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

AIMS/HYPOTHESIS: Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca(2+) and cAMP: exocytosis is triggered by Ca(2+) 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. METHODS: Time-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. RESULTS: Membrane depolarisation-induced Ca(2+) influx caused an increase in cytosolic PKA activity via activating a Ca(2+)-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca(2+) 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/INTERPRETATION: Our findings suggest that depolarisation-induced Ca(2+) 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, Ca(2+) influx may be important for glucose-stimulated insulin secretion.

Real-time endocytosis imaging as a rapid assay of ligand-GPCR binding in single cells.

Most G protein-coupled receptors (GPCRs) do not generate membrane currents in response to ligand-receptor binding (LRB). Here, we describe a novel technique using endocytosis as a bioassay that can detect activation of a GPCR in a way analogous to patch-clamp recording of an ion channel in a living cell. The confocal imaging technique, termed FM endocytosis imaging (FEI), can record ligand-GPCR binding with high temporal (second) and spatial (micrometer) resolution. LRB leads to internalization of an endocytic vesicle, which can be labeled by a styryl FM dye and visualized as a fluorescent spot. Distinct from the green fluorescence protein-labeling method, FEI can detect LRB endocytosis mediated by essentially any receptors (GPCRs or receptors of tyrosine kinase) in a native cell/cell line. Three modified versions of FEI permit promising applications in functional GPCR studies and drug screening in living cells: 1) LRB can be recorded in "real time" (time scale of seconds); 2) internalized vesicles mediated by different GPCRs can be discriminated by different colors; and 3) a high throughput method can screen ligands of a specific GPCR.

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 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.

Thiopental-induced insulin secretion via activation of IP3-sensitive calcium stores in rat pancreatic ß-cells.

While glucose-stimulated insulin secretion depends on Ca(2+) influx through voltage-gated Ca(2+) channels in the cell membrane of the pancreatic ß-cell, there is also ample evidence for an important role of intracellular Ca(2+) stores in insulin secretion, particularly in relation to drug stimuli. We report here that thiopental, a common anesthetic agent, triggers insulin secretion from the intact pancreas and primary cultured rat pancreatic ß-cells. We investigated the underlying mechanisms by measurements of whole cell K(+) and Ca(2+) currents, membrane potential, cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and membrane capacitance. Thiopental-induced insulin secretion was first detected by enzyme-linked immunoassay, then further assessed by membrane capacitance measurement, which revealed kinetics distinct from glucose-induced insulin secretion. The thiopental-induced secretion was independent of cell membrane depolarization and closure of ATP-sensitive potassium (K(ATP)) channels. However, accompanied by the insulin secretion stimulated by thiopental, we recorded a significant intracellular [Ca(2+)] increase that was not from Ca(2+) influx across the cell membrane, but from intracellular Ca(2+) stores. The thiopental-induced [Ca(2+)](i) rise in ß-cells was sensitive to thapsigargin, a blocker of the endoplasmic reticulum Ca(2+) pump, as well as to heparin (0.1 mg/ml) and 2-aminoethoxydiphenyl borate (2-APB; 100 µM), drugs that inhibit inositol 1,4,5-trisphosphate (IP(3)) binding to the IP(3) receptor, and to U-73122, a phospholipase C inhibitor, but insensitive to ryanodine. Thapsigargin also diminished thiopental-induced insulin secretion. Thus, we conclude that thiopental-induced insulin secretion is mediated by activation of the intracellular IP(3)-sensitive Ca(2+) store.

Rhesus macaques develop metabolic syndrome with reversible vascular dysfunction responsive to pioglitazone.

BACKGROUND: The metabolic syndrome (MetS) is a constellation of clinical features that include central obesity, hypertension, atherogenic dyslipidemia, and insulin resistance. However, the concept remains controversial; it has been debated whether MetS represents nothing more than simultaneous co-occurrence of individual risk factors or whether there are common shared pathophysiological mechanisms that link the individual components. METHODS AND RESULTS: To investigate the emergence of metabolic and cardiovascular components during the development of MetS, we identified MetS-predisposed animals (n=35) in a large population of rhesus macaques (Macaca mulatta, 12.7±2.9 years old, n=408), acclimated them to standardized conditions, and monitored the progression of individual component features over 18 months. In 18 MetS animals with recently developed fasting hyperinsulinemia, central obesity, hypertension, and atherogenic dyslipidemia, we found that individual metabolic and cardiovascular components track together during the transition from pre-MetS to onset of MetS; MetS was associated with a 60% impairment of flow-mediated dilation, establishing the mechanistic link with vascular dysfunction. Pioglitazone treatment (3 mg/kg body weight/d for 6 weeks), a peroxisome proliferator-activated receptor γ agonist, reversibly improved atherogenic dyslipidemia and insulin resistance and fully restored flow-mediated dilation with persistent benefits. CONCLUSIONS: Coemergence of metabolic and cardiovascular components during MetS progression and complete normalization of vascular dysfunction with peroxisome proliferator-activated receptor γ agonists suggest shared underlying mechanisms rather than separate processes, arguing for the benefit of early intervention of MetS components. Predictive nonhuman primate (NHP) models of MetS should be highly valuable in mechanistic and translational studies on the pathogenesis of MetS in relation to cardiovascular disease and diabetes mellitus.

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

The somata of dorsal root ganglion (DRG) neurons release neurotransmitters and neuropeptides. In addition to the conventional Ca2+-dependent secretion (CDS), Ca2+-independent but voltage-dependent secretion (CIVDS) also occurs in the somata of DRG neurons. Electrical stimulation induces both CDS and CIVDS, which differ in size and are coupled with different types of endocytosis contributed by CIVDS and CDS, respectively. However, it is unclear whether they use a common vesicle pool, so we investigated the relationship between the vesicle pools of CDS and CIVDS. Membrane capacitance recording and photolysis of a caged-Ca2+ compound showed that, in low external Ca2+ solutions, the depolarization-induced exocytosis contained two (fast and slow) phases, which were contributed by CIVDS and CDS, respectively. Depletion of the CDS readily releasable pool using photolysis did not affect the CIVDS. When the CIVDS and CDS vesicle pools were depleted by electrical stimulation, the pools had different sizes. Their kinetics of exocytosis-coupled endocytosis were also different. Thus, CIVDS and CDS used different vesicle pools in DRG neurons.

Reply to "TRPA1-dependent calcium transients and CGRP release in DRG neurons require extracellular calcium".

In this issue, Gebhardt et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201702151) express interest in our recently published work (Shang et al. 2016. J. Cell Biol.https://doi.org/10.1083/jcb.201603081). Here, we would like to address their concerns regarding the lysosomal TRPA1-mediated intracellular calcium transients in dorsal root ganglion neurons.

An Excitatory Neural Assembly Encodes Short-Term Memory in the Prefrontal Cortex.

Short-term memory (STM) is crucial for animals to hold information for a small period of time. Persistent or recurrent neural activity, together with neural oscillations, is known to encode the STM at the cellular level. However, the coding mechanisms at the microcircuitry level remain a mystery. Here, we performed two-photon imaging on behaving mice to monitor the activity of neuronal microcircuitry. We discovered a neuronal subpopulation in the medial prefrontal cortex (mPFC) that exhibited emergent properties in a context-dependent manner underlying a STM-like behavior paradigm. These neuronal subpopulations exclusively comprise excitatory neurons and mainly represent a group of neurons with stronger functional connections. Microcircuitry plasticity was maintained for minutes and was absent in an animal model of Alzheimer's disease (AD). Thus, these results point to a functional coding mechanism that relies on the emergent behavior of a functionally defined neuronal assembly to encode STM.

Loss of FMRP Impaired Hippocampal Long-Term Plasticity and Spatial Learning in Rats.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene that inactivate expression of the gene product, the fragile X mental retardation 1 protein (FMRP). In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate Fmr1 knockout (KO) rats by disruption of the fourth exon of the Fmr1 gene. Western blotting analysis confirmed that the FMRP was absent from the brains of the Fmr1 KO rats (Fmr1exon4-KO ). Electrophysiological analysis revealed that the theta-burst stimulation (TBS)-induced long-term potentiation (LTP) and the low-frequency stimulus (LFS)-induced long-term depression (LTD) were decreased in the hippocampal Schaffer collateral pathway of the Fmr1exon4-KO rats. Short-term plasticity, measured as the paired-pulse ratio, remained normal in the KO rats. The synaptic strength mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) was also impaired. Consistent with previous reports, the Fmr1exon4-KO rats demonstrated an enhanced 3,5-dihydroxyphenylglycine (DHPG)-induced LTD in the present study, and this enhancement is insensitive to protein translation. In addition, the Fmr1exon4-KO rats showed deficits in the probe trial in the Morris water maze test. These results demonstrate that deletion of the Fmr1 gene in rats specifically impairs long-term synaptic plasticity and hippocampus-dependent learning in a manner resembling the key symptoms of FXS. Furthermore, the Fmr1exon4-KO rats displayed impaired social interaction and macroorchidism, the results consistent with those observed in patients with FXS. Thus, Fmr1exon4-KO rats constitute a novel rat model of FXS that complements existing mouse models.

PGC-1α/ERRα-Sirt3 Pathway Regulates DAergic Neuronal Death by Directly Deacetylating SOD2 and ATP Synthase ß.

AIMS: Parkinson's disease (PD) heavily affects humans and little is known about its cause and pathogenesis. Sirtuin 3 (Sirt3) plays a key role in regulating mitochondrial dysfunction, which is the main cause of DAergic neuronal loss in PD. We investigated the mechanisms of neuroprotective role of Sirt3 in DAergic neuronal survival. RESULTS: Sirt3 was reduced in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-treated neurons with its overexpression being neuroprotective. We identified that Sirt3 interacted with manganese superoxide dismutase (SOD2) and adenosine triphosphate (ATP) synthase ß and modulated their activities by deacetylating SOD2 (K130) and ATP synthase ß (K485) to prevent reactive oxygen species accumulation and ATP depletion, and to alleviate DAergic neuronal death upon MPTP treatment. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) interacted with estrogen-related receptor alpha (ERRα) that bound to the Sirt3 promoter as its transcription factor to regulate Sirt3 expression and DAergic neuronal death. In the mouse midbrain, MPTP administration led to the loss of PGC-1α and Sirt3, high acetylation level of SOD2 and ATP synthase ß, and the specific loss of DAergic neurons, while Sirt3 overexpression could protect against DAergic neuronal loss. Sirt3 knockout mice exhibited more sensitive and more DAergic neuronal loss to MPTP treatment. INNOVATION: The study provides new insights into a critical PGC-1α/ERRα-Sirt3 pathway, linking regulation of mitochondrial protein acetylation and DAergic neuronal death in PD pathogenesis, which provide a potential therapeutic strategy and target in PD treatment. CONCLUSION: These results provide a vital PGC-1α/ERRα-Sirt3 pathway that protects against DAergic neuronal death by directly deacetylating SOD2 (K130) and ATP synthase ß (K485) in PD.

Deacetylation of TFEB promotes fibrillar Aß degradation by upregulating lysosomal biogenesis in microglia.

Microglia play a pivotal role in clearance of Aß by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aß clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aß (fAß) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAß degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAß degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAß degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.

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

Transient receptor potential A1 (TRPA1) is a nonselective cation channel implicated in thermosensation and inflammatory pain. In this study, we show that TRPA1 (activated by allyl isothiocyanate, acrolein, and 4-hydroxynonenal) elevates the intracellular Ca2+ concentration ([Ca2+]i) in dorsal root ganglion (DRG) neurons in the presence and absence of extracellular Ca2+ Pharmacological and immunocytochemical analyses revealed the presence of TRPA1 channels both on the plasma membrane and in endolysosomes. Confocal line-scan imaging demonstrated Ca2+ signals elicited from individual endolysosomes ("lysosome Ca2+ sparks") by TRPA1 activation. In physiological solutions, the TRPA1-mediated endolysosomal Ca2+ release contributed to ∼40% of the overall [Ca2+]i rise and directly triggered vesicle exocytosis and calcitonin gene-related peptide release, which greatly enhanced the excitability of DRG neurons. Thus, in addition to working via Ca2+ influx, TRPA1 channels trigger vesicle release in sensory neurons by releasing Ca2+ from lysosome-like organelles.

The formation and extinction of fear memory in tree shrews.

Fear is an emotion that is well-studied due to its importance for animal survival. Experimental animals, such as rats and mice, have been widely used to model fear. However, higher animals such as nonhuman primates have rarely been used to study fear due to ethical issues and high costs. Tree shrews are small mammals that are closely related to primates; they have been used to model human-related psychosocial conditions such as stress and alcohol tolerance. Here, we describe an experimental paradigm to study the formation and extinction of fear memory in tree shrews. We designed an experimental apparatus of a light/dark box with a voltage foot shock. We found that tree shrews preferred staying in the dark box in the daytime without stimulation and showed avoidance to voltage shocks applied to the footplate in a voltage-dependent manner. Foot shocks applied to the dark box for 5 days (10 min per day) effectively reversed the light-dark preference of the tree shrews, and this memory lasted for more than 50 days without any sign of memory decay (extinction) in the absence of further stimulation. However, this fear memory was reversed with 4 days of reverse training by applying the same stimulus to the light box. When reducing the stimulus intensity during the training period, a memory extinction and subsequently reinstatement effects were observed. Thus, our results describe an efficient method of monitoring fear memory formation and extinction in tree shrews.

Extracellular Ca²âº 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.

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 α6ß2* 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 nicotine's effects on striatal DA release.

Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals.

The mitochondrion is essential for energy metabolism and production of reactive oxygen species (ROS). In intact cells, respiratory mitochondria exhibit spontaneous "superoxide flashes", the quantal ROS-producing events consequential to transient mitochondrial permeability transition (tMPT). Here we perform the first in vivo imaging of mitochondrial superoxide flashes and tMPT activity in living mice expressing the superoxide biosensor mt-cpYFP, and demonstrate their coupling to whole-body glucose metabolism. Robust tMPT/superoxide flash activity occurred in skeletal muscle and sciatic nerve of anesthetized transgenic mice. In skeletal muscle, imaging tMPT/superoxide flashes revealed labyrinthine three-dimensional networks of mitochondria that operate synchronously. The tMPT/superoxide flash activity surged in response to systemic glucose challenge or insulin stimulation, in an apparently frequency-modulated manner and involving also a shift in the gating mode of tMPT. Thus, in vivo imaging of tMPT-dependent mitochondrial ROS signals and the discovery of the metabolism-tMPT-superoxide flash coupling mark important technological and conceptual advances for the study of mitochondrial function and ROS signaling in health and disease.