Direct interrogation of context-dependent GPCR activity with a universal biosensor platform.

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins encoded in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically relevant contexts. Here, we developed a collection of compact ONE vector G-protein Optical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform.

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically-relevant contexts. Here, we developed a collection of compact ONE vector G-protein Optical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed new insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally-occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.

Detecting GPCR Signals With Optical Biosensors of Gα-GTP in Cell Lines and Primary Cell Cultures.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and mediate a wide variety of physiological processes. GPCRs respond to a plethora of extracellular ligands and initiate signaling pathways inside cells via heterotrimeric G proteins (Gαßγ). Because of the critical role GPCRs play in regulating biological processes and as pharmacological targets, the availability of tools to measure their signaling activity are of high interest. Live-cell biosensors that detect the activity of G proteins in response to GPCR stimulation have emerged as a powerful approach to investigate GPCR/G protein signaling. Here, we detail methods to monitor G protein activity through direct measurement of GTP-bound Gα subunits using optical biosensors based on bioluminescence resonance energy transfer (BRET). More specifically, this article describes the use of two types of complementary biosensors. The first protocol explains how to use a multicomponent BRET biosensor that relies on expression of exogenous G proteins in cell lines. This protocol yields robust responses that are compatible with endpoint measurements of dose-dependent ligand effects or with kinetic measurements of subsecond resolution. The second protocol describes the implementation of unimolecular biosensors that detect the activation of endogenous G proteins in cell lines expressing exogenous GPCRs or in primary cells upon stimulation of endogenous GPCRs. Overall, using the biosensors as described in this article will help users characterize the mechanisms of action of many pharmacological agents and natural ligands that modulate GPCR and G protein signaling with high precision. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Using bimolecular BRET biosensors to monitor Gα-GTP formation of tagged Gα in live cells Alternate Protocol 1: Measuring GPCR dose-dependent Gα-GTP responses in endpoint format Basic Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity Alternate Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity in mouse cortical neurons.

Antiseizure effect of 2-deoxyglucose is not dependent on the presynaptic vacuole ATP pump or the somatic ATP-sensitive K+ channel.

Inhibition of glycolysis with 2-deoxyglucose (2-DG) produces antiseizure effects in brain slices and animal models, yet the mechanisms remain elusive. Here, we examined two glycolysis-derived ATP-associated mechanisms: vacuole ATP pump (V-ATPase) and ATP-sensitive K+ channel (KATP). Epileptiform bursts were generated in the CA3 area of hippocampal slices by 0 Mg2+ and 4-aminopyridine. 2-DG consistently abolished epileptiform bursts in the presence of pyruvate (to sustain tricarboxylic acid cycle for oxidative ATP production) at 30-33°C but not at room temperature (22°C). Under physiological conditions, 2-DG did not reduce the amplitude of evoked excitatory postsynaptic currents (EPSCs) or the paired-pulse ratio in CA3 neurons. During repetitive high-frequency (20 Hz, 20-50 pulses) stimulation, 2-DG did not accelerate the decline of EPSCs (i.e., depletion of transmitter release), even when preincubated with 8 mM K+ to enhance activity-dependent uptake of 2-DG. In addition, in 2-DG tetanic stimulation (200 Hz, 1 s) dramatically increased rather than diminished the occurrence of spontaneous EPSCs immediately after stimulation (i.e., no transmitter depletion). Moreover, a V-ATPase blocker (concanamycin) failed to block epileptiform bursts that were subsequently abolished by 2-DG. Furthermore, 2-DG did not induce detectable KATP current in hippocampal neurons. Finally, epileptiform bursts were not affected by either a KATP opener (diazoxide) or a KATP blocker (glibenclamide) but were blocked by 2-DG in the same slices. Altogether, these data suggest that 2-DG's antiseizure action is temperature dependent and achieved exclusively by inhibition of glycolysis and is not likely to be mediated by the two membrane-bound ATP-associated machinery mechanisms, V-ATPase and KATP.NEW & NOTEWORTHY Inhibition of glycolysis with 2-deoxyglucose (2-DG) represents a novel metabolic antiseizure approach, yet the mechanisms remain elusive. Here, we show that 2-DG's antiseizure action is both glycolysis and temperature dependent but not mediated by the vacuole ATP pump (V-ATPase) or ATP-sensitive K+ channel (KATP). Our data provide new insights to understand 2-DG's cellular mechanisms of action and, more broadly, neuronal metabolism and excitability.

2-deoxyglucose and ß-hydroxybutyrate fail to attenuate seizures in the betamethasone-NMDA model of infantile spasms.

Infantile spasms (IS) is an epileptic encephalopathy with a poor neurodevelopmental prognosis, and limited, often ineffective treatment options. The effectiveness of metabolic approaches to seizure control is being increasingly shown in a wide variety of epilepsies. This study investigates the efficacy of the glycolysis inhibitor 2-deoxyglucose (2-DG) and the ketone body ß-hydroxybutyrate (BHB) in the betamethasone-NMDA model of rat IS. Prenatal rats were exposed to betamethasone on gestational day 15 (G15) and NMDA on postnatal day 15 (P15). Video-electroencephalography (v-EEG) was used to monitor spasms. NMDA consistently induced hyperflexion spasms associated with interictal sharp-slow wave EEG activity and ictal flattening of EEG signals, reminiscent of hypsarrhythmia and electrodecrement, respectively. 2-DG (500 mg/kg, i.p), BHB (200 mg/kg, i.p.), or both were administered immediately after occurrence of the first spasm. No experimental treatment altered significantly the number, severity, or progression of spasms compared with saline treatment. These data suggest that metabolic inhibition of glycolysis or ketogenesis does not reduce infantile spasms in the NMDA model. The study further validates the betamethasone-NMDA model in terms of its behavioral and electrographic resemblance to human IS and supports its use for preclinical drug screening.

Na+-K+-ATPase functions in the developing hippocampus: regional differences in CA1 and CA3 neuronal excitability and role in epileptiform network bursting.

The Na+-K+-ATPase (Na+-K+ pump) is essential for setting resting membrane potential and restoring transmembrane Na+ and K+ gradients after neuronal firing, yet its roles in developing neurons are not well understood. This study examined the contribution of the Na+-K+ pump to resting membrane potential and membrane excitability of developing CA1 and CA3 neurons and its role in maintaining synchronous network bursting. Experiments were conducted in postnatal day (P)9 to P13 rat hippocampal slices using whole cell patch-clamp and extracellular field-potential recordings. Blockade of the Na+-K+ pump with strophanthidin caused marked depolarization (23.1 mV) in CA3 neurons but only a modest depolarization (3.3 mV) in CA1 neurons. Regarding other membrane properties, strophanthidin differentially altered the voltage-current responses, input resistance, action-potential threshold and amplitude, rheobase, and input-output relationship in CA3 vs. CA1 neurons. At the network level, strophanthidin stopped synchronous epileptiform bursting in CA3 induced by 0 Mg2+ and 4-aminopyridine. Furthermore, dual whole cell recordings revealed that strophanthidin disrupted the synchrony of CA3 neuronal firing. Finally, strophanthidin reduced spontaneous excitatory postsynaptic current (sEPSC) bursts (i.e., synchronous transmitter release) and transformed them into individual sEPSC events (i.e., nonsynchronous transmitter release). These data suggest that the Na+-K+ pump plays a more profound role in membrane excitability in developing CA3 neurons than in CA1 neurons and that the pump is essential for the maintenance of synchronous network bursting in CA3. Compromised Na+-K+ pump function leads to cessation of ongoing synchronous network activity, by desynchronizing neuronal firing and neurotransmitter release in the CA3 synaptic network. These findings have implications for the regulation of network excitability and seizure generation in the developing brain.NEW & NOTEWORTHY Despite the extensive literature showing the importance of the Na+-K+ pump in various neuronal functions, its roles in the developing brain are not well understood. This study reveals that the Na+-K+ pump differentially regulates the excitability of CA3 and CA1 neurons in the developing hippocampus, and the pump activity is crucial for maintaining network activity. Compromised Na+-K+ pump activity desynchronizes neuronal firing and transmitter release, leading to cessation of ongoing epileptiform network bursting.

The efficacy of fructose-1,6-bisphosphate in suppressing status epilepticus in developing rats.

PURPOSE: Treatment of pediatric status epilepticus (SE) remains challenging as up to 50 % of patients are refractory to conventional anti-seizure medications. The glycolytic intermediate, fructose-1,6-bisphosphate (FBP), has been reported to exert significant anticonvulsant effects in both adult animals and in in vitro models of seizures. This study aims to examine FBP efficacy in controlling seizures in a rat model of juvenile SE. METHODS: Sprague Dawley rats (P11-P17) were injected with pilocarpine (300 mg/kg, i.p.) to induce SE, which was monitored by video-electroencephalography (v-EEG). Thirty minutes into SE, FBP was administrated (500 or 1000 mg/kg, i.p.). v-EEG recording was continued for ∼60 additional minutes to assess the anticonvulsant effect of FBP, compared with vehicle (saline) treatment. RESULTS: SE consistently occurred in rat pups 10-15 min after pilocarpine injection and persisted over the 90-min recording period. Neither saline nor a lower dose of FBP (500 mg/kg) treatment stopped behavioral and electrographic seizures. At higher doses (1000 mg/kg), FBP terminated SE in ∼15 min in 60 % (6 of 10) of the rat pups. CONCLUSION: The endogenous glycolytic metabolite, FBP, promptly suppresses ongoing seizure activity and represents a potential alternative metabolic therapy to improve the treatment of SE in the juvenile age range.

2-Deoxyglucose terminates pilocarpine-induced status epilepticus in neonatal rats.

OBJECTIVE: Neonatal status epilepticus (SE) is a life-threatening medical emergency. Unfortunately, up to 50% of neonates with SE are resistant to current antiseizure drugs, highlighting the need for better treatments. This study aims to explore a novel metabolic approach as a potential alternative treatment to control neonatal SE, using the glycolytic inhibitor 2-deoxyglucose (2-DG). METHODS: SE was induced by pilocarpine (300 mg/kg, intraperitoneally [ip]) in neonatal Sprague Dawley rats (postnatal day 10 [P10]-P17) and was monitored by video-electroencephalography (V-EEG). After 30 minutes of SE, 2-DG or one of two conventional antiseizure drugs with different mechanisms of action, phenobarbital or levetiracetam, was administrated ip, and V-EEG recording was continued for ~60 additional minutes. The time to seizure cessation after drug injection, EEG scores, and power spectra before and after drug or saline treatment were used to assess drug effects. RESULTS: Once SE became sustained, administration of 2-DG (50, 100, or 500 mg/kg, ip) consistently stopped behavioral and electrographic seizures within 10-15 minutes; lower doses took longer (25-30 minutes) to stop SE, demonstrating a dose-dependent effect. Administration of phenobarbital (30 mg/kg, ip) or levetiracetam (100 mg/kg, ip) also stopped SE within 10-15 minutes in neonatal rats. SIGNIFICANCE: Our results suggest that the glycolysis inhibitor 2-DG acts quickly to reduce neuronal hyperexcitability and effectively suppress ongoing seizure activity, which may provide translational value in the treatment of neonatal SE.

Infantile Spasms: An Update on Pre-Clinical Models and EEG Mechanisms.

Infantile spasms (IS) is an epileptic encephalopathy with unique clinical and electrographic features, which affects children in the middle of the first year of life. The pathophysiology of IS remains incompletely understood, despite the heterogeneity of IS etiologies, more than 200 of which are known. In particular, the neurobiological basis of why multiple etiologies converge to a relatively similar clinical presentation has defied explanation. Treatment options for this form of epilepsy, which has been described as "catastrophic" because of the poor cognitive, developmental, and epileptic prognosis, are limited and not fully effective. Until the pathophysiology of IS is better clarified, novel treatments will not be forthcoming, and preclinical (animal) models are essential for advancing this knowledge. Here, we review preclinical IS models, update information regarding already existing models, describe some novel models, and discuss exciting new data that promises to advance understanding of the cellular mechanisms underlying the specific EEG changes seen in IS-interictal hypsarrhythmia and ictal electrodecrement.

Identification of MAGUK scaffold proteins as intracellular binding partners of synaptic adhesion protein Slitrk2.

Synaptic adhesion proteins play a critical role in the formation and maintenance of synapses in the developing nervous system. Errors in synaptic adhesion constitute the molecular basis of many neuropsychiatric disorders, including schizophrenia, bipolar disorder, Tourette syndrome, and autism. Slit- and Trk-like proteins (Slitrks) are a family of leucine-rich repeat containing transmembrane proteins that promote synaptogenesis. These proteins localize to the postsynaptic density, where they induce synapse formation via trans-synaptic interactions with receptor protein tyrosine phosphatases. While trans-synaptic binding partners of Slitrks have been reported, little is known about the intracellular proteins that associate with Slitrks. Here we report an interaction between Slitrk2 and members of the PSD-95 subfamily of membrane associated guanylate kinases (MAGUKs). Coimmunoprecipitation from postnatal mouse brain indicates that PSD-93 and PSD-95 associate with Slitrk2 in vivo. Mapping analysis in yeast demonstrates that Slitrk2 interacts directly with PSD-95 via a non-canonical Src homology 3 (SH3) domain binding motif that associates with the SH3 domain of PSD-95. We also show that PSD-95 induces robust clustering of Slitrk2 in 293T cells, and deletion of the SH3 domain in PSD-95 or the SH3 domain binding motif in Slitrk2 reduces this clustering. These data confirm PSD-95 as the first known intracellular binding partner of Slitrk2. Future studies will examine if Slitrk-MAGUK interactions mediate localization of Slitrks to synaptic sites and facilitate recruitment of additional intracellular signaling molecules involved in postsynaptic differentiation.