RESUMO
Platelet activation of protease-activated receptor 4 (PAR4) and thrombin are at the top of a chain of events leading to fibrin deposition, microinfarcts, blood-brain barrier disruption, and inflammation. We evaluated mRNA expression of the PAR4 gene F2RL3 in human brain and global cognitive performance in participants with and without cognitive impairment or dementia. Data were acquired from the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP). F2RL3 mRNA was elevated in AD cases and was associated with worse retrospective longitudinal cognitive performance. Moreover, F2RL3 expression interacted with clinical AD diagnosis on longitudinal cognition whereas this relationship was attenuated in individuals without cognitive impairment. Additionally, when adjusting for the effects of AD neuropathology, F2RL3 expression remained a significant predictor of cognitive decline. F2RL3 expression correlated positively with transcript levels of proinflammatory markers including TNFα, IL-1ß, NFκB, and fibrinogen α/ß/γ. Together, these results reveal that F2RL3 mRNA expression is associated with multiple AD-relevant outcomes and its encoded product, PAR4, may play a role in disease pathogenesis.
Assuntos
Doença de Alzheimer , Expressão Gênica , RNA Mensageiro , Receptores de Trombina , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Masculino , Feminino , Idoso de 80 Anos ou mais , RNA Mensageiro/metabolismo , Expressão Gênica/genética , Idoso , Disfunção Cognitiva/genética , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Encéfalo/metabolismo , Cognição , Inflamação/genética , NF-kappa B/metabolismo , NF-kappa B/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Mediadores da Inflamação/metabolismoRESUMO
Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF.
RESUMO
Protease-activated receptor 4 (PAR4) is a G protein-coupled receptor activated by thrombin. In the platelet, response to thrombin PAR4 contributes to the predominant procoagulant microparticle formation, increased fibrin deposition, and initiation of platelet-stimulated inflammation. In addition, PAR4 is expressed in other cell types, including endothelial cells. Under inflammatory conditions, PAR4 is overexpressed via epigenetic demethylation of the PAR4 gene, F2RL3. PAR4 knockout (KO) studies have determined a role for PAR4 in ischemia-reperfusion injury in the brain, and PAR4 KO mice display normal cardiac function but present less myocyte death and cardiac dysfunction in response to acute myocardial infarction. Although PAR4 has been reported to be expressed within the kidney, the contribution of PAR4 to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 KO mice are protected against kidney injury in two mouse models. First, PAR4 KO mice are protected against induction of markers of both fibrosis and inflammation in two different models of kidney injury: 1) 7 days following unilateral ureter obstruction (UUO) and 2) an AKI-CKD model of ischemia-reperfusion followed by 8 days of contralateral nephrectomy. We further show that PAR4 expression in the kidney is low in the control mouse kidney but induced over time following UUO. PAR4 KO mice are protected against blood urea nitrogen (BUN) and glomerular filtration rate (GFR) kidney function pathologies in the AKI-CKD model. Following the AKI-CKD model, PAR4 is expressed in the collecting duct colocalizing with Dolichos biflorus agglutinin (DBA), but not in the proximal tubule with Lotus tetragonolobus lectin (LTL). Collectively, the results reported in this study implicate PAR4 as contributing to the pathology in mouse models of acute and chronic kidney injury.NEW & NOTEWORTHY The contribution of the thrombin receptor protease-activated receptor 4 (PAR4) to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 expression is upregulated after kidney injury and PAR4 knockout (KO) mice are protected against fibrosis following kidney injury in two mouse models. First, PAR4 KO mice are protected against unilateral ureter obstruction. Second, PAR4 KO mice are protected against an AKI-CKD model of ischemia-reperfusion followed by contralateral nephrectomy.
Assuntos
Injúria Renal Aguda , Insuficiência Renal Crônica , Animais , Camundongos , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Células Endoteliais/metabolismo , Fibrose , Inflamação/patologia , Isquemia/patologia , Rim/metabolismo , Camundongos Knockout , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/patologia , Trombina/metabolismo , Trombina/farmacologiaRESUMO
Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o GPCRs by Gßγ subunit inhibition of 2 mechanisms: decreased calcium entry and direct interaction of Gßγ with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) that decreased Gßγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact and removing GPCR-Gßγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. Snap25Δ3/Δ3 mice exhibited enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in Snap25Δ3/Δ3 mice challenged with a high-fat diet. Glucose homeostasis, whole-body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in Snap25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting that basal autonomic activity was required. Direct electrode stimulation of sympathetic neuron exocytosis from Snap25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gßγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combating metabolic disease.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Insulinas , Camundongos , Animais , Cálcio/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Exocitose/fisiologia , Proteínas SNARE/genética , Dieta , Obesidade/genética , Adipócitos/metabolismo , Insulinas/metabolismo , Insulina/metabolismoRESUMO
The nucleus accumbens (NAc) guides reward-related motivated behavior implicated in pathological behavioral states, including addiction and depression. These behaviors depend on the precise neuromodulatory actions of Gi/o-coupled G-protein-coupled receptors (GPCRs) at glutamatergic synapses onto medium spiny projection neurons (MSNs). Previous work has shown that discrete classes of Gi/o-coupled GPCR mobilize Gßγ to inhibit vesicular neurotransmitter release via t-SNARE protein, SNAP25. However, it remains unknown which Gαi/o systems in the NAc utilize Gßγ-SNARE signaling to dampen glutamatergic transmission. Utilizing patch-clamp electrophysiology and pharmacology in a transgenic mouse line with a C-terminal three-residue deletion of SNAP25 (SNAP25Δ3) weaking the Gßγ-SNARE interaction, we surveyed a broad cohort of Gi/o-coupled GPCRs with robust inhibitory actions at glutamatergic synapses in the NAc. We find that basal presynaptic glutamate release probability is reduced in SNAP25Δ3 mice. While κ opioid, CB1, adenosine A1, group II metabotropic glutamate receptors, and histamine H3 receptors inhibit glutamatergic transmission onto MSNs independent of SNAP25, we report that SNAP25 contributes significantly to the actions of GABAB, 5-HT1B/D, and µ opioid receptors. These findings demonstrate that presynaptic Gi/o-coupled GPCRs recruit heterogenous effector mechanisms at glutamatergic synapses in the NAc, with a subset requiring SNA25-dependent Gßγ signaling.
RESUMO
Inhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gßγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gßγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gßγ targets at the presynaptic terminal.
Assuntos
Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de GABA-B/metabolismo , Transmissão Sináptica/fisiologia , Animais , Feminino , Ácido Glutâmico/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismoRESUMO
Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the base and diffuses to the tip and that it expedites flash response recovery, there should be an axial concentration gradient for bicarbonate that would accentuate the base-to-tip SPR differences. Seeking to understand how ROS geometry and bicarbonate affect SPR variability, we used mathematical modeling and made electrophysiological recordings of single rods. Modeling predicted and our experiments confirmed minor radial SPR variability in large diameter, salamander rods that was essentially unchanged by bicarbonate. SPRs elicited at the base and tip of salamander rods were similar in the absence of bicarbonate, but when treated with 30 mM bicarbonate, SPRs at the base became slightly faster than those at the tip, verifying the existence of an axial gradient for bicarbonate. The differences were small and unlikely to undermine visual signaling. However, in toad rods with longer ROSs, bicarbonate somehow suppressed the substantial, axial SPR variability that is naturally present in the absence of bicarbonate. Modeling suggested that the axial gradient of bicarbonate might dampen the primary phototransduction cascade at the base of the ROS. This novel effect of bicarbonate solves a mystery as to how toad vision is able to function effectively in extremely dim light.
RESUMO
Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, rods operate over many log units of light intensity. Several processes inside photoreceptor cells make this incredible light adaptation possible. Here, we added to our previously developed, fully space resolved biophysical model of rod phototransduction, some of the mechanisms that play significant roles in shaping the rod response under high illumination levels: the function of RGS9 in shutting off G protein transducin, and calcium dependences of the phosphorylation rates of activated rhodopsin, of the binding of cGMP to the light-regulated ion channel, and of two membrane guanylate cyclase activities. A well stirred version of this model captured the responses to bright, saturating flashes in WT and mutant mouse rods and was used to explain "Pepperberg plots," that graph the time during which the response is saturated against the natural logarithm of flash strength for bright flashes. At the lower end of the range, saturation time increases linearly with the natural logarithm of flash strength. The slope of the relation (τD) is dictated by the time constant of the rate-limiting (slowest) step in the shutoff of the phototransduction cascade, which is the hydrolysis of GTP by transducin. We characterized mathematically the X-intercept ( Φ o ) which is the number of photoisomerizations that just saturates the rod response. It has been observed that for flash strengths exceeding a few thousand photoisomerizations, the curves depart from linearity. Modeling showed that the "upward bend" for very bright flash intensities could be explained by the dynamics of RGS9 complex and further predicted that there would be a plateau at flash strengths giving rise to more than ~107 photoisomerizations due to activation of all available PDE. The model accurately described alterations in saturation behavior of mutant murine rods resulting from transgenic perturbations of the cascade targeting membrane guanylate cyclase activity, and expression levels of GRK, RGS9, and PDE. Experimental results from rods expressing a mutant light-regulated channel purported to lack calmodulin regulation deviated from model predictions, suggesting that there were other factors at play.
RESUMO
The detailed pharmacology and therapeutic potential of the central PAR4 receptors are poorly understood due to a lack of potent, selective, and brain-penetrant tool compounds. Despite this, robust data with biochemical and genetic tools show the therapeutic potential of PAR4 antagonists in traumatic brain injury, Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders with a neuroinflammatory component. Thus, we performed a functional HTS campaign, identified a fundamentally new PAR4 competitive inhibitor chemotype, optimized this new series (increased potency >45-fold), discovered enantiospecific activity (though opposing preference for human versus mouse PAR4), and engendered high central nervous system penetration (rat Kp's of 0.52 to 4.2 and Kp,uu's of 0.52 to 1.2).
Assuntos
Sistema Nervoso Central , Receptores de Trombina , Animais , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Camundongos , Ratos , Receptores de Trombina/metabolismoRESUMO
Ligand binding to G proteincoupled receptors (GPCRs), such as the α2a-adrenergic receptor (α2aAR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gßγ dimers. α2aAR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gßγ effector. To understand how the Gßγ-SNARE complex underlies the α2aAR-dependent inhibition of synaptic transmission, we examined the specificity of Gßγ subunits for the SNARE complex in adrenergic neurons, in which auto-α2aARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α2aARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gß and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α2aARs, Gß1 and Gγ3 interacted with the SNARE complex. However, Gß1, Gß2, and Gγ3 were found in the complex when auto-α2aARs were activated by epinephrine. Further understanding of the specific usage of distinct Gßγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas SNARE , Animais , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Masculino , Camundongos Transgênicos , Proteínas SNARE/metabolismoRESUMO
In daylight, cone photoreceptors in the retina are responsible for the bulk of visual perception, yet compared to rods, far less is known quantitatively about their biochemistry. This is partly because it is hard to isolate and purify cone proteins. The issue is also complicated by the synergistic interaction of these parameters in producing systems biology outputs, such as photoresponse. Using a 3-D resolved, finite element model of cone outer segments, here we conducted a study of parameter significance using global sensitivity analysis, by Sobol indices, which was contextualized within the uncertainty surrounding these parameters in the available literature. The analysis showed that a subset of the parameters influencing the circulating dark current, such as the turnover rate of cGMP in the dark, may be most influential for variance with experimental flash response, while the shut-off rates of photoexcited rhodopsin and phosphodiesterase also exerted sizable effect. The activation rate of transducin by rhodopsin and the light-induced hydrolysis rate of cGMP exerted measurable effects as well but were estimated as relatively less significant. The results of this study depend on experimental ranges currently described in the literature and should be revised as these become better established. To that end, these findings may be used to prioritize parameters for measurement in future investigations.
Assuntos
GMP Cíclico/metabolismo , Transdução de Sinal Luminoso , Luz , Células Fotorreceptoras Retinianas Cones/fisiologia , Transducina/fisiologia , Visão Ocular/fisiologia , Animais , Camundongos , Camundongos KnockoutRESUMO
Retinal rods function as accurate photon counters to provide for vision under very dim light. To do so, rods must generate highly amplified, reproducible responses to single photons, yet outer segment architecture and randomness in the location of rhodopsin photoisomerization on the surface of an internal disk introduce variability to the rising phase of the photon response. Soon after a photoisomerization at a disk rim, depletion of cGMP near the plasma membrane closes ion channels and hyperpolarizes the rod. But with a photoisomerization in the center of a disk, local depletion of cGMP is distant from the channels in the plasma membrane. Thus, channel closure is delayed by the time required for the reduction of cGMP concentration to reach the plasma membrane. Moreover, the local fall in cGMP dissipates over a larger volume before affecting the channels, so response amplitude is reduced. This source of variability increases with disk radius. Using a fully space-resolved biophysical model of rod phototransduction, we quantified the variability attributable to randomness in the location of photoisomerization as a function of disk structure. In mouse rods that have small disks bearing a single incisure, this variability was negligible in the absence of the incisure. Variability was increased slightly by the incisure, but randomness in the shutoff of rhodopsin emerged as the main source of single photon response variability at all but the earliest times. Variability arising from randomness in the transverse location of photoisomerization increased in magnitude and persisted over a longer period in the photon response of large salamander rods. A symmetric arrangement of multiple incisures in the disks of salamander rods greatly reduced this variability during the rising phase, but the incisures had the opposite effect on variability arising from randomness in rhodopsin shutoff at later times.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/metabolismo , Visão Ocular/fisiologia , Animais , Isomerismo , Camundongos , Modelos Teóricos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , UrodelosRESUMO
Although new drug discoveries are revolutionizing cancer treatments, repurposing existing drugs would accelerate the timeline and lower the cost for bringing treatments to cancer patients. Our goal was to repurpose CPI211, a potent and selective antagonist of the thromboxane A2-prostanoid receptor (TPr), a G-protein-coupled receptor that regulates coagulation, blood pressure, and cardiovascular homeostasis. To identify potential new clinical indications for CPI211, we performed a phenome-wide association study (PheWAS) of the gene encoding TPr, TBXA2R, using robust deidentified health records and matched genomic data from more than 29,000 patients. Specifically, PheWAS was used to identify clinical manifestations correlating with a TBXA2R single-nucleotide polymorphism (rs200445019), which generates a T399A substitution within TPr that enhances TPr signaling. Previous studies have correlated 200445019 with chronic venous hypertension, which was recapitulated by this PheWAS analysis. Unexpectedly, PheWAS uncovered an rs200445019 correlation with cancer metastasis across several cancer types. When tested in several mouse models of metastasis, TPr inhibition using CPI211 potently blocked spontaneous metastasis from primary tumors, without affecting tumor cell proliferation, motility, or tumor growth. Further, metastasis following intravenous tumor cell delivery was blocked in mice treated with CPI211. Interestingly, TPr signaling in vascular endothelial cells induced VE-cadherin internalization, diminished endothelial barrier function, and enhanced transendothelial migration by tumor cells, phenotypes that were decreased by CPI211. These studies provide evidence that TPr signaling promotes cancer metastasis, supporting the study of TPr inhibitors as antimetastatic agents and highlighting the use of PheWAS as an approach to accelerate drug repurposing.
Assuntos
Antineoplásicos/farmacologia , Reposicionamento de Medicamentos , Estudo de Associação Genômica Ampla/métodos , Receptores de Tromboxanos/antagonistas & inibidores , Receptores de Tromboxanos/genética , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metástase Neoplásica , Fenótipo , Polimorfismo de Nucleotídeo Único , Receptores de Tromboxanos/metabolismoRESUMO
Behavioral assays in the mouse can show marked differences between male and female animals of a given genotype. These differences identified in such preclinical studies may have important clinical implications. We recently made a mouse model with impaired presynaptic inhibition through Gßγ-SNARE signaling. Here, we examine the role of sexual dimorphism in the severity of the phenotypes of this model, the SNAP25Δ3 mouse. In males, we already reported that SNAP25Δ3 homozygotes demonstrated phenotypes in motor coordination, nociception, spatial memory and stress processing. We now report that while minimal sexually dimorphic effects were observed for the nociceptive, motor or memory phenotypes, large differences were observed in the stress-induced hyperthermia paradigm, with male SNAP25Δ3 homozygotes exhibiting an increase in body temperature subsequent to handling relative to wild-type littermates, while no such genotype-dependent effect was observed in females. This suggests sexually dimorphic mechanisms of Gßγ-SNARE signaling for stress processing or thermoregulation within the mouse. Second, we examined the effects of heterozygosity with respect to the SNAP25Δ3 mutation. Heterozygote SNAP25Δ3 animals were tested alongside homozygote and wild-type littermates in all of the aforementioned paradigms and displayed phenotypes similar to wild-type animals or an intermediate state. From this, we conclude that the SNAP25Δ3 mutation does not behave in an autosomal dominant manner, but rather displays incomplete dominance for many phenotypes.
Assuntos
Hipertermia , Caracteres Sexuais , Animais , Modelos Animais de Doenças , Exocitose , Feminino , Masculino , Camundongos , Memória EspacialRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Assuntos
Cálcio/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Neurotransmissores/metabolismo , Proteínas SNARE/metabolismo , Animais , Humanos , Ligação Proteica/fisiologiaRESUMO
The single photon response (SPR) in vertebrate photoreceptors is inherently variable due to several stochastic events in the phototransduction cascade, the main one being the shutoff of photoactivated rhodopsin. Deactivation is driven by a random number of steps, each of random duration with final quenching occurring after a random delay. Nevertheless, variability of the SPR is relatively low, making the signal highly reliable. Several biophysical and mathematical mechanisms contributing to variability suppression have been examined by the authors. Here we investigate the contribution of local depletion of cGMP by PDE*, the non linear dependence of the photocurrent on cGMP, Ca2+ feedback by making use of a fully space resolved (FSR) mathematical model, applied to two species (mouse and salamander), by varying the cGMP diffusion rate severalfold and rod outer segment diameter by an order of magnitude, and by introducing new, more refined, and time dependent variability functionals. Globally well stirred (GWS) models, and to a lesser extent transversally well stirred models (TWS), underestimate the role of nonlinearities and local cGMP depletion in quenching the variability of the circulating current with respect to fully space resolved models (FSR). These distortions minimize the true extent to which SPR is stabilized by locality in cGMP depletion, nonlinear effects linking cGMP to current, and Ca2+ feedback arising from the physical separation of E* from the ion channels located on the outer shell, and the diffusion of these second messengers in the cytoplasm.
Assuntos
Cálcio/metabolismo , GMP Cíclico/metabolismo , Modelos Biológicos , Fótons , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais , Algoritmos , Animais , Biomarcadores , Camundongos , Segmento Externo da Célula Bastonete/fisiologiaRESUMO
Complex circuit interactions within the nucleus accumbens (NAc) facilitate goal-directed behavior. Medium spiny neurons (MSNs) mediate NAc output by projecting to functionally divergent brain regions, a property conferred, in part, by the differential projection patterns of D1- and D2 dopamine receptor-expressing MSNs. Glutamatergic afferents to the NAc direct MSN output by recruiting feedforward inhibitory microcircuits comprised of parvalbumin (PV)-expressing interneurons (INs). Furthermore, the GABAB heteroreceptor (GABABR), a Gi/o-coupled G-protein-coupled receptor, is expressed at glutamatergic synapses throughout the mesolimbic network, yet its physiological context and synaptic mechanism within the NAc remains unknown. Here, we explored GABABR function at glutamatergic synapses within PV-IN-embedded microcircuits in the NAc core of male mice. We found that GABABR is expressed presynaptically and recruits a noncanonical signaling mechanism to reduce glutamatergic synaptic efficacy at D1(+) and D1(-) (putative D2) MSN subtypes. Furthermore, PV-INs, a robust source of neuronal GABA in the NAc, heterosynaptically target GABABR to selectively modulate glutamatergic transmission onto D1(+) MSNs. These findings elucidate a new mechanism of feedforward inhibition and refine mechanisms by which GABAB heteroreceptors modulate mesolimbic circuit function.SIGNIFICANCE STATEMENT Glutamatergic transmission in the nucleus accumbens (NAc) critically contributes to goal-directed behaviors. However, intrinsic microcircuit mechanisms governing the integration of these synapses remain largely unknown. Here, we show that parvalbumin-expressing interneurons within feedforward microcircuits heterosynaptically target GABAB heteroreceptors (GABABR) on glutamate terminals. Activation of presynaptically-expressed GABABR decreases glutamatergic synaptic strength by engaging a non-canonical signaling pathway that interferes with vesicular exocytotic release machinery. These findings offer mechanistic insight into the role of GABAB heteroreceptors within reward circuitry, elucidate a novel arm to feedforward inhibitory networks, and inform the growing use of GABABR-selective pharmacotherapy for various motivational disorders, including addiction, major depressive disorder, and autism (Cousins et al., 2002; Kahn et al., 2009; Jacobson et al., 2018; Stoppel et al., 2018; Pisansky et al., 2019).
Assuntos
Ácido Glutâmico/metabolismo , Rede Nervosa/metabolismo , Núcleo Accumbens/metabolismo , Receptores de GABA-B/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Agonistas dos Receptores de GABA-B/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Receptores de GABA-B/genética , Sinapses/efeitos dos fármacos , Sinapses/genética , Transmissão Sináptica/efeitos dos fármacosRESUMO
Three SNARE proteins, SNAP-25, syntaxin 1A, and VAMP2 or synaptobrevin 2, constitute the minimal functional machinery needed for the regulated secretion of neurotransmitters. Dynamic changes in the regulated release of neurotransmitters are associated with the induction of long-term plasticity at central synapses. In-vitro studies have validated the C-terminus of SNAP-25 as a target for inhibitory Gi/o-coupled G-protein coupled receptors at a number of synapses. The physiological consequences of the interaction between Gi/o proteins and SNAP-25 in the context of activity-dependent long-term synaptic plasticity are not well understood. Here, we report direct ex-vivo evidence of the involvement of the C-terminus of SNAP-25 in inducing long-term potentiation of synaptic strength at Schaffer collateral-CA1 synapses using a gene-targeted mouse model with truncated C-terminus (carboxyl terminus) of SNAP-25. It has been shown previously that truncation of the three extreme C-terminal residues in SNAP-25[INCREMENT]3 homozygote mice reduces its interaction with the inhibitory Gßγ subunits two-fold. In in-vitro hippocampal slices, we show that these SNAP-25[INCREMENT]3 mice express significantly larger magnitude of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses.
Assuntos
Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciação de Longa Duração/genética , Camundongos Transgênicos , Plasticidade Neuronal/genética , Sinapses/genética , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Lobo Temporal/metabolismoRESUMO
Throughout the past five decades, tremendous advancements have been made in our understanding of G protein signaling and presynaptic inhibition, many of which were published in the Journal of Biological Chemistry under the tenure of Herb Tabor as Editor-in-Chief. Here, we identify these critical advances, including the formulation of the ternary complex model of G protein-coupled receptor signaling and the discovery of Gßγ as a critical signaling component of the heterotrimeric G protein, along with the nature of presynaptic inhibition and its physiological role. We provide an overview for the discovery and physiological relevance of the two known Gßγ-mediated mechanisms for presynaptic inhibition: first, the action of Gßγ on voltage-gated calcium channels to inhibit calcium influx to the presynaptic active zone and, second, the direct binding of Gßγ to the SNARE complex to displace synaptotagmin downstream of calcium entry, which has been demonstrated to be important in neurons and secretory cells. These two mechanisms act in tandem with each other in a synergistic manner to provide more complete spatiotemporal control over neurotransmitter release.