The expression system influences stability, maturation efficiency, and oligomeric properties of the potassium-chloride co-transporter KCC2.

The neuron-specific K+/Cl- co-transporter 2, KCC2, which is critical for brain development, regulates γ-aminobutyric acid-dependent inhibitory neurotransmission. Consistent with its function, mutations in KCC2 are linked to neurodevelopmental disorders, including epilepsy, schizophrenia, and autism. KCC2 possesses 12 transmembrane spans and forms an intertwined dimer. Based on its complex architecture and function, reduced cell surface expression and/or activity have been reported when select disease-associated mutations are present in the gene encoding the protein, SLC12A5. These data suggest that KCC2 might be inherently unstable, as seen for other complex polytopic ion channels, thus making it susceptible to cellular quality control pathways that degrade misfolded proteins. To test these hypotheses, we examined KCC2 stability and/or maturation in five model systems: yeast, HEK293 cells, primary rat neurons, and rat and human brain synaptosomes. Although studies in yeast revealed that KCC2 is selected for endoplasmic reticulum-associated degradation (ERAD), experiments in HEK293 cells supported a more subtle role for ERAD in maintaining steady-state levels of KCC2. Nevertheless, this system allowed for an analysis of KCC2 glycosylation in the ER and Golgi, which serves as a read-out for transport through the secretory pathway. In turn, KCC2 was remarkably stable in primary rat neurons, suggesting that KCC2 folds efficiently in more native systems. Consistent with these data, the mature glycosylated form of KCC2 was abundant in primary rat neurons as well as in rat and human brain. Together, this work details the first insights into the influence that the cellular and membrane environments have on several fundamental KCC2 properties, acknowledges the advantages and disadvantages of each system, and helps set the stage for future experiments to assess KCC2 in a normal or disease setting.

Copper induces neuron-sparing, ferredoxin 1-independent astrocyte toxicity mediated by oxidative stress.

Copper is an essential enzyme cofactor in oxidative metabolism, anti-oxidant defenses, and neurotransmitter synthesis. However, intracellular copper, when improperly buffered, can also lead to cell death. Given the growing interest in the use of copper in the presence of the ionophore elesclomol (CuES) for the treatment of gliomas, we investigated the effect of this compound on the surround parenchyma-namely neurons and astrocytes in vitro. Here, we show that astrocytes were highly sensitive to CuES toxicity while neurons were surprisingly resistant, a vulnerability profile that is opposite of what has been described for zinc and other toxins. Bolstering these findings, a human astrocytic cell line was similarly sensitive to CuES. Modifications of cellular metabolic pathways implicated in cuproptosis, a form of copper-regulated cell death, such as inhibition of mitochondrial respiration or knock-down of ferredoxin 1 (FDX1), did not block CuES toxicity to astrocytes. CuES toxicity was also unaffected by inhibitors of apoptosis, necrosis or ferroptosis. However, we did detect the presence of lipid peroxidation products in CuES-treated astrocytes, indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Indeed, treatment with anti-oxidants mitigated CuES-induced cell death in astrocytes indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Lastly, prior induction of metallothioneins 1 and 2 in astrocytes with zinc plus pyrithione was strikingly protective against CuES toxicity. As neurons express high levels of metallothioneins basally, these results may partially account for their resistance to CuES toxicity. These results demonstrate a unique toxic response to copper in glial cells which contrasts with the cell selectivity profile of zinc, another biologically relevant metal.

GluA1-Shank3 interaction decreases in response to chronic neuronal depolarization.

Interactions between AMPA receptors and synaptic scaffolding proteins are key regulators of synaptic receptor density and, thereby, synapse strength. Shank3 is one such scaffolding protein with high clinical relevance, as genetic variants and deletions of this protein have been linked to autism spectrum disorder. Shank3 acts as a master regulator of the postsynaptic density of glutamatergic synapses, interacting with ionotropic and metabotropic glutamate receptors and cytoskeletal elements to modulate synaptic structure. Notably, Shank3 has been shown to interact directly with the AMPAR subunit GluA1, and Shank3 knockout animals show deficits in AMPAR-mediated synaptic transmission. In this study, we sought to characterize the stability of GluA1-Shank3 interaction in response to chronic stimuli using a highly sensitive and specific proximity ligation assay. We found that GluA1-Shank3 interactions decrease in response to prolonged neuronal depolarization induced by elevated extracellular potassium, and that this reduced interaction is blocked by NMDA receptor antagonism. These results firmly establish the close interaction of GluA1 and Shank3 in cortical neurons in vitro, and that this select interaction is subject to modulation by depolarization.

Neurodevelopmental Consequences of Dietary Zinc Deficiency: A Status Report.

Zinc is a tightly regulated trace mineral element playing critical roles in growth, immunity, neurodevelopment, and synaptic and hormonal signaling. Although severe dietary zinc deficiency is relatively uncommon in the United States, dietary zinc deficiency is a substantial public health concern in low- and middle-income countries. Zinc status may be a key determinant of neurodevelopmental processes. Indeed, limited cohort studies have shown that serum zinc is lower in people diagnosed with autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), and depression. These observations have sparked multiple studies investigating the mechanisms underlying zinc status and neurodevelopmental outcomes. Animal models of perinatal and adult dietary zinc restriction yield distinct behavioral phenotypes reminiscent of features of ASD, ADHD, and depression, including increased anxiety and immobility, repetitive behaviors, and altered social behaviors. At the cellular and molecular level, zinc has demonstrated roles in neurogenesis, regulation of cellular redox status, transcription factor trafficking, synaptogenesis, and the regulation of synaptic architecture via the Shank family of scaffolding proteins. Although mechanistic questions remain, the current evidence suggests that zinc status is important for adequate neuronal development and may be a yet overlooked factor in the pathogenesis of several psychiatric conditions. This review aims to summarize current knowledge of the role of zinc in the neurophysiology of the perinatal period, the many cellular targets of zinc in the developing brain, and the potential consequences of alterations in zinc homeostasis in early life.

Microglial reprogramming by Hv1 antagonism protects neurons from inflammatory and glutamate toxicity.

Although the precise mechanisms determining the neurotoxic or neuroprotective activation phenotypes in microglia remain poorly characterized, metabolic changes in these cells appear critical for these processes. As cellular metabolism can be tightly regulated by changes in intracellular pH, we tested whether pharmacological targeting of the microglial voltage-gated proton channel 1 (Hv1), an important regulator of intracellular pH, is critical for activated microglial reprogramming. Using a mouse microglial cell line and mouse primary microglia cultures, either alone, or co-cultured with rat cerebrocortical neurons, we characterized in detail the microglial activation profile in the absence and presence of Hv1 inhibition. We observed that activated microglia neurotoxicity was mainly attributable to the release of tumor necrosis factor alpha, reactive oxygen species, and zinc. Strikingly, pharmacological inhibition of Hv1 largely abrogated inflammatory neurotoxicity not only by reducing the production of cytotoxic mediators but also by promoting neurotrophic molecule production and restraining excessive phagocytic activity. Importantly, the Hv1-sensitive change from a pro-inflammatory to a neuroprotective phenotype was associated with metabolic reprogramming, particularly via a boost in NADH availability and a reduction in lactate. Most critically, Hv1 antagonism not only reduced inflammatory neurotoxicity but also promoted microglia-dependent neuroprotection against a separate excitotoxic injury. Our results strongly suggest that Hv1 blockers may provide an important therapeutic tool against a wide range of inflammatory neurodegenerative disorders.

Intracellular zinc signaling influences NMDA receptor function by enhancing the interaction of ZnT1 with GluN2A.

Zinc, loaded into glutamate-containing presynaptic vesicles and released into the synapse in an activity-dependent manner, modulates neurotransmission through its actions on postsynaptic targets, prominently via high-affinity inhibition of GluN2A-containing NMDA receptors. Recently, we identified a postsynaptic transport mechanism that regulates endogenous zinc inhibition of NMDARs. In this new model of zinc regulation, the postsynaptic transporter ZnT1 mediates zinc inhibition of NMDARs by binding to GluN2A. Through this interaction, ZnT1, a transporter that moves zinc from the cytoplasm to the extracellular domain, generates a zinc microdomain that modulates NMDAR-mediated neurotransmission. As ZnT1 expression is transcriptionally driven by the metal-responsive transcription factor 1 (MTF-1), we found that intracellular zinc strongly drives MTF-1 in cortical neurons in vitro and increases the number of GluN2A-ZnT1 interactions, thereby enhancing tonic zinc inhibition of NMDAR-mediated currents. Importantly, this effect is absent when the interaction between GluN2A and ZnT1 is disrupted by a cell-permeable peptide. These results suggest that zinc-regulated gene expression can dynamically regulate NMDAR-mediated synaptic processes.

SNAP23 regulates KCC2 membrane insertion and activity following mZnR/GPR39 activation in hippocampalneurons.

Modulation of the neuronal K+/Cl- cotransporter 2 (KCC2) activity, which mediates Cl- export, is critical to neuronal function. Here, we demonstrate that KCC2 interacts with the SNARE protein synaptosome-associated protein 23, SNAP23, an essential component of membrane insertion machinery. Using KCC2 truncated mutants, we show that KCC2 C-terminal domain is essential for membrane targeting and SNAP23-dependent upregulation of KCC2 activity triggered by activation of the Zn2+-sensitive receptor mZnR/GPR39 in HEK293 cells. Expression of SNAP23 phosphorylation-insensitive mutants or inhibition of its upstream activator IκB kinase (IKK) prevents mZnR/GPR39 upregulation of KCC2 activity in mouse hippocampal neurons. We further find that SNAP23 interacts with Syntaxin 1A and KCC2, and that all three proteins exhibit increased membrane insertion following mZnR/GPR39 activation in neurons. Our results elucidate a G-protein-coupled receptor-dependent pathway for regulation of KCC activity, mediated via interaction with SNARE proteins.

The ZIP3 Zinc Transporter Is Localized to Mossy Fiber Terminals and Is Required for Kainate-Induced Degeneration of CA3 Neurons.

Tight regulation of neuronal Zn2+ is critical for physiological function. Multiple Zn2+ transporters are expressed in the brain, yet their spatial distribution and distinct roles are largely unknown. Here, we show developmental regulation of the expression of Zn2+ transporters ZIP1 and ZIP3 in mouse hippocampal neurons, corresponding to previously described increase in neuronal vesicular Zn2+ during the first postnatal month. Rates of Zn2+ uptake in cultured mouse hippocampal neurons, monitored using FluoZin-3 fluorescence, were higher in mature neurons, which express higher levels of ZIP1 and ZIP3. Zn2+ uptake was attenuated by ∼50% following silencing of either ZIP1 or ZIP3. Expression of both ZIP1 and ZIP3 was ubiquitous on somas and most neuronal processes in the cultured neurons. In contrast, we observed distinct localization of the transporters in adult mouse hippocampal brain, with ZIP1 predominantly expressed in the CA3 stratum pyramidale, and ZIP3 primarily localized to the stratum lucidum. Consistent with their localization, silencing of ZIP1 expression in vivo reduced Zn2+ uptake in CA3 neurons while ZIP3 silencing reduced Zn2+ influx into dentate gyrus (DG) granule cells in acute hippocampal slices. Strikingly, in vivo silencing of ZIP3, but not ZIP1, protected CA3 neurons from neurodegeneration following kainate-induced seizures. Our results indicate that distinct Zn2+ transporters control Zn2+ accumulation and toxicity in different neuronal populations in the hippocampus and suggest that selective regulation of Zn2+ transporters can prevent seizure induced brain damage.SIGNIFICANCE STATEMENT Zinc plays a major role in neuronal function and its dysregulation is associated with neurodegeneration. Multiple zinc transporters are expressed in neurons, yet little is known on their distinct roles. Here, we show that the plasma membrane ZIP1 and ZIP3 zinc transporters are expressed on distinct neuronal populations in the CA3 region of the hippocampus. We show that ZIP1 mediates zinc influx into postsynaptic cells, while ZIP3 is responsible for zinc re-uptake from this synapse into dentate granule cells. We further show that silencing of ZIP3, but not ZIP1, can rescue the postsynaptic cells from kainate-induced neurodegeneration. This suggests that neuronal zinc toxicity and degeneration can be modulated by regulation of specific zinc transporters function.

ZnT1 is a neuronal Zn2+/Ca2+ exchanger.

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn2+ neurotoxicity. The mechanism mediating Zn2+ transport by ZnT1, however, has remained elusive. Here, we investigated ZnT1-dependent Zn2+ transport by measuring intracellular changes of this ion using the fluorescent indicator FluoZin-3. In primary mouse cortical neurons, which express ZnT1, transient addition of extracellular Zn2+ triggered a rise in cytosolic Zn2+, followed by its removal. Knockdown of ZnT1 by adeno associated viral (AAV)-short hairpin RNA (shZnT1) markedly increased rates of Zn2+ rise, and decreased rates of its removal, suggesting that ZnT1 is a primary route for Zn2+ efflux in neurons. Although Zn2+ transport by other members of the SLC30A family is dependent on pH gradients across cellular membranes, altered H+ gradients were not coupled to ZnT1-dependent transport. Removal of cytoplasmic Zn2+, against a large inward gradient during the initial loading phase, suggests that Zn2+ efflux requires a large driving force. We therefore asked if Ca2+ gradients across the membrane can facilitate Zn2+ efflux. Elimination of extracellular Ca2+ abolished Zn2+ efflux, while increased extracellular Ca2+ levels enhanced Zn2+ efflux. Intracellular Ca2+ rises, measured in GCaMP6 expressing neurons, closely paralleled cytoplasmic Zn2+ removal. Taken together, these results strongly suggest that ZnT1 functions as a Zn2+/Ca2+ exchanger, thereby regulating the transport of two ions of fundamental importance in neuronal signaling.

Imprecision in Precision Medicine: Differential Response of a Disease-Linked GluN2A Mutant to NMDA Channel Blockers.

Mutations in N-methyl-d-aspartate receptors (NMDAR) subunits have been implicated in a growing number of human neurodevelopmental disorders. Previously, a de novo mutation in GRIN2A, encoding the GluN2A subunit, was identified in a patient with severe epilepsy and developmental delay. This missense mutation, which leads to GluN2A-P552R, produces significant dendrotoxicity in transfected rodent cortical neurons, as evidenced by pronounced dendritic blebbing. This injurious process can be prevented by treatment with the NMDA antagonist memantine. Given the increasing use of FDA approved NMDA antagonists to treat patients with GRIN mutations, who may have seizures refractory to traditional anti-epileptic drugs, we investigated whether additional NMDA antagonists were effective in attenuating neurotoxicity associated with GluN2A-P552R expression. Intriguingly, we found that while treatment with memantine can effectively block GluN2A-P552R-mediated dendrotoxicity, treatment with ketamine does not, despite the fact that both drugs work as open NMDAR channel blockers. Interestingly, we found that neurons expressing GluN2A-P552R were more vulnerable to an excitotoxic insult-an effect that, in this case, could be equally rescued by both memantine and ketamine. These findings suggest that GluN2A-P552R induced dendrotoxicity and increased vulnerability to excitotoxic stress are mediated through two distinct mechanisms. The differences between memantine and ketamine in halting GluN2A-P552R dendrotoxicity could not be explained by NMDA antagonist induced changes in MAP or Src kinase activation, previously shown to participate in NMDA-induced excitotoxicity. Our findings strongly suggest that not all NMDA antagonists may be of equal clinical utility in treating GRIN2A-mediated neurological disorders, despite a shared mechanism of action.

Evolutionary rate covariation identifies SLC30A9 (ZnT9) as a mitochondrial zinc transporter.

Recent advances in genome sequencing have led to the identification of new ion and metabolite transporters, many of which have not been characterized. Due to the variety of subcellular localizations, cargo and transport mechanisms, such characterization is a daunting task, and predictive approaches focused on the functional context of transporters are very much needed. Here we present a case for identifying a transporter localization using evolutionary rate covariation (ERC), a computational approach based on pairwise correlations of amino acid sequence evolutionary rates across the mammalian phylogeny. As a case study, we find that poorly characterized transporter SLC30A9 (ZnT9) coevolves with several components of the mitochondrial oxidative phosphorylation chain, suggesting mitochondrial localization. We confirmed this computational finding experimentally using recombinant human SLC30A9. SLC30A9 loss caused zinc mishandling in the mitochondria, suggesting that under normal conditions it acts as a zinc exporter. We therefore propose that ERC can be used to predict the functional context of novel transporters and other poorly characterized proteins.

The Multifaceted Roles of Zinc in Neuronal Mitochondrial Dysfunction.

Zinc is a highly abundant cation in the brain, essential for cellular functions, including transcription, enzymatic activity, and cell signaling. However, zinc can also trigger injurious cascades in neurons, contributing to the pathology of neurodegenerative diseases. Mitochondria, critical for meeting the high energy demands of the central nervous system (CNS), are a principal target of the deleterious actions of zinc. An increasing body of work suggests that intracellular zinc can, under certain circumstances, contribute to neuronal damage by inhibiting mitochondrial energy processes, including dissipation of the mitochondrial membrane potential (MMP), leading to ATP depletion. Additional consequences of zinc-mediated mitochondrial damage include reactive oxygen species (ROS) generation, mitochondrial permeability transition, and excitotoxic calcium deregulation. Zinc can also induce mitochondrial fission, resulting in mitochondrial fragmentation, as well as inhibition of mitochondrial motility. Here, we review the known mechanisms responsible for the deleterious actions of zinc on the organelle, within the context of neuronal injury associated with neurodegenerative processes. Elucidating the critical contributions of zinc-induced mitochondrial defects to neurotoxicity and neurodegeneration may provide insight into novel therapeutic targets in the clinical setting.

The Function and Regulation of Zinc in the Brain.

Nearly sixty years ago Fredrich Timm developed a histochemical technique that revealed a rich reserve of free zinc in distinct regions of the brain. Subsequent electron microscopy studies in Timm- stained brain tissue found that this "labile" pool of cellular zinc was highly concentrated at synaptic boutons, hinting a possible role for the metal in synaptic transmission. Although evidence for activity-dependent synaptic release of zinc would not be reported for another twenty years, these initial findings spurred decades of research into zinc's role in neuronal function and revealed a diverse array of signaling cascades triggered or regulated by the metal. Here, we delve into our current understanding of the many roles zinc plays in the brain, from influencing neurotransmission and sensory processing, to activating both pro-survival and pro-death neuronal signaling pathways. Moreover, we detail the many mechanisms that tightly regulate cellular zinc levels, including metal binding proteins and a large array of zinc transporters.

Targeted disruption of Kv2.1-VAPA association provides neuroprotection against ischemic stroke in mice by declustering Kv2.1 channels.

Kv2.1 channels mediate cell death-enabling loss of cytosolic potassium in neurons following plasma membrane insertion at somatodendritic clusters. Overexpression of the carboxyl terminus (CT) of the cognate channel Kv2.2 is neuroprotective by disrupting Kv2.1 surface clusters. Here, we define a seven-amino acid declustering domain within Kv2.2 CT (DP-2) and demonstrate its neuroprotective efficacy in a murine ischemia-reperfusion model. TAT-DP-2, a membrane-permeable derivative, induces Kv2.1 surface cluster dispersal, prevents post-injurious pro-apoptotic potassium current enhancement, and is neuroprotective in vitro by disrupting the association of Kv2.1 with VAPA. TAT-DP-2 also induces Kv2.1 cluster dispersal in vivo in mice, reducing infarct size and improving long-term neurological function following stroke. We suggest that TAT-DP-2 induces Kv2.1 declustering by disrupting Kv2.1-VAPA association and scaffolding sites required for the membrane insertion of Kv2.1 channels following injury. We present the first evidence of targeted disruption of Kv2.1-VAPA association as a neuroprotective strategy following brain ischemia.

Synaptic zinc inhibition of NMDA receptors depends on the association of GluN2A with the zinc transporter ZnT1.

The NMDA receptor (NMDAR) is inhibited by synaptically released zinc. This inhibition is thought to be the result of zinc diffusion across the synaptic cleft and subsequent binding to the extracellular domain of the NMDAR. However, this model fails to incorporate the observed association of the highly zinc-sensitive NMDAR subunit GluN2A with the postsynaptic zinc transporter ZnT1, which moves intracellular zinc to the extracellular space. Here, we report that disruption of ZnT1-GluN2A association by a cell-permeant peptide strongly reduced NMDAR inhibition by synaptic zinc in mouse dorsal cochlear nucleus synapses. Moreover, synaptic zinc inhibition of NMDARs required postsynaptic intracellular zinc, suggesting that cytoplasmic zinc is transported by ZnT1 to the extracellular space in close proximity to the NMDAR. These results challenge a decades-old dogma on how zinc inhibits synaptic NMDARs and demonstrate that presynaptic release and a postsynaptic transporter organize zinc into distinct microdomains to modulate NMDAR neurotransmission.

The Redox Biology of Excitotoxic Processes: The NMDA Receptor, TOPA Quinone, and the Oxidative Liberation of Intracellular Zinc.

This special issue of Frontiers in Neuroscience-Neurodegeneration celebrates the 50th anniversary of John Olney's seminal work introducing the concept of excitotoxicity as a mechanism for neuronal cell death. Since that time, fundamental research on the pathophysiological activation of glutamate receptors has played a central role in our understanding of excitotoxic cellular signaling pathways, leading to the discovery of many potential therapeutic targets in the treatment of acute or chronic/progressive neurodegenerative disorders. Importantly, excitotoxic signaling processes have been found repeatedly to be closely intertwined with oxidative cellular cascades. With this in mind, this review looks back at long-standing collaborative efforts by the authors linking cellular redox status and glutamate neurotoxicity, focusing first on the discovery of the redox modulatory site of the N-methyl-D-aspartate (NMDA) receptor, followed by the study of the oxidative conversion of 3,4-dihydroxyphenylalanine (DOPA) to the non-NMDA receptor agonist and neurotoxin 2,4,5-trihydroxyphenylalanine (TOPA) quinone. Finally, we summarize our work linking oxidative injury to the liberation of zinc from intracellular metal binding proteins, leading to the uncovering of a signaling mechanism connecting excitotoxicity with zinc-activated cell death-signaling cascades.

Lessons from Recent Advances in Ischemic Stroke Management and Targeting Kv2.1 for Neuroprotection.

Achieving neuroprotection in ischemic stroke patients has been a multidecade medical challenge. Numerous clinical trials were discontinued in futility and many were terminated in response to deleterious treatment effects. Recently, however, several positive reports have generated the much-needed excitement surrounding stroke therapy. In this review, we describe the clinical studies that significantly expanded the time window of eligibility for patients to receive mechanical endovascular thrombectomy. We further summarize the results available thus far for nerinetide, a promising neuroprotective agent for stroke treatment. Lastly, we reflect upon aspects of these impactful trials in our own studies targeting the Kv2.1-mediated cell death pathway in neurons for neuroprotection. We argue that recent changes in the clinical landscape should be adapted by preclinical research in order to continue progressing toward the development of efficacious neuroprotective therapies for ischemic stroke.

Heterogeneous clinical and functional features of GRIN2D-related developmental and epileptic encephalopathy.

N-methyl d-aspartate receptors are ligand-gated ionotropic receptors mediating a slow, calcium-permeable component of excitatory synaptic transmission in the CNS. Variants in genes encoding NMDAR subunits have been associated with a spectrum of neurodevelopmental disorders. Here we report six novel GRIN2D variants and one previously-described disease-associated GRIN2D variant in two patients with developmental and epileptic encephalopathy. GRIN2D encodes for the GluN2D subunit protein; the GluN2D amino acids affected by the variants in this report are located in the pre-M1 helix, transmembrane domain M3, and the intracellular carboxyl terminal domain. Functional analysis in vitro reveals that all six variants decreased receptor surface expression, which may underline some shared clinical symptoms. In addition the GluN2D(Leu670Phe), (Ala675Thr) and (Ala678Asp) substitutions confer significantly enhanced agonist potency, and/or increased channel open probability, while the GluN2D(Ser573Phe), (Ser1271Phe) and (Arg1313Trp) substitutions result in a mild increase of agonist potency, reduced sensitivity to endogenous protons, and decreased channel open probability. The GluN2D(Ser573Phe), (Ala675Thr), and (Ala678Asp) substitutions significantly decrease current amplitude, consistent with reduced surface expression. The GluN2D(Leu670Phe) variant slows current response deactivation time course and increased charge transfer. GluN2D(Ala678Asp) transfection significantly decreased cell viability of rat cultured cortical neurons. In addition, we evaluated a set of FDA-approved NMDAR channel blockers to rescue functional changes of mutant receptors. This work suggests the complexity of the pathological mechanisms of GRIN2D-mediated developmental and epileptic encephalopathy, as well as the potential benefit of precision medicine.

Defining the Kv2.1-syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant.

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.

Molecular Neuroprotection Induced by Zinc-Dependent Expression of Hepatitis C-Derived Protein NS5A Targeting Kv2.1 Potassium Channels.

We present the design of an innovative molecular neuroprotective strategy and provide proof-of-concept for its implementation, relying on the injury-mediated activation of an ectopic gene construct. As oxidative injury leads to the intracellular liberation of zinc, we hypothesize that tapping onto the zinc-activated metal regulatory element (MRE) transcription factor 1 system to drive expression of the Kv2.1-targeted hepatitis C protein NS5A (hepatitis C nonstructural protein 5A) will provide neuroprotection by preventing cell death-enabling cellular potassium loss in rat cortical neurons in vitro. Indeed, using biochemical and morphologic assays, we demonstrate rapid expression of MRE-driven products in neurons. Further, we report that MRE-driven NS5A expression, induced by a slowly evolving excitotoxic stimulus, functionally blocks injurious, enhanced Kv2.1 potassium whole-cell currents and improves neuronal viability. We suggest this form of "on-demand" neuroprotection could provide the basis for a tenable therapeutic strategy to prevent neuronal cell death in neurodegeneration.