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

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.National Institutes of HealthOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

N-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs

Summary of pharmacological and functional properties of pre-M1 mutations.

<p>Summary of pharmacological and functional properties of pre-M1 mutations.</p

Potential interaction between the pre-M1 and M3 helices.

<p><b>A</b>,<b>B</b>, Ribbon structures of the GluN1/GluN2A (<b><i>A</i></b>) and GluN1/GluN2B (<b><i>B</i></b>) receptors without the amino terminal domain is shown. GluN1 is tan and GluN2 is light blue; regions with an OE-ratio below the 5^th percentile are colored purple, and indicate the regions under the strongest purifying selection. <b>C</b>, Side and top down view of the pore forming elements M1, M3, M4 in GluN1/GluN2A receptors colored as in (<b><i>A</i></b>), with regions of purifying selection shown in purple. <b>D</b>, Expanded view of the pre-M1 helix for GluN1 (<i>left panel</i>) and for GluN2A (<i>right panel</i>).</p

Agonist potency of GluN2A-P552R equivalent mutations in GluN2B, GluN2C, and GluN2D.

<p>Agonist potency of GluN2A-P552R equivalent mutations in GluN2B, GluN2C, and GluN2D.</p

Neurotoxic consequences of GluN2A-P552R expression and rescue pharmacology.

<p><b>A</b>, Schematic of experimental timeline indicates the relative dates of neuronal cell culture from embryonic day 16/17 (E16/17), transfection along with memantine/vehicle treatment, and toxicity studies (luciferase assays, cell counts, and confocal imaging). <b>B</b>, Confocal images of cortical neurons transfected with a GFP-expressing construct, with various concentrations of the human GluN2A-P552R-expressing plasmid illustrate the cell morphology. Images were acquired 24 h post-transfection at 20× magnification (scale bars 10 μm). <b>C</b>, Confocal images of cortical neurons transfected with GFP-expressing construct with either 0.6 μg cDNA per well (40% of total transfection cDNA) of WT GluN2A or GluN2A-P552R, the latter in the absence or presence of memantine (50 μM). Images were acquired 24 h post-transfection at 20× magnification, with the exception of the bottom left panel (40×), which highlights GluN2A-P552R-induced dendritic swelling and blebs (scale bar 10 μm). <b>D</b>, The mean cell viability values are shown as a percent of control. Luciferase assays: neuronal cultures were transfected with GFP-N1 plasmid (0.525 μg or 0.825 μg per well) luciferase cDNA (0.375 μg/well) for cell viability assaying, with varied concentrations (0.3 μg or 0.6 μg per well) of pCIneo-vector, WT GluN2A, or GluN2A-P552R cDNA (1.5 μg total DNA per well). Each transfection condition was performed in pairs, either supplemented with vehicle (–) or memantine (20 μM for 0.3 μg; 50 μM for 0.6 μg) treatment (+). Luciferase assays were performed 48 h following transfection and treatment. Experiments were performed in quadruplicate, and independent experiments were repeated (0.3 μg cDNA, n = 7; 0.6 μg cDNA, n = 8). Each condition was normalized to its relevant vector-transfected group to obtain relative viability values, expressed as % control. Data are mean ± SEM of viability (% control) for each condition (ANOVA/Bonferroni; *p <0.05, **p < 0.01, ***p < 0.001). Cell counts: Neuronal cultures were transfected with GFP-N1 plasmid for cell visualization, with either 0.6 μg pCIneo including vector, WT GluN2A, or GluN2A-P552R cDNA (40% of total transfection cDNA). Each transfection condition was performed in duplicate. Cell counts were performed 48 h post-transfection (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#sec013" target="_blank">Methods</a>). Data are mean ± SEM of viability (% control) for each condition in 6 independent experiments. ANOVA/Bonferroni (**p < 0.01). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.s017" target="_blank">S9 Table</a> for statistics.</p