2-Methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine, an edaravone analog, exerts neuroprotective effects against acute ischemic injury via inhibiting oxidative stress

Oxidative stress plays an indispensable role in the pathogenesis of cerebral ischemia.Inhibiting oxidative stress has been considered as an effective approach for stroke treatment.Edaravone,a free radical scavenger,has been shown to prevent cerebral ischemic injury.However,the clinical efficacy of edaravone is limited because it has a low scavenging activity for superoxide anions（O_2-（·-））.Here,we report that 2-methyl-5 H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine,a novel small-molecule compound structurally related to edaravone,showed a stronger inhibitory effect on oxidative stress in vitro.In vivo,2-methyl-5 H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine reversed transient middle cerebral artery occlusion-induced dysfunctions of superoxide dismutases and malondialdehyde,two proteins crucial for oxidative stress,suggesting a strengthened antioxidant system.Moreover,2-methyl-5 H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine decreased blood brain barrier permeability.Then,we found that 2-methyl-5 Hbenzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine had a stronger neuroprotective effect than edaravone.More importantly,2-methyl-5 H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine decreased not only infarct size and neurological deficits in the acute phase but also modified neurological severity score and escape latency in Morris water maze task in the delayed period,indicating enhanced neuroprotection,sensorimotor function and spatial memory.Together,these findings suggest that 2-methyl-5 H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine could be a preferable option for stroke treatment.

Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

Many efforts have been made to understand excitotoxicity and develop neuroprotectants for the therapy of ischemic stroke.The narrow treatment time window is still to be solved.Given that the ischemic core expanded over days,treatment with an extended time window is anticipated.Bestrophin1(BEST1)belongs to a bestrophin family of calcium-activated chloride channels.We revealed an increase in neuronal BEST1 expression and function within the peri-infarct from 8 to 48 h after ischemic stroke in mice.Interfering the protein expression or inhibiting the channel function of BEST1 by genetic manipulation displayed neuroprotective effects and improved motor functional deficits.Using electrophysiological recordings,we demonstrated that extrasynaptic glutamate release through BEST1 channel resulted in delayed excitotoxicity.Finally,we confirmed the therapeutic efficacy of pharmacological inhibition of BEST1 during 6—72 h post-ischemia in rodents.This delayed treatment prevented the expansion of infarct volume and the exacerbation of neurological functions.Our study identifies the glutamatereleasing BEST1 channel as a potential therapeutic target against ischemic stroke with a wide time window.

Preclinical evaluation of ZL006-05,a new antistroke drug with fast-onset antidepressant and anxiolytic effects

Background Poststroke depression and anxiety,independent predictor of poor functional outcomes,are common in the acute phase of stroke.Up to now,there is no fast-onset antidepressive and anxiolytic agents suitable for the management of acute stroke.ZL006-05,a dual-target analgesic we developed,dissociates nitric oxide synthase from postsynaptic density-95 while potentiatesα2-containingγ-aminobutyric acid type A receptor.This study aims to determine whether ZL006-05 can be used as an antistroke agent with fast-onset antidepressant and anxiolytic effects.Methods Photothrombotic stroke and transient middle cerebral artery occlusion were induced in rats and mice.Infarct size was measured by TTC(2,3,5-Triphenyltetrazolium chloride)staining or Nissl staining.Neurological defects were assessed by four-point scale neurological score or modified Neurological Severity Scores.Grid-walking,cylinder and modified adhesive removal tasks were conducted to assess sensorimotor functions.Spatial learning was assessed using Morris water maze task.Depression and anxiety were induced by unpredictable chronic mild stress.Depressive behaviours were assessed by tail suspension,forced swim and sucrose preference tests.Anxiety behaviours were assessed by novelty-suppressed feeding and elevated plus maze tests.Pharmacokinetics,toxicokinetics and long-term toxicity studies were performed in rats.Results Administration of ZL006-05 in the acute phase of stroke attenuated transient and permanent ischaemic injury and ameliorated long-term functional impairments significantly,with a treatment window of 12 hours after ischemia,and reduced plasminogen activato-induced haemorrhagic transformation.ZL006-05 produced fast-onset antidepressant and anxiolytic effects with onset latency of 1 hour in the normal and CMS mice,had antidepressant and anxiolytic effects in stroke mice.ZL006-05 crossed the blood-brain barrier and distributed into the brain rapidly,and had a high safety profile in toxicokinetics and long-term toxicological studies.Conclusion ZL006-05 is a new neuroprotectant with fast-onset antidepressant and anxiolytic effects and has translational properties in terms of efficacy,safety and targeting of clinical issues.

Phosphofructokinase-1 Negatively Regulates Neurogenesis from Neural Stem Cells

Phosphofructokinase-1（PFK-1）,a major regulatory glycolytic enzyme,has been implicated in the functions of astrocytes and neurons.Here,we report that PFK-1 negatively regulates neurogenesis from neural stem cells（NSCs）by targeting pro-neural transcriptional factors.Using in vitro assays,we found that PFK-1 knockdown enhanced,and PFK-1 overexpression inhibited the neuronal differentiation of NSCs,which was consistent with the findings from NSCs subjected to 5 h of hypoxia.Meanwhile,the neurogenesis induced by PFK-1 knockdown was attributed to the increased proliferation of neural progenitors and the commitment of NSCs to the neuronal lineage.Similarly,in vivo knockdown of PFK-1 also increased neurogenesis in the dentate gyrus of the hippocampus.Finally,we demonstrated that the neurogenesis mediated by PFK-1 was likely achieved by targeting mammalian achaete-scute homologue-1（Mash 1）,neuronal differentiation factor（NeuroD）,and sex-determining region Y（SRY）-related HMG box 2（Sox2）.All together,our results reveal PFK-1 as an important regulator of neurogenesis.