Immunocytoprotection after reperfusion with Kv1.3 inhibitors has an extended treatment window for ischemic stroke.

Introduction: Mechanical thrombectomy has improved treatment options and outcomes for acute ischemic stroke with large artery occlusion. However, as the time window of endovascular thrombectomy is extended there is an increasing need to develop immunocytoprotective therapies that can reduce inflammation in the penumbra and prevent reperfusion injury. We previously demonstrated, that by reducing neuroinflammation, KV1.3 inhibitors can improve outcomes not only in young male rodents but also in female and aged animals. To further explore the therapeutic potential of KV1.3 inhibitors for stroke therapy, we here directly compared a peptidic and a small molecule KV1.3 blocker and asked whether KV1.3 inhibition would still be beneficial when started at 72 hours after reperfusion. Methods: Transient middle cerebral artery occlusion (tMCAO, 90-min) was induced in male Wistar rats and neurological deficit assessed daily. On day-8 infarction was determined by T2-weighted MRI and inflammatory marker expression in the brain by quantitative PCR. Potential interactions with tissue plasminogen activator (tPA) were evaluated in-vitro with a chromogenic assay. Results: In a direct comparison with administration started at 2 hours after reperfusion, the small molecule PAP-1 significantly improved outcomes on day-8, while the peptide ShK-223 failed to reduce infarction and neurological deficits despite reducing inflammatory marker expression. PAP-1 still provided benefits when started 72 hours after reperfusion. PAP-1 does not reduce the proteolytic activity of tPA. Discussion: Our studies suggest that KV1.3 inhibition for immunocytoprotection after ischemic stroke has a wide therapeutic window for salvaging the inflammatory penumbra and requires brain-penetrant small molecules.

Repurposing the KCa3.1 Blocker Senicapoc for Ischemic Stroke.

Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC50 of 7 nM, reduced Ca2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke.

The seizure-inducing plastic explosive RDX inhibits the α1ß2γ2 GABAA receptor.

OBJECTIVE: Royal demolition explosive (RDX) can induce seizures in wildlife and humans following release into the environment or after voluntary consumption. During the Vietnam War, RDX intoxication was the most common cause of generalized seizures in US service personnel, and in some sections of the armed forces, eating of RDX has continued as "a dare" to this day. After its mechanism of action was long unknown, RDX was recently shown to be a GABAA receptor antagonist. We here determined the GABAA receptor subtype-selectivity of RDX and mapped its functional binding site. METHODS: We used whole-cell patch-clamp to determine the potency of RDX on 10 recombinantly expressed GABAA receptors and mapped the RDX binding site using a combination of Rosetta molecular modeling and site-directed mutagenesis. RESULTS: RDX was found to reversibly inhibit the α1ß2γ2 GABAA receptor with an IC50 of 23 µmol/L (95% CI 15.1-33.3 µmol/L), whereas α4 and α6 containing GABAA receptor combinations were 4-10-fold less sensitive. RDX is binding to the noncompetitive antagonist (NCA) site in the pore. In a molecular model based on the cryo-EM structure of the resting state of the α1ß2γ2 receptor, RDX forms two hydrogen bonds with the threonines at the T6' ring and makes hydrophobic interactions with the valine and alanine in 2' position of the α1 or ß2 subunits. INTERPRETATION: Our findings characterize the mechanism of action of RDX at the atomistic level and suggest that RDX-induced seizures should be susceptible to treatment with GABAA modulating drugs such as benzodiazepines, barbiturates, propofol, or neurosteroids.

An in vivo Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective in vivo delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection. Secreted ZF activated the imprinted paternal Ube3a in a transgenic reporter mouse and ameliorated motor deficits in a Ube3a deletion Angelman Syndrome (AS) mouse. Intrathecally administered autologous rhesus MSCs were well-tolerated for 3 weeks following administration and secreted ZF protein was detectable within the cerebrospinal fluid (CSF), midbrain, and spinal cord. This approach is less invasive when compared to direct intracranial injection which requires a surgical procedure.

Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α 2 ß 3 γ 2 GABAA Receptor.

Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABAA) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α 2 ß 3 γ 2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α 2 ß 3 γ 2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6' ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α 2 or ß 3 subunit reduces the IC50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABAA receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT: Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α2ß3γ2 GABA receptor type A receptor at the so-called T6' ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.

Draft Genome Sequences of Two Pseudoalteromonas porphyrae Strains Isolated from Seagrass Sediment.

Here, we present the draft genome sequences of Pseudoalteromonas porphyrae UCD-SED9 and UCD-SED14 (phylum Proteobacteria). These strains were isolated from sediment surrounding the roots of the seagrass, Zostera marina, collected near the UC, Davis Bodega Marine Laboratory (Bodega Bay, California). The assemblies contain 4,847,456 bp and 4,817,752 bp, respectively.

Draft Genome Sequences of Two Vibrio splendidus Strains, Isolated from Seagrass Sediment.

Here, we present the draft genome sequences of Vibrio splendidus UCD-SED7 and UCD-SED10 (phylum Proteobacteria). These strains were isolated from sediment surrounding Zostera marina roots near the UC Davis Bodega Marine Laboratory (Bodega, Bay, California). These assemblies contain 5,334,236 bp and 5,904,824 bp, respectively.

Draft Genome Sequence of Bacillus vietnamensis Strain UCD-SED5 (Phylum Firmicutes).

Here, we present the draft genome sequence of Bacillus vietnamensis UCD-SED5 (phylum Firmicutes). This strain was isolated from sediment surrounding Zostera marina roots near the UC Davis Bodega Marine Laboratory (Bodega Bay, California) and represents the second genome of this species. The assembly consists of 4,325,707 bp, in 108 contigs.

Draft Genome Sequence of Pseudoalteromonas tetraodonis Strain UCD-SED8 (Phylum Gammaproteobacteria).

Here, we present the draft genome sequence of Pseudoalteromonas tetraodonis UCD-SED8, a marine bacterium normally associated with the production of tetrodotoxin in pufferfish. This strain was isolated from sediment samples surrounding Zostera marina roots collected from Bodega Marine, California. The assembly consists of 4,017,727 bp contained in 35 contigs.

Draft Genome Sequence of the Endosymbiont "Candidatus Ruthia magnifica" UCD-CM (Phylum Proteobacteria).

Here, we present the draft genome of the endosymbiont "Candidatus Ruthia magnifica" UCD-CM, a member of the phylum Proteobacteria, found from the gills of a deep-sea giant clam, Calyptogena magnifica. The assembly consists of 1,160,249 bp contained in 18 contigs.