute stress secretagogue PACAP leads to catecholamine release, but the source of calcium necessary for secretion is unknown. Results: PACAP stimulation increases LVA Cav3.2 influx in a PKC-dependent process. Conclusion: PACAP-mediated acute sympathetic stress functionally recruits a pool of latent Cav3.2 channels to supply calcium for secretion. Significance: Native sympathoadrenal stimulation elicits catecholamine release through a non-canonical mechanism. Low voltage-activated T-type Cav3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Cav3.2-dependent conductance. However, Cav3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Cav3.2 exists, there are no reports testing the specific role of Cav3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Cav3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca2+-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Cav3.2 Ca2+ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.

Activity-dependent fusion pore expansion regulated by a calcineurin-dependent dynamin-syndapin pathway in mouse adrenal chromaffin cells.

Neuroendocrine chromaffin cells selectively secrete a variety of transmitter molecules into the circulation as a function of sympathetic activation. Activity-dependent release of transmitter species is controlled through regulation of the secretory fusion pore. Under sympathetic tone, basal synaptic excitation drives chromaffin cells to selectively secrete modest levels of catecholamine through a restricted secretory fusion pore. In contrast, elevated sympathetic activity, experienced under stress, results in fusion pore expansion to evoke maximal catecholamine release and to facilitate release of copackaged peptide transmitters. Therefore, fusion pore expansion is a key control point for the activation of the sympatho-adrenal stress response. Despite the physiological importance of this process, the molecular mechanism by which it is regulated remains unclear. Here we employ fluorescence imaging with electrophysiological and electrochemical-based approaches to investigate the role of dynamin I in the regulation of activity-mediated fusion pore expansion in mouse adrenal chromaffin cells. We show that under elevated stimulation, dynamin I is dephosphorylated at Ser-774 by calcineurin. We also demonstrate that disruption of dynamin I-syndapin binding, an association regulated by calcineurin-dependent dynamin dephosphorylation, limits fusion pore expansion. Last, we show that perturbation of N-WASP function (a syndapin substrate) limits activity-mediated fusion pore expansion. Our results suggest that fusion pore expansion is regulated by a calcineurin-dependent dephosphorylation of dynamin I. Dephosphorylated dynamin I acts via a syndapin/N-WASP signaling cascade to mediate pore expansion.

Dynamin and myosin regulate differential exocytosis from mouse adrenal chromaffin cells

Neuroendocrine chromaffin cells of the adrenal medulla represent a primary output for the sympathetic nervous system. Chromaffin cells release catecholamine as well as vaso- and neuro-active peptide transmitters into the circulation through exocytic fusion of large dense-core secretory granules. Under basal sympathetic activity, chromaffin cells selectively release modest levels of catecholamines, helping to set the “rest and digest” status of energy storage. Under stress activation, elevated sympathetic firing leads to increased catecholamine as well as peptide transmitter release to set the “fight or flight” status of energy expenditure. While the mechanism for catecholamine release has been widely investigated, relatively little is known of how peptide transmitter release is regulated to occur selectively under elevated stimulation. Recent studies have shown selective catecholamine release under basal stimulation is accomplished through a transient, restricted exocytic fusion pore between granule and plasma membrane, releasing a soluble fraction of the small, diffusible molecules. Elevated cell firing leads to the active dilation of the fusion pore, leading to the release of both catecholamine and the less diffusible peptide transmitters. Here we propose a molecular mechanism regulating the activity-dependent dilation of the fusion pore. We review the immediate literature and provide new data to formulate a working mechanistic hypothesis whereby calcium-mediated dephosphorylation of dynamin I at Ser-774 leads to the recruitment of the molecular motor myosin II to actively dilate the fusion pore to facilitate release of peptide transmitters. Thus, activity-dependent dephosphorylation of dynamin is hypothesized to represent a key molecular step in the sympatho-adrenal stress response.

Enhanced dense core granule function and adrenal hypersecretion in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a progressive developmental disorder resulting from loss‐of‐function mutations in the gene encoding methyl‐CpG‐binding protein 2 (MeCP2), a transcription regulatory protein. The RTT phenotype is complex and includes severe cardiorespiratory abnormalities, dysautonomia and behavioral symptoms of elevated stress. These findings have been attributed to an apparent hyperactivity of the sympathetic nervous system due to defects in brainstem development; however, the possibility that the peripheral sympathoadrenal axis itself is abnormal has not been explored. The present study demonstrates that the adrenal medulla and sympathetic ganglia of Mecp2 null mice exhibit markedly reduced catecholamine content compared with wild‐type controls. Despite this, null animals exhibit significantly higher plasma epinephrine levels, suggesting enhanced secretory granule function in adrenal chromaffin cells. Indeed, we find that Mecp2 null chromaffin cells exhibit a cell autonomous hypersecretory phenotype characterized by significant increases in the speed and size of individual secretory granule fusion events in response to electrical stimulation. These findings appear to indicate accelerated formation and enhanced dilation of the secretory granule fusion pore, resulting in elevated catecholamine release. Our data therefore highlight abnormal catecholamine function in the sympathoadrenal axis as a potential source of autonomic dysfunction in RTT. These findings may help to explain the apparent ‘overactivity’ of the sympathetic nervous system reported in patients with RTT.

Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells.

Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and the plasma membrane. Upon activation of the sympathoadrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of copackaged peptide hormones. Thus regulated expansion of the secretory fusion pore is a control point for differential hormone release of the sympathoadrenal stress response. Previous work has shown that syndapin 1 deletion alters transmitter release and that the dynamin 1-syndapin 1 interaction is necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contributes to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin and syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show that all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.