Synaptotagmin 4: A New Antiobesity Target?

In this issue of Neuron, Zhang et?al. show that Synaptotagmin 4 (Syt4) is specifically induced in adult hypothalamic oxytocin neurons by high-fat diet. Evidence is provided to support a critical role for Syt4 in negative regulation of oxytocin release, which in turn is responsible for diet-induced obesity, raising the possibility of using Syt4 as a new antiobesity target.     

Circadian intervention of obesity development via resting-stage feeding manipulation or oxytocin treatment

The obesity pandemic can be viewed as a result of an imbalanced reaction to changing environmental factors. Recent research has linked circadian arrhythmicity to obesity and related diseases; however, the underlying mechanisms are still unclear. In this study, we found that high-fat diet (HFD) feeding strikingly promoted daytime rather than nighttime caloric intake in mice, leading to feeding circadian arrhythmicity. Using scheduled feeding with a defined amount of daily HFD intake, we found that an increase in the ratio of daytime to nighttime feeding promoted weight gain, whereas a decrease of this ratio rebalanced energy expenditure to counteract obesity. In identifying the underlying mechanism, we found that hypothalamic release of anorexigenic neuropeptide oxytocin displayed a diurnal rhythm of daytime rise and nighttime decline, which negatively correlated with the diurnal feeding activities of normal chow-fed mice. In contrast, chronic HFD feeding abrogated oxytocin diurnal rhythmicity, primarily by suppressing daytime oxytocin rise. Using pharmacological experiments with hypothalamic injection of oxytocin or oxytocin antagonist, we showed that daytime manipulation of oxytocin can change feeding circadian patterns to reprogram energy expenditure, leading to attenuation or induction of obesity independently of 24-h caloric intake. Also importantly, we found that peripheral injection of oxytocin activated hypothalamic oxytocin neurons to release oxytocin, and exerted metabolic effects similar to central oxytocin injection, thus offering a practical clinical avenue to use oxytocin in obesity control. In conclusion, resting-stage oxytocin release and feeding activity represent a critical circadian mechanism and therapeutic target for obesity.     

Neuronal calcium sensor synaptotagmin-9 is not involved in the regulation of glucose homeostasis or insulin secretion

Background

Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7.

Methodology/Principal Findings

In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X) mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice.

Conclusions

Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic β-cells.     

Postsynaptic complexin controls AMPA receptor exocytosis during LTP

Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest?a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.     

Synaptotagmin-1, -2, and -9: Ca(2+) sensors for fast release that specify distinct presynaptic properties in subsets of neurons

Synaptotagmin-1 and -2 are known Ca(2+) sensors for fast synchronous neurotransmitter release, but the potential Ca(2+)-sensor functions of other synaptotagmins in release remain uncharacterized. We now show that besides synaptotagmin-1 and -2, only synaptotagmin-9 (also called synaptotagmin-5) mediates fast Ca(2+) triggering of release. Release induced by the three different synaptotagmin Ca(2+) sensors exhibits distinct kinetics and apparent Ca(2+) sensitivities, suggesting that the synaptotagmin isoform expressed by a neuron determines the release properties of its synapses. Conditional knockout mice producing GFP-tagged synaptotagmin-9 revealed that synaptotagmin-9 is primarily expressed in the limbic system and striatum. Acute deletion of synaptotagmin-9 in striatal neurons severely impaired fast synchronous release without changing the size of the readily-releasable vesicle pool. These data show that in mammalian brain, only synaptotagmin-1, -2, and -9 function as Ca(2+) sensors for fast release, and that these synaptotagmins are differentially expressed to confer distinct release properties onto synapses formed by defined subsets of neurons.     

Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca²⁺ binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca²⁺ binding to an unidentified secondary Ca²⁺ sensor. To test whether CaM could be the enigmatic Ca²⁺ sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca²⁺ binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca²⁺/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.     

Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance

Macroautophagy is a lysosomal degradative pathway that maintains cellular homeostasis by turning over cellular components. Here we demonstrate a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in the regulation of food intake and energy balance. We show that starvation-induced hypothalamic autophagy mobilizes neuron-intrinsic lipids to generate endogenous free fatty acids, which in turn regulate AgRP levels. The functional consequences of inhibiting autophagy are the failure to upregulate AgRP in response to starvation, and constitutive increases in hypothalamic levels of pro-opiomelanocortin and its cleavage product α-melanocyte-stimulating hormone that typically contribute to a lean phenotype. We propose a conceptual framework for considering how autophagy-regulated lipid metabolism within hypothalamic neurons may modulate neuropeptide levels to have immediate effects on food intake, as well as long-term effects on energy homeostasis. Regulation of hypothalamic autophagy could become an effective intervention in conditions such as obesity and the metabolic syndrome.     

A role for beta-melanocyte-stimulating hormone in human body-weight regulation

Pro-opiomelanocortin (POMC) expressing neurons mediate the regulation of orexigenic drive by peripheral hormones such as leptin, cholecystokinin, ghrelin, and insulin. Most research effort has focused on alpha-melanocyte-stimulating hormone (alpha-MSH) as the predominant POMC-derived neuropeptide in the central regulation of human energy balance and body weight. Here we report a missense mutation within the coding region of the POMC-derived peptide beta-MSH (Y5C-beta-MSH) and its association with early-onset human obesity. In vitro and in vivo data as well as postmortem human brain studies indicate that the POMC-derived neuropeptide beta-MSH plays a critical role in the hypothalamic control of body weight in humans.     

Increased lipolysis and energy expenditure in a mouse model with severely impaired glucagon secretion

Background

Secretion of insulin and glucagon is triggered by elevated intracellular calcium levels. Although the precise mechanism by which the calcium signal is coupled to insulin and glucagon granule exocytosis is unclear, synaptotagmin-7 has been shown to be a positive regulator of calcium-dependent insulin and glucagon secretion, and may function as a calcium sensor for insulin and glucagon granule exocytosis. Deletion of synaptotagmin-7 leads to impaired glucose-stimulated insulin secretion and nearly abolished Ca²⁺-dependent glucagon secretion in mice. Under non-stressed resting state, however, synaptotagmin-7 KO mice exhibit normal insulin level but severely reduced glucagon level.

Methodology/Principal Findings

We studied energy expenditure and metabolism in synaptotagmin-7 KO and control mice using indirect calorimetry and biochemical techniques. Synaptotagmin-7 KO mice had lower body weight and body fat content, and exhibited higher oxygen consumption and basal metabolic rate. Respiratory exchange ratio (RER) was lower in synaptotagmin-7 KO mice, suggesting an increased use of lipid in their energy production. Consistent with lower RER, gene expression profiles suggest enhanced lipolysis and increased capacity for fatty acid transport and oxidation in synaptotagmin-7 KO mice. Furthermore, expression of uncoupling protein 3 (UCP3) in skeletal muscle was approximately doubled in the KO mice compared with control mice.

Conclusions

These results show that the lean phenotype in synaptotagmin-7 KO mice was mostly attributed to increased lipolysis and energy expenditure, and suggest that reduced glucagon level may have broad influence on the overall metabolism in the mouse model.     

Antisense oligodeoxynucleotide effects on the hypothalamic-neurohypophysial system and the hypothalamic-pituitary-adrenal axis

The possibility of sequence-dependent, transient, and local inhibition of neuropeptide or neuropeptide receptor expression within the brain makes antisense targeting an attractive approach for those interested in the involvement of brain neuropeptide systems in behavioral and neuroendocrine regulation. Here, I describe our attempts to manipulate the synthetic activity of peptidergic systems of the hypothalamic-neurohypophysial system, i.e. , oxytocin and vasopressin, and the hypothalamic-pituitary-adrenal (HPA) axis by antisense oligodeoxynucleotides. Detailed experimental protocols including different approaches for intracerebral antisense application in anesthetized or conscious rats are provided. As a consequence of local oxytocin or vasopressin antisense treatment within the hypothalamic supraoptic nucleus, various aspects of the neuronal activity are already altered after a few hours. Thus, we monitored electrophysiological parameters of oxytocinergic and vasopressinergic neurons, stimulus-induced expression of the Fos protein in oxytocin neurons, and stimulated release of oxytocin or vasopressin into blood as well as within the hypothalamus by dendrites and cell bodies as measured by simultaneous microdialysis in blood and brain, shortly after a single acute antisense infusion. We also employed chronic antisense infusion via osmotic minipumps or by repeated local infusion into the targeted brain region; for example, septal vasopressin receptor downregulation impairs the ability of male rats to discriminate between juvenile rats. Further, reduction of the amount of available CRH, vasopressin, and oxytocin within the hypothalamic paraventricular nuclei alters the neuroendocrine stress response of the HPA axis.     

Phosphatidylinositol 4,5-bisphosphate increases Ca2+ affinity of synaptotagmin-1 by 40-fold

Synaptotagmin-1 is the main Ca(2+) sensor of neuronal exocytosis. It binds to both Ca(2+) and the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), but the precise cooperativity of this binding is still poorly understood. Here, we used microscale thermophoresis to quantify the cooperative binding of PIP(2) and Ca(2+) to synaptotagmin-1. We found that PIP(2) bound to the well conserved polybasic patch of the C2B domain with an apparent dissociation constant of ～20 μM. PIP(2) binding reduced the apparent dissociation constant for Ca(2+) from ～250 to <5 μM. Thus, our data show that PIP(2) makes synaptotagmin-1 >40-fold more sensitive to Ca(2+). This interplay between Ca(2+), synaptotagmin-1, and PIP(2) is crucial for neurotransmitter release.     

Effect of cytokines on hypothalamic neuropeptide Y release in vitro

Studies involving altered energy balance states in rodents have demonstrated that hypothalamic neuropeptide Y (NPY) activity is strongly activated in states of negative energy balance, such as periods of dietary restriction or starvation. However, in cancer cachexia, when there is a significant reduction in body weight as a result of appetite loss, leading to loss in fat and lean tissue mass, there is no augmentation in the activity of the hypothalamic NPY system. Therefore, we have examined whether cytokines, interleukin (IL)-1, IL-1beta, IL-6, and tumor-necrosis factor-alpha (TNF-alpha; cachectin), which are elevated in cancer patients, can attenuate NPY release from hypothalamic slices in vitro. None of the cytokines altered either the basal or stimulated NPY release from the hypothalamic slices. However, we were able to measure a significant reduction in potassium-stimulated NPY release (-60%) by using the nonselective voltage-dependent calcium channel blocker NiCl (30 microM) without any effect on basal release, as a positive control. Therefore, we suggest that the failure to activate the hypothalamic NPY system in states of cancer cachexia cannot be attributed to a cytokine-induced reduction in neurotransmitter release.     

Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release

Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine(97), and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine(97) by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca(2+)-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.     

Structural and Mutational Analysis of Functional Differentiation between Synaptotagmins-1 and -7

Synaptotagmins are known to mediate diverse forms of Ca²⁺-triggered exocytosis through their C₂ domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca²⁺ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca²⁺ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C₂B domain replaced by the synaptotagmin-7 C₂B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C₂B domain, we determined its crystal structure at 1.44 Å resolution. The synaptotagmin-7 C₂B domain structure is very similar to that of the synaptotagmin-1 C₂B domain and contains three Ca²⁺-binding sites. Two of the Ca²⁺-binding sites of the synaptotagmin-7 C₂B domain are also present in the synaptotagmin-1 C₂B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C₂B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.     

下丘脑Kiss1神经元在能量代谢调节中的作用

代谢是机体生存和延续的基础,机体通过影响行为并诱发一系列的生理反应,调节代谢状态。能量代谢失衡可能导致机体消瘦或肥胖,甚至会造成生长发育和生殖功能的障碍等。因此,维持机体的能量平衡至关重要,而这一状态的维持受中枢神经系统的严格控制。中枢神经系统,特别是下丘脑,在调节机体生理功能和能量平衡中发挥着重要的作用。下丘脑Kisspeptin被认为在调节性腺轴、营养性发育和生殖中发挥重要作用。近些年来,关于其在能量代谢调控中的作用也引起广泛关注。本文将从能量摄入和能量消耗两个方面对下丘脑Kisspeptin在能量代谢调控中的作用进行综述,以期为防治因能量失衡诱发的代谢性疾病提供新的研究思路和依据。     

Effects of hypothalamic neurodegeneration on energy balance

Normal aging in humans and rodents is accompanied by a progressive increase in adiposity. To investigate the role of hypothalamic neuronal circuits in this process, we used a Cre-lox strategy to create mice with specific and progressive degeneration of hypothalamic neurons that express agouti-related protein (Agrp) or proopiomelanocortin (Pomc), neuropeptides that promote positive or negative energy balance, respectively, through their opposing effects on melanocortin receptor signaling. In previous studies, Pomc mutant mice became obese, but Agrp mutant mice were surprisingly normal, suggesting potential compensation by neuronal circuits or genetic redundancy. Here we find that Pomc-ablation mice develop obesity similar to that described for Pomc knockout mice, but also exhibit defects in compensatory hyperphagia similar to what occurs during normal aging. Agrp-ablation female mice exhibit reduced adiposity with normal compensatory hyperphagia, while animals ablated for both Pomc and Agrp neurons exhibit an additive interaction phenotype. These findings provide new insight into the roles of hypothalamic neurons in energy balance regulation, and provide a model for understanding defects in human energy balance associated with neurodegeneration and aging.     

The role of nitric oxide in the hypothalamic control of LHRH and oxytocin release, sexual behavior and aging of the LHRH and oxytocin neurons

Nitric oxide (NO) affects reproductive processes both at the level of the brain and reproductive tract and this review is focused on its role as an essential regulator of the hypothalamic control of reproduction. The data gathered indicate that glutamate stimulates noradrenergic neurons which subsequently activate NO-ergic cells via alpha1-adrenergic receptors. The released NO diffuses into luteinizing hormone-releasing hormone (LHRH) terminals where it triggers LHRH secretion by activation of guanylyl cyclase and cyclooxygenase. The NO released by estrogen-stimulated NO-ergic ventromedial neurons plays a crucial role in the regulation of sexual behavior. Furthermore, an increased expression of inducible nitric oxide synthase in the LHRH and oxytocin neurons underlies the destructive action of NO on the aging of the hypothalamic neuroendocrine pathways. Within the hypothalamo-hypophyseal system, NO exerts an inhibitory effect in the control of oxytocin secretion. This action seems to employ an indirect mechanism by which NO may modulate the release of GABA. This review provides an overview of the role of NO in hypothalamic control of LHRH and oxytocin release, aging of the LHRH and oxytocin neurons and sexual behavior.     

Evoked axonal oxytocin release in the central amygdala attenuates fear response

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.