Evidence Implicating Blood-Brain Barrier Impairment in the Pathogenesis of Acquired Epilepsy following Acute Organophosphate Intoxication.

Organophosphate (OP) poisoning can trigger cholinergic crisis, a life-threatening toxidrome that includes seizures and status epilepticus. These acute toxic responses are associated with persistent neuroinflammation and spontaneous recurrent seizures (SRS), also known as acquired epilepsy. Blood-brain barrier (BBB) impairment has recently been proposed as a pathogenic mechanism linking acute OP intoxication to chronic adverse neurologic outcomes. In this review, we briefly describe the cellular and molecular components of the BBB, review evidence of altered BBB integrity following acute OP intoxication, and discuss potential mechanisms by which acute OP intoxication may promote BBB dysfunction. We highlight the complex interplay between neuroinflammation and BBB dysfunction that suggests a positive feedforward interaction. Lastly, we examine research from diverse models and disease states that suggest mechanisms by which loss of BBB integrity may contribute to epileptogenic processes. Collectively, the literature identifies BBB impairment as a convergent mechanism of neurologic disease and justifies further mechanistic research into how acute OP intoxication causes BBB impairment and its role in the pathogenesis of SRS and potentially other long-term neurologic sequelae. Such research is critical for evaluating BBB stabilization as a neuroprotective strategy for mitigating OP-induced epilepsy and possibly seizure disorders of other etiologies. SIGNIFICANCE STATEMENT: Clinical and preclinical studies support a link between blood-brain barrier (BBB) dysfunction and epileptogenesis; however, a causal relationship has been difficult to prove. Mechanistic studies to delineate relationships between BBB dysfunction and epilepsy may provide novel insights into BBB stabilization as a neuroprotective strategy for mitigating epilepsy resulting from acute organophosphate (OP) intoxication and non-OP causes and potentially other adverse neurological conditions associated with acute OP intoxication, such as cognitive impairment.

Selective Neuronal Knockout of STAT3 Function Inhibits Epilepsy Progression, Improves Cognition, and Restores Dysregulated Gene Networks in a Temporal Lobe Epilepsy Model.

OBJECTIVE: Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and hippocampal neural circuit remodeling that results in spontaneous seizures and cognitive dysfunction. Targeting these cascades may provide disease-modifying treatments for TLE patients. Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) inhibitors have emerged as potential disease-modifying therapies; a more detailed understanding of JAK/STAT participation in epileptogenic responses is required, however, to increase the therapeutic efficacy and reduce adverse effects associated with global inhibition. METHODS: We developed a mouse line in which tamoxifen treatment conditionally abolishes STAT3 signaling from forebrain excitatory neurons (nSTAT3KO). Seizure frequency (continuous in vivo electroencephalography) and memory (contextual fear conditioning and motor learning) were analyzed in wild-type and nSTAT3KO mice after intrahippocampal kainate (IHKA) injection as a model of TLE. Hippocampal RNA was obtained 24 h after IHKA and subjected to deep sequencing. RESULTS: Selective STAT3 knock-out in excitatory neurons reduced seizure progression and hippocampal memory deficits without reducing the extent of cell death or mossy fiber sprouting induced by IHKA injection. Gene expression was rescued in major networks associated with response to brain injury, neuronal plasticity, and learning and memory. We also provide the first evidence that neuronal STAT3 may directly influence brain inflammation. INTERPRETATION: Inhibiting neuronal STAT3 signaling improved outcomes in an animal model of TLE, prevented progression of seizures and cognitive co-morbidities while rescuing pathogenic changes in gene expression of major networks associated with epileptogenesis. Specifically targeting neuronal STAT3 may be an effective disease-modifying strategy for TLE. ANN NEUROL 2023;94:106-122.

Down-regulation of AMPA receptors and long-term potentiation during early epileptogenesis.

Epilepsy is a brain disorder characterized by the occurrence of recurrent spontaneous seizures. Behavioral disorders and altered cognition are frequent comorbidities affecting the quality of life of people with epilepsy. These impairments are undoubtedly multifactorial and the specific mechanisms underlying these comorbidities are largely unknown. Long-lasting alterations in synaptic strength due to changes in expression, phosphorylation, or function of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) have been associated with alterations in neuronal synaptic plasticity. In particular, alterations in hippocampal long-term potentiation (LTP), a well-accepted model of learning and memory, have been associated with altered cognition in epilepsy. Here, we analyzed the effects of pilocarpine-induced status epilepticus (SE) on AMPARs to determine if alterations in AMPAR signaling might be one of the mechanisms contributing to altered cognition during epilepsy. We found alterations in the phosphorylation and plasma membrane expression of AMPARs. In addition, we detected altered expression of GRIP, a key scaffolding protein involved in the proper distribution of AMPARs at the neuronal cell surface. Interestingly, a functional analysis revealed that these molecular changes are linked to impaired LTP. Together, these observations suggest that seizure-induced alterations in the molecular machinery regulating AMPARs likely impact the neuron's ability to support synaptic plasticity that is required for learning and memory.

Effects of a potassium channel opener on brain injury and neurologic outcomes in an animal model of neonatal hypoxic-ischemic injury.

BACKGROUND: Hypoxia-ischemia (HI) is the most common cause of brain injury in newborns and the survivors often develop cognitive and sensorimotor disabilities that undermine the quality of life. In the current study, we examined the effectiveness of flupirtine, a potassium channel opener, shown previously in an animal model to have strong anti-neonatal-seizure efficacy, to provide neuroprotection and alleviate later-life disabilities caused by neonatal hypoxic-ischemic injury. METHODS: The rats were treated with a single dose of flupirtine for 4 days following HI induction in 7-day-old rats. The first dose of flupirtine was given after the induction of HI and during the reperfusion period. The effect of treatment was examined on acute and chronic brain injury, motor functions, and cognitive abilities. RESULTS: Flupirtine treatment significantly reduced HI-induced hippocampal and cortical tissue loss at acute time point. Furthermore, at chronic time point, flupirtine reduced contralateral hippocampal volume loss and partially reversed learning and memory impairments but failed to improve motor deficits. CONCLUSION: The flupirtine treatment regimen used in the current study significantly reduced brain injury at acute time point in an animal model of neonatal hypoxic-ischemic encephalopathy. However, these neuroprotective effects were not persistent and only modest improvement in functional outcomes were observed at chronic time points.

Calpain activation and neuronal death during early epileptogenesis.

Epilepsy is a brain disorder characterized by a predisposition to suffer epileptic seizures. Acquired epilepsy might be the result of brain insults like head trauma, stroke, brain infection, or status epilepticus (SE) when one of these triggering injuries starts a transformative process known as epileptogenesis. There is some data to suggest that, during epileptogenesis, seizures themselves damage the brain but there is no conclusive evidence to demonstrate that spontaneous recurrent seizures themselves injure the brain. Our recent evidence indicates that calpain overactivation might be relevant for epileptogenesis. Here, we investigated if spontaneous recurrent seizures that occur during an early period of epileptogenesis show any correlation with the levels of calpain activation and/or expression. In addition, we also investigated a possible association between the occurrence of spontaneous seizures and increased levels of cell death, gliosis and inflammation (typical markers associated with epileptogenesis). We found that the number of spontaneous seizures detected prior to sample collection was correlated with altered calpain activity and expression. Moreover, the levels of hippocampal neurodegeneration were also correlated with seizure occurrence. Our findings suggest that, at least during early epileptogenesis, there is a correlation between seizure occurrence, calpain activity and neurodegeneration. Thus, this study opens the possibility that aberrant calpain reactivation by spontaneous seizures might contribute to the manifestation of future spontaneous seizures.

A calpain inhibitor ameliorates seizure burden in an experimental model of temporal lobe epilepsy.

In this study, we used the pilocarpine model of epilepsy to evaluate the involvement of calpain dysregulation on epileptogenesis. Detection of spectrin breakdown products (SBDPs, a hallmark of calpain activation) after induction of pilocarpine-induced status epilepticus (SE) and before appearance of spontaneous seizure suggested the existence of sustained calpain activation during epileptogenesis. Acute treatment with a cell permeable inhibitor of calpain, MDL-28170, resulted in a partial but significant reduction on seizure burden. The reduction on seizure burden was associated with a limited reduction on the generation of SBDPs but was correlated with a reduction in astrocytosis, microglia activation and cell sprouting. Together, these observations provide evidence for the role of calpain in epileptogenesis. In addition, provide proof-of-principle for the use of calpain inhibitors as a novel strategy to prevent epileptic seizures and its associated pathologies.

Seizure-related regulation of GABAA receptors in spontaneously epileptic rats.

In this study, we analyzed the impact that spontaneous seizures might have on the plasma membrane expression, composition and function of GABAA receptors (GABAARs). For this, the tissue of chronically epileptic rats was collected within 3h of seizure occurrence (≤3h group) or at least 24h after seizure occurrence (≥24h group). A retrospective analysis of seizure frequency revealed that selecting animals on the bases of seizure proximity also grouped animals in terms of overall seizure burden with a higher seizure burden observed in the ≤3h group. A biochemical analysis showed that although animals with more frequent/recent seizures (≤3h group) had similar levels of GABAAR at the plasma membrane they showed deficits in inhibitory neurotransmission. By contrast, the tissue obtained from animals experiencing infrequent seizures (≥24h group) had increased plasma membrane levels of GABAAR and showed no deficit in inhibitory function. Together, our findings offer an initial insight into the molecular changes that might help to explain how alterations in GABAAR function can be associated with differential seizure burden. Our findings also suggest that increased plasma membrane levels of GABAAR might act as a compensatory mechanism to more effectively maintain inhibitory function, repress hyperexcitability and reduce seizure burden. This study is an initial step towards a fuller characterization of the molecular events that trigger alterations in GABAergic neurotransmission during chronic epilepsy.

Effect of spontaneous seizures on GABAA receptor α4 subunit expression in an animal model of temporal lobe epilepsy.

OBJECTIVE: Temporal lobe epilepsy (TLE) is frequently medically intractable and often progressive. Compromised inhibitory neurotransmission due to altered γ-aminobutyric acid (GABA)A receptor α4 subunit (GABAA Rα4) expression has been emphasized as a potential contributor to the initial development of epilepsy following a brain insult (primary epileptogenesis), but the regulation of GABAA Rα4 during chronic epilepsy, specifically, how expression is altered following spontaneous seizures, is less well understood. METHODS: Continuous video-electroencephalography (EEG) recordings from rats with pilocarpine-induced TLE were used to capture epileptic animals within 3 h of a spontaneous seizure (SS), or >24 h after the last SS, to determine whether recent occurrence of a seizure was associated with altered levels of GABAA Rα4 expression. We further evaluated whether this GABAA Rα4 plasticity is regulated by signaling mechanisms active in primary epileptogenesis, specifically, increases in brain-derived neurotrophic factor (BDNF) and early growth response factor 3 (Egr3). RESULTS: Elevated levels of GABAA Rα4 messenger RNA (mRNA) and protein were observed following spontaneous seizures, and were associated with higher levels of BDNF and Egr3 mRNA. SIGNIFICANCE: These data suggest that spontaneous, recurrent seizures that define chronic epilepsy may influence changes in GABAA Rα4 expression, and that signaling pathways known to regulate GABAA Rα4 expression after status epilepticus may also be activated after spontaneous seizures in chronically epileptic animals.

Down-regulation of gephyrin and GABAA receptor subunits during epileptogenesis in the CA1 region of hippocampus.

PURPOSE: Epileptogenesis is the process by which a brain becomes hyperexcitable and capable of generating recurrent spontaneous seizures. In humans, it has been hypothesized that following a brain insult there are a number of molecular and cellular changes that underlie the development of spontaneous seizures. Studies in animal models have shown that an injured brain may develop epileptiform activity before appearance of epileptic seizures and that the pathophysiology accompanying spontaneous seizures is associated with a dysfunction of γ-aminobutyric acid (GABA)ergic neurotransmission. Here, we analyzed the effects of status epilepticus on the expression of GABAA receptors (GABAA Rs) and scaffolding proteins involved in the regulation of GABAA R trafficking and anchoring. METHODS: Western blot analysis was used to determine the levels of proteins involved in GABAA R trafficking and anchoring in adult rats subjected to pilocarpine-induced status epilepticus (SE) and controls. Cell surface biotinylation using a cell membrane-impermeable reagent was used to assay for changes in the expression of receptors at the plasma membrane. Finally, immunoprecipitation experiments were used to evaluate the composition of GABAA Rs. We examined for a correlation between total GABAA R subunit expression, plasma membrane expression, and receptor composition. KEY FINDINGS: Analysis of tissue samples from the CA1 region of hippocampus show that SE promotes a loss of GABAA R subunits and of the scaffolding proteins associated with them. We also found a decrease in the levels of receptors located at the plasma membrane and alterations in GABAA R composition. SIGNIFICANCE: The changes in protein expression of GABAA Rs and scaffolding proteins detected in these studies provide a potential mechanism to explain the deficits in GABAergic neurotransmission observed during the epileptogenic period. Our current observations represent an additional step toward the elucidation of the molecular mechanisms underlying GABAA R dysfunction during epileptogenesis.

Altered Protein Profiles During Epileptogenesis in the Pilocarpine Mouse Model of Temporal Lobe Epilepsy.

Epilepsy is characterized by recurrent, spontaneous seizures and is a major contributor to the global burden of neurological disease. Although epilepsy can result from a variety of brain insults, in many cases the cause is unknown and, in a significant proportion of cases, seizures cannot be controlled by available treatments. Understanding the molecular alterations that underlie or are triggered by epileptogenesis would help to identify therapeutics to prevent or control progression to epilepsy. To this end, the moderate throughput technique of Reverse Phase Protein Arrays (RPPA) was used to profile changes in protein expression in a pilocarpine mouse model of acquired epilepsy. Levels of 54 proteins, comprising phosphorylation-dependent and phosphorylation-independent components of major signaling pathways and cellular complexes, were measured in hippocampus, cortex and cerebellum of mice at six time points, spanning 15 min to 2 weeks after induction of status epilepticus. Results illustrate the time dependence of levels of the commonly studied MTOR pathway component, pS6, and show, for the first time, detailed responses during epileptogenesis of multiple components of the MTOR, MAPK, JAK/STAT and apoptosis pathways, NMDA receptors, and additional cellular complexes. Also noted are time- and brain region- specific changes in correlations among levels of functionally related proteins affecting both neurons and glia. While hippocampus and cortex are primary areas studied in pilocarpine-induced epilepsy, cerebellum also shows significant time-dependent molecular responses.

The role of KCC2 in hyperexcitability of the neonatal brain.

BACKGROUND: The hyperpolarizing activity of γ-aminobutyric acid A (GABAA) receptors depends on the intracellular chloride gradient that is developmentally regulated by the activity of the chloride extruder potassium (K) chloride (Cl) cotransporter 2 (KCC2). In humans and rodents, KCC2 expression can be detected at birth. In rodents, KCC2 expression progressively increases and reaches adult-like levels by the second postnatal week of life. Several studies report changes in KCC2 expression levels in response to early-life injuries. However, the functional contribution of KCC2 in maintaining the excitation-inhibition balance in the neonatal brain is not clear. In the current study, we examined the effect of KCC2 antagonism on the neonatal brain activity under hyperexcitable conditions ex vivo and in vivo. METHODS: Ex vivo electrophysiology experiments were performed on hippocampal slices prepared from 7 to 9 days-old (P7-P9) male rats. Excitability of CA1 pyramidal neurons bathed in zero-Mg2+ buffer was measured using single-unit extracellular (loose) or cell-attach protocol before and after application of VU0463271, a specific antagonist of KCC2. To examine the functional role of KCC2 in vivo, the effect of VU0463271 on hypoxia-ischemia (HI)-induced ictal (seizures and brief runs of epileptiform discharges - BREDs), and inter-ictal spike and sharp-wave activity was measured in P7 male rats. A highly sensitive LC-MS/MS method was used to determine the distribution and the concentration of VU0463271 in the brain. RESULTS: Ex vivo blockade of KCC2 by VU0463271 significantly increased the frequency of zero-Mg2+-triggered spiking in CA1 pyramidal neurons. Similarly, in vivo administration of VU0463271 significantly increased the number of ictal events, BREDs duration, and spike and sharp-wave activity in HI rats. LC-MS/MS data revealed that following systemic administration, VU0463271 rapidly reached brain tissues and distributed well among different brain regions. CONCLUSION: The results suggest that KCC2 plays a critical functional role in maintaining the balance of excitation-inhibition in the neonatal brain, and thus it can be used as a therapeutic target to ameliorate injury associated with hyperexcitability in newborns.

Calpain-dependent cleavage of GABAergic proteins during epileptogenesis.

Epileptogenesis is the processes by which a normal brain transforms and becomes capable of generate spontaneous seizures. In acquired epilepsy, it is thought that epileptogenesis can be triggered by a brain injury but the understanding of the cellular or molecular changes unraveling is incomplete. In the CA1 region of hippocampus less GABAergic activity precede the appearance of spontaneous seizures and calpain overactivation has been detected after chemoconvulsant-induced status epilepticus (SE). Inhibition of calpain overactivation following SE ameliorates seizure burden, suggesting a role for calpain dysregulation in epileptogenesis. The current study analyzed if GABAergic proteins (i.e., gephyrin, the vesicular GABA transporter and the potassium chloride co-transporter 2) undergo calpain-dependent cleavage during epileptogenesis. A time-dependent generation of break down products (BDPs) for these proteins was observed in the CA1 region of hippocampus after pilocarpine-induced SE. Generation of these BDPs was partially blocked by treatment with the calpain inhibitor MDL-28170. These findings suggest that calpain-dependent loss of GABAergic proteins might promote the erosion of inhibitory drive and contribute to hyperexcitability during epileptogenesis.

Ubiquitination-mediated internalization and degradation of the astroglial glutamate transporter, GLT-1.

Sodium-dependent glutamate uptake is essential for limiting excitotoxicity, and dysregulation of this process has been implicated in a wide array of neurological disorders. The majority of forebrain glutamate uptake is mediated by the astroglial glutamate transporter, GLT-1. We and others have shown that this transporter undergoes endocytosis and degradation in response to activation of protein kinase C (PKC), however, the mechanisms involved remain unclear. In the current study, transfected C6 glioma cells or primary cortical cultures were used to show that PKC activation results in incorporation of ubiquitin into GLT-1 immunoprecipitates. Mutation of all 11 lysine residues in the amino and carboxyl-terminal domains to arginine (11R) abolished this signal. Selective mutation of the seven lysine residues in the carboxyl terminus (C7K-R) did not eliminate ubiquitination, but it completely blocked PKC-dependent internalization and degradation. Two families of variants of GLT-1 were prepared with various lysine residues mutated to arginine. Analyses of these constructs indicated that redundant lysine residues in the carboxyl terminus were sufficient for the appearance of ubiquitinated product and degradation of GLT-1. Together these data define a novel mechanism by which the predominant forebrain glutamate transporter can be rapidly targeted for degradation.

Constitutive endocytosis and recycling of the neuronal glutamate transporter, excitatory amino acid carrier 1.

The neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), has a diverse array of physiologic and metabolic functions. There is evidence that there is a relatively large intracellular pool of EAAC1 both in vivo and in vitro, that EAAC1 cycles on and off the plasma membrane, and that EAAC1 cell surface expression can be rapidly regulated by intracellular signals. Despite the possible relevance of EAAC1 trafficking to both physiologic and pathologic processes, the cellular machinery involved has not been defined. In the present study, we found that agents that disrupt clathrin-dependent endocytosis or plasma membrane cholesterol increased steady-state levels of biotinylated EAAC1 in C6 glioma cells and primary neuronal cultures. Acute depletion of cholesterol increased the V(max) for EAAC1-mediated activity and had no effect on Na(+)-dependent glycine transport in the same system. These agents also impaired endocytosis as measured using a reversible biotinylating reagent. Co-expression with dominant-negative variants of dynamin or the clathrin adaptor, epidermal growth factor receptor pathway substrate clone 15, increased the steady-state levels of biotinylated myc-EAAC1. EAAC1 immunoreactivity was found in a subcellular fraction enriched in early endosome antigen 1 (EEA1) isolated by differential centrifugation and partially co-localized with EEA1. Co-expression of a dominant-negative variant of Rab11 (Rab11 S25N) reduced steady-state levels of biotinylated myc-EAAC1 and slowed constitutive delivery of myc-EAAC1 to the plasma membrane. Together, these observations suggest that EAAC1 is constitutively internalized via a clathrin- and dynamin-dependent pathway into early endosomes and that EAAC1 is trafficked back to the cell surface via the endocytic recycling compartment in a Rab11-dependent mechanism. As one defines the machinery required for constitutive trafficking of EAAC1, it may be possible to determine how intracellular signals regulate EAAC1 cell surface expression.

Regulation of the cell surface expression of chloride transporters during epileptogenesis.

The process is commonly known as epileptogenesis refers to the cascade of molecular and cellular changes that transform the brain to make it hyperexcitable and capable of generate recurrent spontaneous seizures. Unfortunately, our understanding of the molecular changes that affect the brain during epileptogenesis remains incomplete. Recent evidence suggests that dysfunction of cation-chloride transporters (CCCs) might be one of the factors that contribute to the deficits in inhibitory neurotransmission observed during epileptogenesis. This study analyzed the cell surface expression of CCCs during epileptogenesis and during chronic epilepsy to evaluate if a loss of CCCs from the plasma membrane might contribute to hyperexcitability. Alterations in the plasma membrane expression of CCCs were mostly detected during the early phase of the epileptogenic period, suggesting that dysfunction of CCCs might contribute to the alterations in the chloride gradient previously detected. Together, the findings presented here suggest that aberrant regulation of the plasma membrane levels of CCCs might contribute to the impartment of GABAergic neurotransmission and that CCCs dysfunction might be relevant for the initial appearance of spontaneous seizures.

Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery.

Brain-derived neurotrophic factor (BDNF) levels are elevated after status epilepticus (SE), leading to activation of multiple signaling pathways, including the janus kinase/signal transducer and activator of transcription pathway that mediates a decrease in GABAA receptor α1 subunits in the hippocampus (Lund et al., 2008). While BDNF can signal via its pro or mature form, the relative contribution of these forms to signaling after SE is not fully known. In the current study, we investigate changes in proBDNF levels acutely after SE in C57BL/6J mice. In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d. Immunohistochemistry studies indicate that seizure-induced BDNF localizes to all hippocampal subfields, predominantly in principal neurons and also in astrocytes. Analysis of the proteolytic machinery that cleaves proBDNF to produce mature BDNF demonstrates that acutely after SE there is a decrease in tissue plasminogen activator and an increase in plasminogen activator inhibitor-1 (PAI-1), an inhibitor of extracellular and intracellular cleavage, which normalizes over the first week after SE. In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels. These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.

Phorbol myristate acetate-dependent interaction of protein kinase Calpha and the neuronal glutamate transporter EAAC1.

Sodium-dependent transporters clear extracellular glutamate in the mammalian CNS. Activation of protein kinase C (PKC) rapidly increases the activity of the neuronal glutamate transporter EAAC1 (excitatory amino acid carrier-1). This effect is associated with redistribution of EAAC1 to the cell membrane and appears to be dependent on a particular PKC subtype, PKCalpha. In the present study, we sought to determine whether this specificity for regulation of EAAC1 is associated with the formation of EAAC1-PKCalpha complexes. In C6 glioma cells, activation of PKC with phorbol 12-myristate 13-acetate (PMA) induced formation of EAAC1-PKCalpha complexes but did not induce formation of complexes with PKCdelta, a PKC not thought to regulate EAAC1. Formation of these complexes was blocked by inhibitors of PKC. Confocal microscopy revealed that PMA caused EAAC1 and PKCalpha to colocalize in clusters at or near the cell surface. The EAAC1-PKCalpha complexes were also observed in rat brain synaptosomes, demonstrating that this interaction is not restricted to C6 cells. These data demonstrate that EAAC1 and PKCalpha interact in a PKC-dependent manner that is associated with EAAC1 redistribution. Although PKC activation has been implicated in the regulation of many different neurotransmitter transporters, this study provides the first example of an interaction between a neurotransmitter transporter and PKC. PKCalpha also forms complexes with GluR2 (glutamate receptor subunit 2) and causes a reduction in the levels of GluR2-containing AMPA receptors at the plasma membrane. Together, these data suggest that PKCalpha may simultaneously trigger the redistribution of EAAC1 and glutamate receptors.

Evidence that Akt mediates platelet-derived growth factor-dependent increases in activity and surface expression of the neuronal glutamate transporter, EAAC1.

Previously we have shown that platelet-derived growth factor (PDGF) rapidly increases the activity of the neuronal glutamate transporter, EAAC1. This increase in activity is associated with a rapid (within minutes) redistribution of transporter from a subcellular compartment to the plasma membrane and is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K). Similar effects of PI3K inhibitors have been observed for insulin-dependent up-regulation of the GLUT4 subtype of glucose transporter. Although GLUT4 regulation also depends on the serine-threonine kinase (Akt/protein kinase B), a downstream target of PI3K, the downstream effectors responsible of PDGF-dependent regulation of EAAC1 have not been identified. In the present study, PDGF increased the level of Akt phosphorylation (Ser 473) in C6 glioma cells, a cell line that has been used to study regulated trafficking of endogenous EAAC1. Two inhibitors of PI3K blocked this effect. In transient transfection studies, a dominant negative mutant of Akt-1 blocked PDGF-induced redistribution of epitope-tagged EAAC1 (myc-EAAC1). Conversely, constitutively active Akt-1 (CA Akt-1) increased the cell surface expression of myc-EAAC1. A lentiviral vector engineered to express CA Akt-1 increased Akt activation, cell surface expression of endogenous EAAC1, and Na(+)-dependent glutamate transport activity. Together, these studies suggest that Akt is required for PDGF-induced regulation of EAAC1.

Neurotransmitter transporters: why dance with so many partners?

Plasma membrane transporters terminate the actions of several small molecule neurotransmitters, including glutamate, gamma-aminobutyric acid, glycine, dopamine, serotonin and norepinephrine. The fact that anti-depressants, cocaine and amphetamines can have such profound behavioral effects by inhibiting the activity of some of these transporters underscores the importance of these molecules. Recent studies have begun to define the mechanisms that regulate these transporters. As these studies progress, it is becoming clear that the transporters form complexes both with themselves and with many other proteins that can regulate either transporter localization or activity. In most cases, the physiological and/or pathological relevance of these interactions is only beginning to emerge.

Brain-derived neurotrophic factor promotes gephyrin protein expression and GABAA receptor clustering in immature cultured hippocampal cells.

Fast synaptic inhibition in the adult brain is largely mediated by GABAA receptors (GABAAR). GABAAR are anchored to synaptic sites by gephyrin, a scaffolding protein that appears to be assembled as a hexagonal lattice beneath the plasma membrane. Brain derived neurotrophic factor (BDNF) alters the clustering and synaptic distribution of GABAAR but mechanisms behind this regulation are just starting to emerge. The current study was aimed to examine if BDNF alters the protein levels and/or clustering of gephyrin and to investigate whether the modulation of gephyrin is accompanied by changes in the distribution and/or clustering of GABAAR. Exogenous application of BDNF to immature neuronal cultures from rat hippocampus increased the protein levels and clustering of gephyrin. BDNF also augmented the association of gephyrin with GABAAR and promoted the formation of GABAAR clusters. Together, these observations indicate that BDNF might regulate the assembly of GABAergic synapses by promoting the association of GABAAR with gephyrin.