GSK-3ß inhibitor TWS119 promotes neuronal differentiation after hypoxic-ischemic brain damage in neonatal rats.

Brain injury in preterm infants is a major cause of disability and mortality in children. GSK-3ß is a common pathogenic factor for cognitive dysfunction and involves in neuronal proliferation and differentiation. However, GSK-3ß affected neuronal differentiation and its molecular pathogenesis after hypoxic-ischemic brain damage in neonatal rats remains unclear. This study investigated the effects of GSK-3ß inhibitor (TWS119) on cell cycle regulatory proteins, a neuronal differentiation factor (CEND1), maturation neurons, T-box brain transcription factor 1 (TBR1)-positive neurons to clarify the mechanisms of hypoxic-ischemic brain damage in neonatal rats. We used hypoxic-ischemic Sprague-Dawley neonatal rats with brain damage as models. These rats were used for investigating the effect of GSK-3ß on cell cycle regulatory proteins, neuronal differentiation factor (CEND1), maturation neurons, TBR1-positive neurons by western blot and immunofluorescence. Cyclin D1 (a positive cell cycle regulator) expression decreased, and p21 (a negative cell cycle regulator) expression increased in the TWS119 group compared to the hypoxia-ischemia (HI) group 7 days after HI. Additionally, compared to the HI group, TWS119 treatment up-regulated CEND1 expression and promoted neuronal differentiation and cortex development based on NeuN and TBR1 expression. Our study suggests that the GSK-3ß inhibitor TWS119 promotes neuronal differentiation after hypoxic-ischemic brain damage in neonatal rats by inhibiting cell cycle pathway.

Hispolon inhibits neuronal ferroptosis by promoting the expression of Nrf-2.

Research has shown that neuronal ferroptosis is associated with various central nervous system diseases, including Parkinson's disease, acute brain injury, and spinal cord injury. Inhibiting neuronal ferroptosis can greatly alleviate the progression of these diseases. However, there is currently a lack of effective drugs to inhibit neuronal ferroptosis. In this study, we pretreated neuronal cells with Hispolon and subsequently induced a neuronal ferroptosis model using Erastin. We further assessed the changes in the protein expression levels of SLC7A11, GPX4, ACSL4, Nrf-2, and HO-1 using Western blot and immunofluorescence techniques. Additionally, we measured the intracellular levels of Fe2+, GSH, and MDA using relevant assay kits. The research findings revealed that after Hispolon treatment, the expression of the pro-ferroptosis protein ACSL4 decreased, while the expression of the ferroptosis-regulating proteins GPX4 and SLC7A11 increased. Moreover, the use of an Nrf-2-specific inhibitor was able to reverse the effects of Hispolon as mentioned above. In this study, we discovered that Hispolon can promote the expression of Nrf-2 and inhibit the occurrence of neuronal ferroptosis induced by Erastin.

Poria cocos (Schw.) Wolf, a Traditional Chinese Edible Medicinal Herb, Promotes Neuronal Differentiation, and the Morphological Maturation of Newborn Neurons in Neural Stem/Progenitor Cells.

Neurogenesis in the adult brain comprises the entire set of events of neuronal development. It begins with the division of precursor cells to form a mature, integrated, and functioning neuronal network. Adult neurogenesis is believed to play an important role in animals' cognitive abilities, including learning and memory. In the present study, significant neuronal differentiation-promoting activity of 80% (v/v) ethanol extract of P. cocos (EEPC) was found in Neuro-2a cells and mouse cortical neural stem/progenitor cells (NSPCs). Subsequently, a total of 97 compounds in EEPC were identified by UHPLC-Q-Exactive-MS/MS. Among them, four major compounds-Adenosine; Choline; Ethyl palmitoleate; and L-(-)-arabinitol-were further studied for their neuronal differentiation-promoting activity. Of which, choline has the most significant neuronal differentiation-promoting activity, indicating that choline, as the main bioactive compound in P. cocos, may have a positive effect on learning and memory functions. Compared with similar research literature, this is the first time that the neuronal differentiation-promoting effects of P. cocos extract have been studied.

[Resveratrol improves cognitive function in severely burned rats by inhibiting hippocampal NF-κB/JNK pathway].

Objective To investigate the protective effect of resveratrol (RSV) on improving cognitive function in severely burned rats and its possible mechanism. Methods 18 male SD rats aged 18-20 months were randomly divided into 3 groups: control group, model group and RSV group, with 6 rats in each group. After successful modeling, the rats in RSV group were gavaged once daily with RSV (20 mg/kg). Meanwhile, the rats in control group and model group were gavaged once daily with an equal volume of sodium chloride solution. After 4 weeks, the cognitive function of all rats was estimated by Step-down Test. The concentration of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) protein in serum of rats were detected by ELISA. The expression of IL-6, TNF-α mRNA and protein were estimated by real-time PCR and Western blotting. The apoptosis of hippocampal neurons was tested by terminal deoxynuclectidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). The expression of nuclear transcription factor-κB (NF-κB)/c-Jun N-terminal kinase (JNK) pathway-related proteins in hippocampus were assessed by Western blotting. Results Compared with the rats in model group, rats in RSV group exhibited improved cognitive function. Consistently, the rats in RSV group had a reduced concentration of TNF-α and IL-6 in serum, decreased mRNA and protein expressions of TNF-α and IL-6 in hippocampus, and decreased apoptosis rate and relative expression of p-NF-κB p65/NF-κB p65 and p-JNK/JNK in hippocampal neurons. Conclusion RSV alleviates inflammatory response and hippocampal neuronal apoptosis by inhibiting NF-κB/JNK pathway, thereby improving cognitive function in severely burned rats.

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

Glibenclamide promotes FGF21 secretion in interscapular BAT and attenuates depression-like behaviors in male mice with HFD-induced obesity.

AIMS: Epidemiological evidence suggests that comorbidity of obesity and depression is extremely common and continues to grow in prevalence. However, the mechanisms connecting these two conditions are unknown. In this study, we explored how treatment with KATP channel blocker glibenclamide (GB) or the well-known metabolic regulator FGF21 impact male mice with high-fat diet (HFD)-induced obesity and depressive-like behaviors. MATERIALS AND METHODS: Mice were fed with HFD for 12 weeks and then treated with recombinant FGF21 protein by infusion for 2 weeks, followed by intraperitoneal injection of 3 mg/kg recombinant FGF21 once per day for 4 days. Measurements were made of catecholamine levels, energy expenditure, biochemical endpoints and behavior tests, including sucrose preference and forced swim tests were. Alternatively, animals were infused with GB into brown adipose tissue (BAT). The WT-1 brown adipocyte cell line was used for molecular studies. KEY FINDINGS: Compared to HFD controls, HFD + FGF21 mice exhibited less severe metabolic disorder symptoms, improved depressive-like behaviors, and more extensive mesolimbic dopamine projections. FGF21 treatment also rescued HFD-induced dysregulation of FGF21 receptors (FGFR1 and co-receptor ß-klotho) in the ventral tegmental area (VTA), and it altered dopaminergic neuron activity and morphology in HFD-fed mice. Importantly, we also found that FGF21 mRNA level and FGF21 release were increased in BAT after administration of GB, and GB treatment to BAT reversed HFD-induced dysregulation of FGF21 receptors in the VTA. SIGNIFICANCE: GB administration to BAT stimulates FGF21 production in BAT, corrects HFD-induced dysregulation of FGF21 receptor dimers in VTA dopaminergic neurons, and attenuates depression-like symptoms.

Effectiveness of Albumin-Fused Thioredoxin against 6-Hydroxydopamine-Induced Neurotoxicity In Vitro.

Parkinson's disease (PD) is a neurodegenerative disorder caused by oxidative stress-dependent loss of dopaminergic neurons in the substantia nigra and elevated microglial inflammatory responses. Recent studies show that cell loss also occurs in the hypothalamus in PD. However, effective treatments for the disorder are lacking. Thioredoxin is the major protein disulfide reductase in vivo. We previously synthesized an albumin-thioredoxin fusion protein (Alb-Trx), which has a longer plasma half-life than thioredoxin, and reported its effectiveness in the treatment of respiratory and renal diseases. Moreover, we reported that the fusion protein inhibits trace metal-dependent cell death in cerebrovascular dementia. Here, we investigated the effectiveness of Alb-Trx against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. Alb-Trx significantly inhibited 6-OHDA-induced neuronal cell death and the integrated stress response. Alb-Trx also markedly inhibited 6-OHDA-induced reactive oxygen species (ROS) production, at a concentration similar to that inhibiting cell death. Exposure to 6-OHDA perturbed the mitogen-activated protein kinase pathway, with increased phosphorylated Jun N-terminal kinase and decreased phosphorylated extracellular signal-regulated kinase levels. Alb-Trx pretreatment ameliorated these changes. Furthermore, Alb-Trx suppressed 6-OHDA-induced neuroinflammatory responses by inhibiting NF-κB activation. These findings suggest that Alb-Trx reduces neuronal cell death and neuroinflammatory responses by ameliorating ROS-mediated disruptions in intracellular signaling pathways. Thus, Alb-Trx may have potential as a novel therapeutic agent for PD.

TLR4 activation by lysozyme induces pain without inflammation.

Mostly, pain has been studied in association with inflammation, until recent studies which indicate that during bacterial infections, pain mechanisms could be independent of the inflammation. Chronic pain can sustain long after the healing from the injury, even in the absence of any visible inflammation. However, the mechanism behind this is not known. We tested inflammation in lysozyme-injected mice foot paw. Interestingly, we observed no inflammation in mice foot paw. Yet, lysozyme injections induced pain in these mice. Lysozyme induces pain in a TLR4-dependent manner and TLR4 activation by its ligands such as LPS leads to inflammatory response. We compared the intracellular signaling of MyD88 and TRIF pathways upon TLR4 activation by lysozyme and LPS to understand the underlying mechanism behind the absence of an inflammatory response upon lysozyme treatment. We observed a TLR4 induced selective TRIF and not MyD88 pathway activation upon lysozyme treatment. This is unlike any other previously known endogenous TLR4 activators. A selective activation of TRIF pathway by lysozyme induces weak inflammatory cytokine response devoid of inflammation. However, lysozyme activates glutamate oxaloacetate transaminase-2 (GOT2) in neurons in a TRIF-dependent manner, resulting in enhanced glutamate response. We propose that this enhanced glutaminergic response could lead to neuronal activation resulting in pain sensation upon lysozyme injections. Collectively we identify that TLR4 activation by lysozyme can induce pain in absence of a significant inflammation. Also, unlike other known TLR4 endogenous activators, lysozyme does not activate MyD88 signaling. These findings uncover a mechanism of selective activation of TRIF pathway by TLR4. This selective TRIF activation induces pain with negligible inflammation, constituting a chronic pain homeostatic mechanism.

Metabolic Fingerprinting of Different Sideritis Taxa Infusions and Their Neurogenic Activity.

Over the last years, Sideritis extracts were shown to improve memory. However, their potential to promote the generation of new neurons, starting with the neuronal differentiation of neural stem cells, remains unexplored. Therefore, the present study aimed to evaluate the neurogenic effects of different Sideritis infusions in neural stem and precursor cells and their impact on cell viability. Moreover, the metabolic fingerprints were recorded using LC-DAD, LC-HRESIMS, and GC-MS. The neurogenic potential of infusions of the eight Sideritis taxa tested was as potent as the classical neuronal inducer combination of retinoic acid and valproic acid. Further cytotoxicity assays revealed that the IC50 values of the extracts were between 163 and 322 µg/mL. Hierarchical cluster analyses of the metabolic fingerprints unveiled that the two Sideritis taxa with the lowest IC50 values were the most divergent in the analytical techniques used. As the analysis focused on polyphenols, it is reasonable to assume that these compounds are responsible for the effect on the cell viability of SH-SY5Y neuroblastoma cells. This study is the first report on the neurogenic potential of Sideritis taxa and might support the use of Sideritis herbal preparations in the context of neurodegenerative diseases.

Knocking down Trim47 ameliorated sevoflurane-induced neuronal cell injury and cognitive impairment in rats.

Sevoflurane (SEV), usually causing neuronal damage and cognitive dysfunction, is one of the most commonly used anesthetics in clinical practice. However, the function of Trim47 in SEV-induced neuronal impairment remains elusive. The aim of this study was to study the effect of knocking down Trim47 on the nerve injury induced by SEV. Nerve injury was induced in rats by 3% SEV, and H19-7 was used to establish a pathological model, and sh-Trim47 was transfected into H19-7 to study the function of Trim47. The effects of SEV on the expression of Trim47 in the hippocampus and cognitive function of rats were studied by neurological function score and Moris water maze (MWM). The mRNA and protein expression of TNF-α, IL-1ß and IL-6 in the cells, along with the neuronal apoptosis in the hippocampus of rats in each group were studied by TUNEL or WB. Flow cytometry was used to study the effect of knockdown of Trim47 on cell apoptosis. CCK-8 was used to detect cell viability of H19-7 cells. Finally, the potential signaling pathway affected by knockdown of Trim47 after abrogation of SEV induction was investigated by WB. The results showed that, knockdown of Trim47 ameliorated SEV-induced neurological damage and cognitive deficits, inflammation and neuronal cell apoptosis in rats, and promoted hippocampal neuronal activity. Knockdown of Trim47 can inhibit the NF-κB signaling pathway and improve neuronal cell damage and cognitive impairment induced by SEV in neonatal rats by regulating NF-κB signaling pathway, alleviating inflammatory response, and inhibiting neuronal apoptosis.

Octadecaneuropeptide Ameliorates Cognitive Impairments Through Inhibiting Oxidative Stress in Alzheimer's Disease Models.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-ß peptide (Aß) deposition. Aß accumulation induces oxidative stress, leading to mitochondrial dysfunction, apoptosis, and so forth. Octadecaneuropeptide (ODN), a diazepam-binding inhibitor (DBI)-derived peptide, has been reported to have antioxidant properties. However, it is unclear whether ODN has neuroprotective effects in AD. OBJECTIVE: To profile the potential effects of ODN on AD. METHODS: We established a mouse model of AD via microinjection of Aß in the lateral ventricle. Utilizing a combination of western blotting assays, electrophysiological recordings, and behavioral tests, we investigated the neuroprotective effects of ODN on AD. RESULTS: DBI expression was decreased in AD model mice and cells. Meanwhile, ODN decreased Aß generation by downregulating amyloidogenic AßPP processing in HEK-293 cells stably expressing human Swedish mutant APP695 and BACE1 (2EB2). Moreover, ODN could inhibit Aß-induced oxidative stress in primary cultured cells and mice, as reflected by a dramatic increase in antioxidants and a decrease in pro-oxidants. We also found that ODN could reduce oxidative stress-induced apoptosis by restoring mitochondrial membrane potential, intracellular Ca2+ and cleaved caspase-3 levels in Aß-treated primary cultured cells and mice. More importantly, intracerebroventricular injection of ODN attenuated cognitive impairments as well as long-term potentiation in Aß-treated mice. CONCLUSION: These results suggest that ODN may exert a potent neuroprotective effect against Aß-induced neurotoxicity and memory decline via its antioxidant effects, indicating that ODN may be a potential therapeutic agent for AD.

A Pore Forming Toxin-like Protein Derived from Chinese Red Belly Toad Bombina maxima Triggers the Pyroptosis of Hippomal Neural Cells and Impairs the Cognitive Ability of Mice.

Toxin-like proteins and peptides of skin secretions from amphibians play important physiological and pathological roles in amphibians. ßγ-CAT is a Chinese red-belly toad-derived pore-forming toxin-like protein complex that consists of aerolysin domain, crystalline domain, and trefoil factor domain and induces various toxic effects via its membrane perforation process, including membrane binding, oligomerization, and endocytosis. Here, we observed the death of mouse hippocampal neuronal cells induced by ßγ-CAT at a concentration of 5 nM. Subsequent studies showed that the death of hippocampal neuronal cells was accompanied by the activation of Gasdermin E and caspase-1, suggesting that ßγ-CAT induces the pyroptosis of hippocampal neuronal cells. Further molecular mechanism studies revealed that the pyroptosis induced by ßγ-CAT is dependent on the oligomerization and endocytosis of ßγ-CAT. It is well known that the damage of hippocampal neuronal cells leads to the cognitive attenuation of animals. The impaired cognitive ability of mice was observed after intraperitoneal injection with 10 µg/kg ßγ-CAT in a water maze assay. Taken together, these findings reveal a previously unknown toxicological function of a vertebrate-derived pore-forming toxin-like protein in the nerve system, which triggers the pyroptosis of hippocampal neuronal cells, ultimately leading to hippocampal cognitive attenuation.

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

Sevoflurane induces neurotoxic effects on developing neurons through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway.

Children repeatedly exposed to anaesthesia have a high risk of cognitive impairment, but the mechanism of its regulation in this context is unknown. The objective of this study was to investigate the possible toxic mechanism of sevoflurane through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway. The hippocampal neuronal HT22 cell line was used in this study. The intervention group was treated with the WNK1 inhibitor WNK-463, CaN inhibitor FK506 and Drp-1 inhibitor Mdivi-1 respectively in the medium for 30 min before sevoflurane anaesthesia. The sevofluane group and all intervention group treated with 4.1% sevoflurane for 6 h. Compared with the control group, sevoflurane treatment decreased cell viability and increased cellular apoptosis. Our study found that WNK-463, FK506 and Mdivi-1 can all alleviate the sevoflurane-induced reduction in cell viability, decrease the cell apoptosis. In addition, WNK-463 pretreatment could inhibit the increase of WNK1 kinase and NKCC1 protein concentration caused by sevoflurane. Further, sevoflurane anaesthesia causes intracellular calcium overload, increases the expression of CaN and induces the dephosphorylation of Drp-1 protein at ser637, while CaN inhibitor FK506 pretreatment could reduce the dephosphorylation of Drp-1. Therefore, the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway plays an important role in sevoflurane-related neurotoxicity. Reducing intracellular calcium influx may be one of the important mechanism to ameliorate sevoflurane toxicity.

Neuroprotective Effects of Ethanol Extract of Polyscias fruticosa (EEPF) against Glutamate-Mediated Neuronal Toxicity in HT22 Cells.

In traditional herbal medicine, the Polyscias fruticosa has been frequently used for the treatment of ischemia and inflammation. Oxidative stress mediated by elevated glutamate levels cause neuronal cell death in ischemia and various neurodegenerative diseases. However, so far, the neuroprotective effects of this plant extract against glutamate-mediated cell death have not been investigated in cell models. The current study investigates the neuroprotective effects of ethanol extracts of Polyscias fruticosa (EEPF) and elucidates the underlying molecular mechanisms of EEPFs relevant to neuroprotection against glutamate-mediated cell death. The oxidative stress-mediated cell death was induced by 5 mM glutamate treatment in HT22 cells. The cell viability was measured by a tetrazolium-based EZ-Cytox reagent and Calcein-AM fluorescent dye. Intracellular Ca2+ and ROS levels were measured by fluorescent dyes, fluo-3 AM and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. Protein expressions of p-AKT, BDNF, p-CREB, Bax, Bcl-2, and apoptosis-inducing factor (AIF) were determined by western blot analysis. The apoptotic cell death was measured by flow cytometry. The in vivo efficacy of EEPF was evaluated using the Mongolian gerbil mouse by surgery-induced brain ischemia. EEPF treatment showed a neuroprotective effect against glutamate-induced cell death. The EEPF co-treatment reduced the intracellular Ca2+ and ROS and apoptotic cell death. Furthermore, it recovered the p-AKT, p-CREB, BDNF, and Bcl-2 levels decreased by glutamate. The EEPF co-treatment suppressed the activation of apoptotic Bax, the nuclear translocation of AIF, and mitogen-activated protein kinase (MAPK) pathway proteins (ERK1/2, p38, JNK). Further, EEPF treatment significantly rescued the degenerative neurons in the ischemia-induced Mongolian gerbil in vivo model. EEPF exhibited neuroprotective properties that suppress glutamate-mediated neurotoxicity. The underlying mechanism of EEPF is increasing the level of p-AKT, p-CREB, BDNF, and Bcl-2 associated with cell survival. It has therapeutic potential for the treatment of glutamate-mediated neuropathology.

Protein phosphatase 2A activators reverse age-related behavioral changes by targeting neural cell senescence.

The contribution of cellular senescence to the behavioral changes observed in the elderly remains elusive. Here, we observed that aging is associated with a decline in protein phosphatase 2A (PP2A) activity in the brains of zebrafish and mice. Moreover, drugs activating PP2A reversed age-related behavioral changes. We developed a transgenic zebrafish model to decrease PP2A activity in the brain through knockout of the ppp2r2c gene encoding a regulatory subunit of PP2A. Mutant fish exhibited the behavioral phenotype observed in old animals and premature accumulation of neural cells positive for markers of cellular senescence, including senescence-associated ß-galactosidase, elevated levels cdkn2a/b, cdkn1a, senescence-associated secretory phenotype gene expression, and an increased level of DNA damage signaling. The behavioral and cell senescence phenotypes were reversed in mutant fish through treatment with the senolytic ABT263 or diverse PP2A activators as well as through cdkn1a or tp53 gene ablation. Senomorphic function of PP2A activators was demonstrated in mouse primary neural cells with downregulated Ppp2r2c. We conclude that PP2A reduction leads to neural cell senescence thereby contributing to age-related behavioral changes and that PP2A activators have senotherapeutic properties against deleterious behavioral effects of brain aging.

Use-Dependent Relief of Inhibition of Nav1.8 Channels by A-887826.

Sodium channel inhibitors used as local anesthetics, antiarrhythmics, or antiepileptics typically have the property of use-dependent inhibition, whereby inhibition is enhanced by repetitive channel activation. For targeting pain, Nav1.8 channels are an attractive target because they are prominent in primary pain-sensing neurons, with little or no expression in most other kinds of neurons, and a number of Nav1.8-targeted compounds have been developed. We examined the characteristics of Nav1.8 inhibition by one of the most potent Nav1.8 inhibitors so far described, A-887826, and found that when studied with physiologic resting potentials and physiologic temperatures, inhibition had strong "reverse use dependence", whereby inhibition was relieved by repetitive short depolarizations. This effect was much stronger with A-887826 than with A-803467, another Nav1.8 inhibitor. The use-dependent relief from inhibition was seen in both human Nav1.8 channels studied in a cell line and in native Nav1.8 channels in mouse dorsal root ganglion (DRG) neurons. In native Nav1.8 channels, substantial relief of inhibition occurred during repetitive stimulation by action potential waveforms at 5 Hz, suggesting that the phenomenon is likely important under physiologic conditions. SIGNIFICANCE STATEMENT: Nav1.8 sodium channels are expressed in primary pain-sensing neurons and are a prime current target for new drugs for pain. This work shows that one of the most potent Nav1.8 inhibitors, A-887826, has the unusual property that inhibition is relieved by repeated short depolarizations. This "reverse use dependence" may reduce inhibition during physiological firing and should be selected against in drug development.

Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons.

Efferent modulation of vestibular afferent excitability is linked to muscarinic signaling cascades that close low-voltage-gated potassium channels (i.e., KCNQ). Here, we show that muscarinic signaling cascades also depolarize the activation range of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We compared the voltage activation range and kinetics of HCN channels and induced firing patterns before and after administering the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M) in dissociated vestibular ganglion neurons (VGNs) from rats of either sex using perforated whole-cell patch-clamp methods. Oxo-M depolarized HCN channels' half-activation voltage (V 1/2) and sped up the rate of activation near resting potential twofold. HCN channels in large-diameter and/or transient firing VGN (putative cell bodies of irregular firing neuron from central epithelial zones) had relatively depolarized V 1/2 in control solution and were less sensitive to mAChR activation than those found in small-diameter VGN with sustained firing patterns (putatively belonging to regular firing afferents). The impact of mAChR on HCN channels is not a direct consequence of closing KCNQ channels since pretreating the cells with Linopirdine, a KCNQ channel blocker, did not prevent HCN channel depolarization by Oxo-M. Efferent signaling promoted ion channel configurations that were favorable to highly regular spiking in some VGN, but not others. This is consistent with previous observations that low-voltage gated potassium currents in VGN are conducted by mAChR agonist-sensitive and -insensitive channels. Connecting efferent signaling to HCN channels is significant because of the channel's impact on spike-timing regularity and nonchemical transmission between Type I hair cells and vestibular afferents.SIGNIFICANCE STATEMENT Vestibular afferents express a diverse complement of ion channels. In vitro studies identified low-voltage activated potassium channels and hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Moreover, a network of acetylcholine-releasing efferent neurons controls afferent excitability by closing a subgroup of low-voltage activated potassium channels on the afferent neuron. This work shows that these efferent signaling cascades also enhance the activation of HCN channels by depolarizing their voltage activation range. The size of this effect varies depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system exquisite control over afferent neuron activity.

Cannabidiol Impairs Brain Mitochondrial Metabolism and Neuronal Integrity.

Background: The mechanisms underlying the clinical effects of CBD remain poorly understood. Given the increasing evidence for CBD's effects on mitochondria, we sought to examine in more detail whether CBD impacts mitochondrial function and neuronal integrity. Methods: We utilized BE(2)-M17 neuroblastoma cells or acutely isolated brain mitochondria from rodents using a Seahorse extracellular flux analyzer and a fluorescent spectrofluorophotometer assay. Mitochondrial ion channel activity and hippocampal long-term potentiation were measured using standard cellular electrophysiological methods. Spatial learning/memory function was evaluated using the Morris water maze task. Plasma concentrations of CBD were assessed with liquid chromatography-mass spectrometry, and cellular viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction neuronal injury assay. Results: At low micromolar concentrations, CBD reduced mitochondrial respiration, the threshold for mitochondrial permeability transition, and calcium uptake, blocked a novel mitochondrial chloride channel, and reduced the viability of hippocampal cells. These effects were paralleled by in vitro and in vivo learning/memory deficits. We further found that these effects were independent of cannabinoid receptor 1 and mitochondrial G-protein-coupled receptor 55. Conclusion: Our results provide evidence for concentration- and dose-dependent toxicological effects of CBD, findings that may bear potential relevance to clinical populations.

SATB1/SLC7A11/HO-1 Axis Ameliorates Ferroptosis in Neuron Cells After Ischemic Stroke by Danhong Injection.

Neuronal damage after ischemic stroke (IS) is frequently due to ferroptosis, contributing significantly to ischemic injury. However, the mechanism against ferroptosis in IS remained unclear. The aim of this study was to investigate the potential mechanism of Danhong injection (DHI) and the critical transcription factor SATB1 in preventing neuronal ferroptosis after ischemic stroke in vivo and in vitro. The results showed that DHI treatment significantly reduced the infarct area and associated damage in the brains of the pMCAO mice, and enhanced the viability of OGD-injured neurons. And several characteristic indicators of ferroptosis, such as mitochondrial necrosis and iron accumulation, were regulated by DHI after IS. Importantly, we found that the expression and activity of SATB1 were decreased in the pMCAO mice, especially in neuron cells. Meanwhile, the SATB1/SLC7A11/HO-1 signaling pathway was activated after DHI treatment in ischemic stroke and was found to improve neuronal ferroptosis. Inhibition of SATB1 significantly reduced SLC7A11-HO-1 and significantly attenuated the anti-ferroptosis effects of DHI in the OGD model. These findings indicate that neuronal ferroptosis after IS can be alleviated by DHI through SATB1/SLC7A11/HO-1 pathway, and SATB1 may be an attractive therapeutic target for treating ischemic stroke.