Antiretroviral drugs from multiple classes induce loss of excitatory synapses between hippocampal neurons in culture.

Introduction: Antiretroviral (ARV) drugs have improved prognoses for people living with HIV. However, HIV-associated neurocognitive disorders (HAND) persist despite undetectable viral loads. Some ARVs have been linked to neuropsychiatric effects that may contribute to HAND. Synapse loss correlates with cognitive decline in HAND and synaptic deficits may contribute to the neuropsychiatric effects of ARV drugs. Methods: Using an automated high content assay, rat hippocampal neurons in culture expressing PSD95-eGFP to label glutamatergic synapses and mCherry to fill neuronal structures were imaged before and after treatment with 25 clinically used ARVs. Results and Discussion: At a concentration of 10 µM the protease inhibitors nelfinavir and saquinavir, the non-nucleoside reverse transcriptase inhibitors etravirine and the 8-OH metabolite of efavirenz, the integrase inhibitor bictegravir, and the capsid inhibitor lenacapavir produced synaptic toxicity. Only lenacapavir produced synapse loss at the nanomolar concentrations estimated free in the plasma, although all 4 ARV drugs induced synapse loss at Cmax. Evaluation of combination therapies did not reveal synergistic synaptic toxicity. Synapse loss developed fully by 24 h and persisted for at least 3 days. Bictegravir-induced synapse loss required activation of voltage-gated Ca2+ channels and bictegravir, etravirine, and lenacapavir produced synapse loss by an excitotoxic mechanism. These results indicate that select ARV drugs might contribute to neuropsychiatric effects in combination with drugs that bind serum proteins or in disease states in which synaptic function is altered. The high content imaging assay used here provides an efficient means to evaluate new drugs and drug combinations for potential CNS toxicity.

A novel 3D bilayer hydrogel tri-culture system for studying functional motor units.

BACKGROUND: A motor unit (MU) is formed by a single alpha motor neuron (MN) and the muscle fibers it innervates. The MU is essential for all voluntary movements. Functional deficits in the MU result in neuromuscular disorders (NMDs). The pathological mechanisms underlying most NMDs remain poorly understood, in part due to the lack of in vitro models that can comprehensively recapitulate multistage intercellular interactions and physiological function of the MU. RESULTS: We have designed a novel three-dimensional (3D) bilayer hydrogel tri-culture system where architecturally organized MUs can form in vitro. A sequential co-culture procedure using the three cell types of a MU, MN, myoblast, and Schwann cell was designed to construct a co-differentiating tri-culture on a bilayer hydrogel matrix. We utilized a µ-molded hydrogel with an additional Matrigel layer to form the bilayer hydrogel device. The µ-molded hydrogel layer provides the topological cues for myoblast differentiation. The Matrigel layer, with embedded Schwann cells, not only separates the MNs from myoblasts but also provides a proper micro-environment for MU development. The completed model shows key MU features including an organized MU structure, myelinated nerves, aligned myotubes innervated on clustered neuromuscular junctions (NMJs), MN-driven myotube contractions, and increases in cytosolic Ca2+ upon stimulation. CONCLUSIONS: This organized and functional in vitro MU model provides an opportunity to study pathological events involved in NMDs and peripheral neuropathies, and can serve as a platform for physiological and pharmacological studies such as modeling and drug screening. Technically, the rational of this 3D bilayer hydrogel co-culture system exploits multiple distinct properties of hydrogels, facilitating effective and efficient co-culturing of diverse cell types for tissue engineering.

A Complete Endocannabinoid Signaling System Modulates Synaptic Transmission between Human Induced Pluripotent Stem Cell-Derived Neurons.

The endocannabinoid system (ECS) modulates synaptic function to regulate many aspects of neurophysiology. It adapts to environmental changes and is affected by disease. Thus, the ECS presents an important target for therapeutic development. Despite recent interest in cannabinoid-based treatments, few preclinical studies are conducted in human systems. Human induced pluripotent stem cells (hiPSCs) provide one possible solution to this issue. However, it is not known if these cells have a fully functional ECS. Here, we show that hiPSC-derived neuron/astrocyte cultures exhibit a complete ECS. Using Ca2+ imaging and a genetically encoded endocannabinoid sensor, we demonstrate that they not only respond to exogenously applied cannabinoids but also produce and metabolize endocannabinoids. Synaptically driven [Ca2+]i spiking activity was inhibited (EC50 = 48 ± 13 nM) by the efficacious agonist [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (Win 55,212-2) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC50 = 2.0 ± 0.6 µm). The effects of Win 55212-2 were blocked by a CB1 receptor-selective antagonist. Δ9-Tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47 ± 14% (EC50 = 1.4 ± 1.9 µm). Carbachol stimulated 2-AG production in a manner that was independent of Ca2+ and blocked by selective inhibition of diacylglycerol lipase. 2-AG returned to basal levels via a process mediated by monoacylglycerol lipase as indicated by slowed recovery in cultures treated with 4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL 184). Win 55,212-2 markedly desensitized CB1 receptor function following a 1-day exposure, whereas desensitization was incomplete following 7-day treatment with JZL 184. This human cell culture model is well suited for functional analysis of the ECS and as a platform for drug development. SIGNIFICANCE STATEMENT: Despite known differences between the human response to cannabinoids and that of other species, an in vitro human model demonstrating a fully functional endocannabinoid system has not been described. Human induced pluripotent stem cells (hiPSCs) can be obtained from skin samples and then reprogrammed into neurons for use in basic research and drug screening. Here, we show that hiPSC-derived neuronal cultures exhibit a complete endocannabinoid system suitable for mechanistic studies and drug discovery.

Mild membrane depolarization in neurons induces immediate early gene transcription and acutely subdues responses to a successive stimulus.

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization-mediated by elevated extracellular potassium (K+)-induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor-dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.

HIV Tat Protein Selectively Impairs CB1 Receptor-Mediated Presynaptic Inhibition at Excitatory But Not Inhibitory Synapses.

Despite the success of antiretroviral therapy in suppressing viral load, nearly half of the 37 million people infected with HIV experience cognitive and motor impairments, collectively classified as HIV-associated neurocognitive disorders (HAND). In the CNS, HIV-infected microglia release neurotoxic agents that act indirectly to elicit excitotoxic synaptic injury. HIV trans-activator of transcription (Tat) protein is one such neurotoxin that is thought to play a major role in the neuropathogenesis of HAND. The endocannabinoid (eCB) system provides on-demand neuroprotection against excitotoxicity, and exogenous cannabinoids attenuate neurotoxicity in animal models of HAND. Whether this neuroprotective system is altered in the presence of HIV is unknown. Here, we examined the effects of Tat on the eCB system in rat primary hippocampal cultures. Using whole-cell patch-clamp electrophysiology, we measured changes in retrograde eCB signaling following exposure to Tat. Treatment with Tat significantly reduced the magnitude of depolarization-induced suppression of excitation (DSE) in a graded manner over the course of 48 h. Interestingly, Tat did not alter this form of short-term synaptic plasticity at inhibitory terminals. The Tat-induced decrease in eCB signaling resulted from impaired CB1 receptor (CB1R)-mediated presynaptic inhibition of glutamate release. This novel loss-of-function was particularly dramatic for low-efficacy agonists such as the eCB 2-arachidonoylglycerol (2-AG) and Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive ingredient in marijuana. Our observation that HIV Tat decreases CB1R function in vitro suggests that eCB-mediated neuroprotection may be reduced in vivo; this effect of Tat may contribute to synaptodendritic injury in HAND.

Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures.

Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 µM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 µM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 µM 2-BP or 100 µM glutamate for 24 h, while 300 µM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.

Druggable targets of the endocannabinoid system: Implications for the treatment of HIV-associated neurocognitive disorder.

HIV-associated neurocognitive disorder (HAND) affects nearly half of all HIV-infected individuals. Synaptodendritic damage correlates with neurocognitive decline in HAND, and many studies have demonstrated that HIV-induced neuronal injury results from excitotoxic and inflammatory mechanisms. The endocannabinoid (eCB) system provides on-demand protection against excitotoxicity and neuroinflammation. Here, we discuss evidence of the neuroprotective and anti-inflammatory properties of the eCB system from in vitro and in vivo studies. We examine the pharmacology of the eCB system and evaluate the therapeutic potential of drugs that modulate eCB signaling to treat HAND. Finally, we provide perspective on the need for additional studies to clarify the role of the eCB system in HIV neurotoxicity and speculate that strategies that enhance eCB signaling might slow cognitive decline in HAND.

HIV gp120 upregulates tonic inhibition through α5-containing GABAARs.

HIV-Associated Neurocognitive disorder (HAND) affects nearly half of infected patients. The HIV envelope protein gp120 is shed by infected cells and is a potent neurotoxin in vitro that reproduces many aspects of HAND when expressed in vivo. Here, we show that HIV gp120 increases the amplitude of a tonic current mediated by γ-aminobutyric acid type-A receptors (GABAARs). Treating rat hippocampal cultures with 600 pM gp120IIIB for 4 h increased a tonic bicuculline-sensitive current, which remained elevated for 24 h. The increased current resulted from upregulation of extrasynaptic α5-containing GABAARs, as indicated by inhibition with the selective inverse agonist basmisanil. Treatment with gp120 increased α5-GABAAR immunoreactivity on the cell surface without new protein synthesis. The increase in tonic inhibition was prevented by a C-X-C chemokine receptor type 4 (CXCR4) antagonist or elimination of microglia from the culture. Treatment with interleukin-1ß (IL-1ß) increased the tonic current and an IL-1 receptor antagonist blocked the gp120-evoked response. Pharmacological or genetic inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the gp120-evoked increase in tonic current and direct activation of a mutant form of p38 MAPK expressed in neurons increased the current. Collectively, these data show that gp120 activates CXCR4 to stimulate microglia to release IL-1ß. Subsequent stimulation of IL-1 receptors activates p38 MAPK in neurons leading to the upregulation of α5-containing GABAARs. Increased tonic inhibition impairs neuroplasticity and inhibition of α5-containing GABAARs improves cognitive function in disease models. Thus, gp120-induced upregulation of α5-containing GABAARs presents a novel therapeutic target for HAND.

Scaling Synapses in the Presence of HIV.

A defining feature of HIV-associated neurocognitive disorder (HAND) is the loss of excitatory synaptic connections. Synaptic changes that occur during exposure to HIV appear to result, in part, from a homeostatic scaling response. Here we discuss the mechanisms of these changes from the perspective that they might be part of a coping mechanism that reduces synapses to prevent excitotoxicity. In transgenic animals expressing the HIV proteins Tat or gp120, the loss of synaptic markers precedes changes in neuronal number. In vitro studies have shown that HIV-induced synapse loss and cell death are mediated by distinct mechanisms. Both in vitro and animal studies suggest that HIV-induced synaptic scaling engages new mechanisms that suppress network connectivity and that these processes might be amenable to therapeutic intervention. Indeed, pharmacological reversal of synapse loss induced by HIV Tat restores cognitive function. In summary, studies indicate that there are temporal, mechanistic and pharmacological features of HIV-induced synapse loss that are consistent with homeostatic plasticity. The increasingly well delineated signaling mechanisms that regulate synaptic scaling may reveal pharmacological targets suitable for normalizing synaptic function in chronic neuroinflammatory states such as HAND.

HIV gp120-induced neuroinflammation potentiates NMDA receptors to overcome basal suppression of inhibitory synapses by p38 MAPK.

HIV-associated neurocognitive disorder affects about half of HIV-infected patients. HIV impairs neuronal function through indirect mechanisms mainly mediated by inflammatory cytokines and neurotoxic viral proteins, such as the envelope protein gp120. HIV gp120 elicits a neuroinflammatory response that potentiates NMDA receptor function and induces the loss of excitatory synapses. How gp120 influences neuronal inhibition remains unknown. In this study, we expressed a green fluorescent protein (GFP)-tagged recombinant antibody-like protein that binds to the post-synaptic scaffolding protein gephyrin to label inhibitory synapses in living neurons. Treatment with 600 pM gp120 for 24 h increased the number of labeled inhibitory synapses. HIV gp120 evoked the release of interleukin-1ß (IL-1ß) from microglia to activate IL-1 receptors on neurons. Subsequent activation of the tyrosine kinase Src and GluN2A-containing NMDA receptors increased the number of inhibitory synapses via a process that required protein synthesis. In naïve cultures, inhibition of neuronal p38 mitogen-activated protein kinase (p38 MAPK) increased the number of inhibitory synapses suggesting that p38 MAPK produces a basal suppression of inhibitory synapses that is overcome in the presence of gp120. Direct activation of a mutant form of p38 MAPK expressed in neurons mimicked basal suppression of inhibitory synapses. This study shows for the first time that gp120-induced neuroinflammation increases the number of inhibitory synapses and that this increase overcomes a basal suppression of synaptic inhibition. Increased inhibition may be an adaptive mechanism enabling neurons to counteract excess excitatory input in order to maintain network homeostasis. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Evidence for a role of plasma membrane calcium pumps in neurodegenerative disease: Recent developments.

Plasma membrane Ca2+ ATPases (PMCAs) are a major system for calcium extrusion from all cells. Different PMCA isoforms and splice variants are involved in the precise temporal and spatial handling of Ca2+ signals and the re-establishment of resting Ca2+ levels in the nervous system. Lack or inappropriate expression of specific PMCAs leads to characteristic neuronal phenotypes, which may be reciprocally exacerbated by genetic predisposition through alleles in other genes that modify PMCA interactions, regulation, and function. PMCA dysfunction is often poorly compensated in neurons and may lead to changes in synaptic transmission, altered excitability and, with long-term calcium overload, eventual cell death. Decrease and functional decline of PMCAs are hallmarks of neurodegeneration during aging, and mutations in specific PMCAs are responsible for neuronal dysfunction and accelerated neurodegeneration in many sensory and cognitive diseases.

Monoacylglycerol lipase inhibitor JZL184 prevents HIV-1 gp120-induced synapse loss by altering endocannabinoid signaling.

Monoacylglycerol lipase (MGL) hydrolyzes 2-arachidonoylglycerol to arachidonic acid and glycerol. Inhibition of MGL may attenuate neuroinflammation by enhancing endocannabinoid signaling and decreasing prostaglandin (PG) production. Almost half of HIV infected individuals are afflicted with HIV-associated neurocognitive disorder (HAND), a neuroinflammatory disease in which cognitive decline correlates with synapse loss. HIV infected cells shed the envelope protein gp120 which is a potent neurotoxin that induces synapse loss. Here, we tested whether inhibition of MGL, using the selective inhibitor JZL184, would prevent synapse loss induced by gp120. The number of synapses between rat hippocampal neurons in culture was quantified by imaging clusters of a GFP-tagged antibody-like protein that selectively binds to the postsynaptic scaffolding protein, PSD95. JZL184 completely blocked gp120-induced synapse loss. Inhibition of MGL decreased gp120-induced interleukin-1ß (IL-1ß) production and subsequent potentiation of NMDA receptor-mediated calcium influx. JZL184-mediated protection of synapses was reversed by a selective cannabinoid type 2 receptor (CB2R) inverse agonist/antagonist. JZL184 also reduced gp120-induced prostaglandin E2 (PGE2) production; PG signaling was required for gp120-induced IL-1ß expression and synapse loss. Inhibition of MGL prevented gp120-induced synapse loss by activating CB2R resulting in decreased production of the inflammatory cytokine IL-1ß. Because PG signaling was required for gp120-induced synapse loss, JZL184-induced decreases in PGE2 levels may also protect synapses. MGL presents a promising target for preventing synapse loss in neuroinflammatory conditions such as HAND.

A GluN2B-Selective NMDAR Antagonist Reverses Synapse Loss and Cognitive Impairment Produced by the HIV-1 Protein Tat.

HIV-associated neurocognitive disorder (HAND) affects approximately half of HIV-infected patients. Loss of synaptic connections is a hallmark of many neurocognitive disorders, including HAND. The HIV-1 protein transactivator of transcription (Tat) disrupts synaptic connections both in vitro and in vivo and has been linked to impaired neurocognitive function in humans. In vitro studies have shown that ifenprodil, an antagonist selective for GluN2B-containing NMDARs, reverses synapse loss when applied after Tat. Here, we tested the hypothesis that Tat-induced loss and ifenprodil-mediated rescue of synaptic spines in vivo would predict impairment and rescue of cognitive function. Using intracranial multiphoton imaging, we found that infusion of 100 ng of HIV-1 Tat into the lateral ventricle of yellow fluorescent protein-expressing transgenic mice produced a 17 ± 1% loss of dendritic spines in layer 1 of retrosplenial cortex. Repeated imaging of the same dendrites over 3 weeks enabled longitudinal experiments that demonstrated sustained spine loss after Tat infusion and transient rescue after ifenprodil administration (10 mg/kg, i.p.). Parallel trace fear conditioning experiments showed that spine loss predicted learning deficits and that the time course of ifenprodil-induced rescue of spine density correlated with restoration of cognitive function. These results show for the first time that, during exposure to an HIV-1 neurotoxin in vivo, alteration of GluN2B-containing NMDAR signaling suppresses spine density and impairs learning. Pharmacological inhibition of these NMDARs rescued spines and restored cognitive function. Drugs that rescue synapses may improve neurocognitive function in HAND.SIGNIFICANCE STATEMENT Synaptodendritic damage correlates with cognitive decline in HIV-associated neurocognitive disorder (HAND) patients. We developed an in vivo imaging approach for longitudinal tracking of spine density that enabled correlation of synaptic changes with behavioral outcomes in a model of HAND. We show for the first time that spine loss after exposure to an HIV-1 protein can be reversed pharmacologically and that loss and recovery of dendritic spines predict impairment and restoration of cognitive function, respectively. Therefore, synapse loss, the hallmark of cognitive decline in HAND, is reversible. Drugs that restore spine density may have broad application for improving cognitive function during the early phases of neurodegenerative diseases.

NMDARs Adapt to Neurotoxic HIV Protein Tat Downstream of a GluN2A-Ubiquitin Ligase Signaling Pathway.

HIV-associated neurocognitive disorder (HAND) affects approximately half of HIV-infected patients. Infected non-neuronal cells release neurotoxic factors such as the viral protein transactivator of transcription (Tat) that potentiate NMDAR function. NMDARs regulate synaptic changes observed after exposure to HIV proteins, which may underlie cognitive impairment in HAND patients. Here, we used patch-clamp recording to measure NMDAR-mediated currents in rat hippocampal cultures after exposure to Tat. Tat (4-16 h) potentiated NMDA-evoked whole-cell current and increased the NMDAR:AMPAR ratio of evoked EPSCs. Potentiated currents adapted back to baseline amplitudes after 24 h of exposure to Tat. Pharmacological inhibition of GluN2A-containing NMDARs prevented adaptation, but inhibition of GluN2B-containing NMDARs did not. Pharmacological and genetic approaches determined that potentiated NMDARs activated the kinase Akt, which then activated the E3 ubiquitin ligase Mdm2. Inhibition of protein synthesis prevented adaptation, suggesting that Mdm2 altered gene expression, possibly through its well known target p53. Expression of GFP-tagged GluN1 subunits resulted in fluorescent puncta that colocalized with synaptic markers. Tat (24 h) caused an Mdm2-dependent loss of NMDAR puncta on a timescale similar to adaption of NMDAR function. Activation of the Mdm2 pathway degrades PSD-95, a scaffolding protein that clusters NMDARs at the synapse and enhances their function. Adaptation to the continued presence of excitotoxins that potentiate NMDARs such as HIV Tat may protect from excessive NMDAR activation while also contributing to the synaptic loss that correlates with cognitive decline in HAND. SIGNIFICANCE STATEMENT: Synaptodendritic damage correlates with cognitive decline in HIV-associated neurocognitive disorder (HAND). In a cell culture model, we show that the HIV protein transactivator of transcription (Tat) initially potentiates NMDARs that then adapt to the presence of the toxin. Adaptation of NMDAR function was mediated by a GluN2A/Akt/Mdm2 pathway not previously linked to neuroinflammatory disorders such as HAND. Activation of this pathway caused a loss of synaptic NMDAR clusters. Decreased NMDAR function may result from a homeostatic response gone awry and underlie impaired synaptic function in HAND.

Interleukin-1ß activates an Src family kinase to stimulate the plasma membrane Ca2+ pump in hippocampal neurons.

The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in clearing Ca(2+) from the neuronal cytoplasm. The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, synaptic plasticity, and neurotransmission. Here, we examined the modulation of PMCA activity by PTKs in hippocampal neurons. PMCA-mediated Ca(2+) clearance slowed in the presence of pyrazolopyrimidine 2, an inhibitor of Src family kinases (SFKs), and accelerated in the presence of C2-ceramide, an activator of PTKs. Ca(2+) clearance kinetics were attenuated in cells expressing a dominant-negative Src mutant, suggesting that the pump is tonically stimulated by a PTK. Tonic stimulation was reduced in hippocampal neurons expressing short hairpin (sh)RNA directed to mRNA for Yes. shRNA-mediated knockdown of PMCA isoform 1 (PMCA1) removed tonic stimulation of Ca(2+) clearance, indicating that the kinase stimulates PMCA1. IL-1ß accelerated Ca(2+) clearance in a manner blocked by an IL-1ß receptor antagonist or by an inhibitor of neutral sphingomyelinase, the enzyme that produces ceramide. Thus IL-1ß activates an SFK to stimulate the plasma membrane Ca(2+) pump, decreasing the duration of Ca(2+) transients in hippocampal neurons.

HIV-1 Tat activates a RhoA signaling pathway to reduce NMDA-evoked calcium responses in hippocampal neurons via an actin-dependent mechanism.

HIV-associated neurocognitive disorders afflict approximately half of HIV-infected patients. HIV-infected cells within the CNS release neurotoxic viral proteins such as the transactivator of transcription (Tat). Tat caused a biphasic change in NMDAR function; NMDA-evoked increases in intracellular Ca(2+) were initially potentiated following 16 h exposure to Tat and then adapted by gradually returning to baseline by 24 h. Following Tat-induced NMDAR potentiation, a RhoA/Rho-associated protein kinase (ROCK) signaling pathway was activated; a subsequent remodeling of the actin cytoskeleton reduced NMDA-evoked increases in intracellular Ca(2+) . Pharmacologic or genetic inhibition of RhoA or ROCK failed to affect potentiation, but prevented adaptation of NMDAR function. Activation of RhoA/ROCK signaling increases the formation of filamentous actin. Drugs that prevent changes to filamentous actin blocked adaptation of NMDAR function following Tat-induced potentiation, whereas stimulating either depolymerization or polymerization of actin attenuated NMDAR function. These findings indicate that Tat activates a RhoA/ROCK signaling pathway resulting in actin remodeling and subsequent reduction of NMDAR function. Adaptation of NMDAR function may be a mechanism to protect neurons from excessive Ca(2+) influx and could reveal targets for the treatment of HIV-associated neurocognitive disorders.

Receptor-interacting protein 140 attenuates endoplasmic reticulum stress in neurons and protects against cell death.

Inositol 1, 4, 5-trisphosphate receptor (IP3R)-mediated Ca(2+) release from the endoplasmic reticulum (ER) triggers many physiological responses in neurons, and when uncontrolled can cause ER stress that contributes to neurological disease. Here we show that the unfolded protein response (UPR) in neurons induces rapid translocation of nuclear receptor-interacting protein 140 (RIP140) to the cytoplasm. In the cytoplasm, RIP140 localizes to the ER by binding to the IP3R. The carboxyl-terminal RD4 domain of RIP140 interacts with the carboxyl-terminal gate-keeping domain of the IP3R. This molecular interaction disrupts the IP3R's 'head-tail' interaction, thereby suppressing channel opening and attenuating IP3R-mediated Ca(2+) release. This contributes to a rapid suppression of the ER stress response and provides protection from apoptosis in both hippocampal neurons in vitro and in an animal model of ER stress. Thus, RIP140 translocation to the cytoplasm is an early response to ER stress and provides protection against neuronal death.

HIV-1 protein Tat produces biphasic changes in NMDA-evoked increases in intracellular Ca2+ concentration via activation of Src kinase and nitric oxide signaling pathways.

HIV-associated neurocognitive disorders afflict about half of HIV-infected patients. HIV-infected cells shed viral proteins, such as the transactivator of transcription (Tat), which can cause neurotoxicity by over activation of NMDA receptors. Here, we show that Tat causes a time-dependent, biphasic change in NMDA-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)]i). NMDA-evoked responses were potentiated following 2-h exposure to Tat (50 ng/mL). Tat-induced potentiation of NMDA-evoked increases in [Ca(2+)]i peaked by 8 h and then adapted by gradually reversing to baseline by 24 h and eventually dropping below control by 48 h. Tat-induced potentiation of NMDA-evoked responses was blocked by inhibition of lipoprotein receptor-related protein (LRP) or Src tyrosine kinase. Potentiation was unaffected by inhibition of nitric oxide synthase (NOS). However, NOS activity was required for adaptation. Adaptation was also prevented by inhibition of soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate-dependent protein kinase G (PKG). Together, these findings indicate that Tat potentiates NMDA-evoked increases in [Ca(2+)]i via LRP-dependent activation of Src and that this potentiation adapts via activation of the NOS/sGC/PKG pathway. Adaptation may protect neurons from excessive Ca(2+) influx and could reveal targets for the treatment of HIV-associated neurocognitive disorders. HIV-associated neurocognitive disorders (HAND) afflict about half of HIV-infected patients. HIV-infected cells shed viral proteins, such as the transactivator of transcription (Tat), which can cause neurotoxicity by over activation of NMDA receptors (NMDARs). We show that HIV-1 Tat evoked biphasic changes in NMDA-evoked [Ca(2+) ]i responses. Initially, Tat potentiated NMDA-evoked responses following LRP-mediated activation of Src kinase. Subsequently, Tat-induced NMDAR potentiation adapted by activation of a NOS/sGC/PKG pathway that attenuated NMDA-evoked increases in [Ca(2+)]i . Adaptation may be a novel neuroprotective mechanism to prevent excessive Ca(2+) influx. Solid and dashed arrows represent direct and potentially indirect connections, respectively.

HIV-1 Tat-induced changes in synaptically-driven network activity adapt during prolonged exposure.

HIV-associated neurocognitive disorders (HAND) afflict approximately half of HIV-infected patients. The HIV-1 transactivator of transcription (Tat) protein is released by infected cells and contributes to the pathogenesis of HAND, but many of the underlying mechanisms remain poorly understood. Here we used fura-2-based Ca(2+) imaging and whole-cell patch-clamp recording to study the effects of Tat on the spontaneous synaptic activity that occurs in networked rat hippocampal neurons in culture. Tat triggered aberrant network activity that exhibited a decrease in the frequency of spontaneous action potential bursts and Ca(2+) spikes with a simultaneous increase in burst duration and Ca(2+) spike amplitude. These network changes were apparent after 4 h treatment with Tat and required the low-density lipoprotein receptor-related protein (LRP). Interestingly, Tat-induced changes in network activity adapted during 24 h exposure. The activity returned to control levels in the maintained presence of Tat for 24 h. These observations indicate that Tat causes aberrant network activity, which is dependent on LRP, and adapts following prolonged exposure. Changes in network excitability may contribute to Tat-induced neurotoxicity in vitro and seizure disorders in vivo. Adaptation of neural networks may be a neuroprotective response to the sustained presence of the neurotoxic protein Tat and could underlie the behavioral and electrophysiological changes observed in HAND.

Human immunodeficiency virus-1 Tat protein increases the number of inhibitory synapses between hippocampal neurons in culture.

Synaptodendritic damage correlates with cognitive decline in many neurodegenerative diseases, including human immunodeficiency virus-1 (HIV-1)-associated neurocognitive disorders (HAND). Because HIV-1 does not infect neurons, viral-mediated toxicity is indirect, resulting from released neurotoxins such as the HIV-1 protein transactivator of transcription (Tat). We compared the effects of Tat on inhibitory and excitatory synaptic connections between rat hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding proteins gephyrin or PSD95 fused to GFP. Tat (24 h) increased the number of GFP-gephyrin puncta and decreased the number of PSD95-GFP puncta. The effects of Tat on inhibitory and excitatory synapse number were mediated via the low-density lipoprotein receptor-related protein and subsequent Ca(2+) influx through GluN2A-containing NMDA receptors (NMDARs). The effects of Tat on synapse number required cell-autonomous activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Ca(2+) buffering experiments suggested that loss of excitatory synapses required activation of CaMKII in close apposition to the NMDAR, whereas the increase in inhibitory synapses required Ca(2+) diffusion to a more distal site. The increase in inhibitory synapses was prevented by inhibiting the insertion of GABAA receptors into the membrane. Synaptic changes induced by Tat (16 h) were reversed by blocking either GluN2B-containing NMDARs or neuronal nitric oxide synthase, indicating changing roles for pathways activated by NMDAR subtypes during the neurotoxic process. Compensatory changes in the number of inhibitory and excitatory synapses may serve as a novel mechanism to reduce network excitability in the presence of HIV-1 neurotoxins; these changes may inform the development of treatments for HAND.