Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behaviors in mice.

BACKGROUND: Major depressive disorder (MDD) is a common but severe psychiatric illness characterized by depressive mood and diminished interest. Both nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 1 (NLRP1) inflammasome and autophagy have been reported to implicate in the pathological processes of depression. However, the mechanistic interplay between NLRP1 inflammasome, autophagy, and depression is still poorly known. METHODS: Animal model of depression was established by chronic social defeat stress (CSDS). Depressive-like behaviors were determined by social interaction test (SIT), sucrose preference test (SPT), open field test (OFT), forced swim test (FST), and tail-suspension test (TST). The protein expression levels of NLRP1 inflammasome complexes, pro-inflammatory cytokines, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K)/PI3K, phosphorylated-AKT (p-AKT)/AKT, phosphorylated-mechanistic target of rapamycin (p-mTOR)/mTOR, brain-derived neurotrophic factor (BDNF), phosphorylated-tyrosine kinase receptor B (p-TrkB)/TrkB, Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl2) and cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3) were examined by western blotting. The mRNA expression levels of pro-inflammatory cytokines were tested by quantitative real-time PCR. The interaction between proteins was detected by immunofluorescence and coimmunoprecipitation. Neuronal injury was assessed by Nissl staining. The autophagosomes were visualized by transmission electron microscopy. Nlrp1a knockdown was performed using an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. RESULTS: CSDS exposure caused a bidirectional change in hippocampal autophagy function, which was activated in the initial period but impaired at the later stage. In addition, CSDS exposure increased the expression levels of hippocampal NLRP1 inflammasome complexes, pro-inflammatory cytokines, p-PI3K, p-AKT and p-mTOR in a time-dependent manner. Interestingly, NLRP1 is immunoprecipitated with mTOR but not PI3K/AKT and CSDS exposure facilitated the immunoprecipitation between them. Hippocampal Nlrp1a knockdown inhibited the activity of PI3K/AKT/mTOR signaling, rescued the impaired autophagy and ameliorated depressive-like behavior induced by CSDS. In addition, rapamycin, an autophagy inducer, abolished NLRP1 inflammasome-driven inflammatory reactions, alleviated depressive-like behavior and exerted a neuroprotective effect. CONCLUSIONS: Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behavior in mice and the regulation of autophagy could be a valuable therapeutic strategy for the management of depression.

Aging Promotes Chronic Stress-Induced Depressive-Like Behavior by Activating NLRP1 Inflammasome-Driven Inflammatory Signaling in Mice.

NLRP1 inflammasome has been reported to participate in many neurological disorders. Our previous study has demonstrated that NLRP1 inflammasome is implicated in chronic stress-induced depressive-like behaviors in mice. Age has been reported to be related to depression. Here we examine whether NLRP1 inflammasome is involved in the effect of age on depressive disorder. Two chronic stress stimuli, chronic social defeat stress (CSDS) and repeat social defeat stress (RSDS), were used to establish a depression model in mice of different ages. We found that aged mice exhibited worse depressive-like behaviors and locomotor activity compared to young mice. Interestingly, the expression of hippocampal NLRP1 inflammasome complexes and the levels of the inflammatory cytokines were increased in an age-dependent manner. Also, chronic stress-induced increase in the expression of the hippocampal chemokine C-X-C motif ligand 1 (CXCL1), and its cognate receptor, CXC-motif receptor 2 (CXCR2), was more remarkable in aged mice than that in young mice. Moreover, aged mice exhibited lower hippocampal BDNF levels compared to young mice. Hippocampal Nlrp1a knockdown reduced the levels of pro-inflammatory cytokines and the expression of CXCL1/CXCR2, restored BDNF levels, and alleviated chronic stress-induced depressive-like behaviors in aged mice. Our results suggest that NLRP1 inflammasome-CXCL1/CXCR2-BDNF signaling contributes to the effect of age on chronic stress-induced depressive-like behavior in mice.

Life extension factor klotho regulates behavioral responses to stress via modulation of GluN2B function in the nucleus accumbens.

Klotho is a life extension factor that has the ability to regulate the function of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs), whose dysfunction in the nucleus accumbens (NAc) underlies critical aspects of the pathophysiology of major depression. Here, we study the functional relevance of klotho in the pathogenesis of depression. A chronic social defeat stress paradigm, in which mice are categorized as either susceptible or unsusceptible based on their performance in a social interaction test, was used in this study. We found that the expression of klotho was largely decreased in the NAc of susceptible mice compared to control or unsusceptible mice. Genetic knockdown of klotho in the NAc induced behavioral alterations relevant to depression in naive mice, while overexpression of klotho produced an antidepressive effect in normal mice and ameliorated the behavioral responses to stress in susceptible mice. Molecularly, knockdown of klotho in the NAc resulted in selective decreases in total and synaptic GluN2B expression that were identical to those in susceptible mice. Elevation of klotho in the NAc reversed the reductions in GluN2B expressions and altered synaptic transmission and spine density in the NAc of susceptible mice. Furthermore, blockade of GluN2B with a specific antagonist abolished the beneficial effects of klotho elevation in susceptible mice. Collectively, we demonstrated that klotho in the NAc modulates behavioral responses to stress by regulating the function of GluN2B-containing NMDARs. These results reveal a novel role for klotho in the pathogenesis of depression, providing new insights into the molecular basis of major depression.

The variations of endophilin A2-FoxO3a-autophagy signal in angiotensin II-induced dopaminergic neuron injury mouse model and by biochanin A.

Biochanin A (Bioch A) is a natural plant estrogen, with various biological activities such as anti-apoptosis, anti-oxidation, and suppression of inflammation. In this study, we investigated the protective effects of Bioch A on angiotensin II (AngII) - induced dopaminergic (DA) neuron damage in vivo and on molecular mechanisms. Spontaneous activity and motor ability of mice among groups was detected by open-field test and swim-test. The expression of TH, microtubule-associated proteins light chain 3B II (LC3BII)/LC3BI, beclin-1, P62, forkhead box class O3 (FoxO3), phosphorylated (p) FoxO3a/FoxO3a, FoxO3, and endophilin A2 were determined by Western blot and immunohistochemistry or immunofluorescence staining. Our results showed that AngII treatment significantly increased the behavioral dysfunction of mice and DA neuron damage. Meanwhile, AngII treatment increased the expression of LC3BII/LC3BI, beclin-1, P62, and FoxO3a and decreased the expression of endophilin A2 and p-FoxO3a/FoxO3a, however, Bioch A treatment alleviate these changes. In summary, these results suggest that Bioch A exerts protective effects on AngII-induced mouse model may be related to regulating endophilin A2, FoxO3a, and autophagy-related proteins; however, the specific mechanism is not yet clear and needs further study.

NLRP1 inflammasome contributes to chronic stress-induced depressive-like behaviors in mice.

BACKGROUND: Major depressive disorder (MDD) is a highly prevalent psychiatric disorder, and inflammation has been considered crucial components of the pathogenesis of depression. NLRP1 inflammasome-driven inflammatory response is believed to participate in many neurological disorders. However, it is unclear whether NLRP1 inflammasome is implicated in the development of depression. METHODS: Animal models of depression were established by four different chronic stress stimuli including chronic unpredictable mild stress (CUMS), chronic restrain stress (CRS), chronic social defeat stress (CSDS), and repeat social defeat stress (RSDS). Depressive-like behaviors were determined by sucrose preference test (SPT), forced swim test (FST), tail-suspension test (TST), open-field test (OFT), social interaction test (SIT), and light-dark test (LDT). The expression of NLRP1 inflammasome complexes, BDNF, and CXCL1/CXCR2 were tested by western blot and quantitative real-time PCR. The levels of inflammatory cytokines were tested by enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. RESULTS: Chronic stress stimuli activated hippocampal NLRP1 inflammasome and promoted the release of pro-inflammatory cytokines IL-1ß, IL-18, IL-6, and TNF-α in mice. Hippocampal Nlrp1a knockdown prevented NLRP1 inflammasome-driven inflammatory response and ameliorated stress-induced depressive-like behaviors. Also, chronic stress stimuli caused the increase in hippocampal CXCL1/CXCR2 expression and low BDNF levels in mice. Interestingly, Nlrp1a knockdown inhibited the up-regulation of CXCL1/CXCR2 expression and restored BDNF levels in the hippocampus. CONCLUSIONS: NLRP1 inflammasome-driven inflammatory response contributes to chronic stress induced depressive-like behaviors and the mechanism may be related to CXCL1/CXCR2/BDNF signaling pathway. Thus, NLRP1 inflammasome could become a potential antidepressant target.

Spinal cord NLRP1 inflammasome contributes to dry skin induced chronic itch in mice.

BACKGROUND: Dry skin itch is one of the most common skin diseases and elderly people are believed to be particularly prone to it. The inflammasome has been suggested to play an important role in chronic inflammatory disorders including inflammatory skin diseases such as psoriasis. However, little is known about the role of NLRP1 inflammasome in dry skin-induced chronic itch. METHODS: Dry skin-induced chronic itch model was established by acetone-ether-water (AEW) treatment. Spontaneous scratching behavior was recorded by video monitoring. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes, transient receptor potential vanilloid type 1 (TRPV1), and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. H.E. staining was used to evaluate skin lesion. RESULTS: AEW treatment triggers spontaneous scratching and significantly increases the expression of NLRP1, ASC, and caspase-1 and the levels of IL-1ß, IL-18, IL-6, and TNF-α in the spinal cord and the skin of mice. Spinal cord Nlrp1a knockdown prevents AEW-induced NLRP1 inflammasome assembly, TRPV1 channel activation, and spontaneous scratching behavior. Capsazepine, a specific antagonist of TRPV1, can also inhibit AEW-induced inflammatory response and scratching behavior. Furthermore, elderly mice and female mice exhibited more significant AEW-induced scratching behavior than young mice and male mice, respectively. Interestingly, AEW-induced increases in the expression of NLRP1 inflammasome complex and the levels of inflammatory cytokines were more remarkable in elderly mice and female mice than in young mice and male mice, respectively. CONCLUSIONS: Spinal cord NLRP1 inflammasome-mediated inflammatory response contributes to dry skin-induced chronic itch by TRPV1 channel, and it is also involved in age and sex differences of chronic itch. Inhibition of NLRP1 inflammasome may offer a new therapy for dry skin itch.

Chronic unpredictable mild stress accelerates lipopolysaccharide- induced microglia activation and damage of dopaminergic neurons in rats.

Parkinson's disease (PD) is a neurodegenerative disorder, which is characterized by microglia activation and dopaminergic neurons affected by inflammatory processes. Inflammation has been recognized to be necessary for initiation and progress of PD. Emerging evidence indicates that NLRP3 inflammasome complex is involved in the recognition and execution of host inflammatory response. Stress is acknowledged to be a predisposing and precipitating factor in some neurodegenerative diseases. However, it is unknown whether chronic unpredictable mild stress (CUMS) sensitized microglia to pro-inflammatory stimuli. In this study, in vivo experiments are used to evaluate the effects of CUMS on lipopolysaccharide (LPS)-induced microglia activation and NLRP3 inflammasome activation. The results showed that CUMS pretreatment for 14 days significantly aggravated the behavioral dysfunction of PD rats, increased the activation of microglia. Pretreatment with CUMS for 14 days increased the levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) in the serum, and increased the expression of NLRP-3, ASC, Casepase-1 in the substantia nigra of PD rats. Our data showed that pretreatment with CUMS for 14 days increased the microglia activation and the DA neurons damage, and the mechanisms may be associated with the acceleration of the inflammatory response and activation of NLRP3 inflammasome.

GYY4137 Promotes Mice Feeding Behavior via Arcuate Nucleus Sulfur-Sulfhydrylation and AMPK Activation.

Hydrogen sulfide (H2S) is an endogenous gaseous molecule and plays important biological and neurochemical roles in many processes such as the neural activity and immunity. The arcuate nucleus (ARC) of hypothalamus is a control center for appetite and energy metabolism. AMPK is a gage kinase in the monitoring of energy status and regulation of energy metabolism, and it can be activated by H2S via CaMKKß/AMPK pathway. But the role of H2S in ARC and appetite has not been reported. Here we studied the orexigenic effect of H2S and the mechanisms by means of GYY4137, a water soluble and slow-releasing donor of H2S, and protein sulfur-sulfhydrylation analysis. We demonstrated that GYY4137-derived H2S increased food intake of mice, augmented the production of neuropeptide Y (NPY), and elevated the protein sulfur-sulfhydrylation level and the activation of AMPK and CaMKKß in ARC. Blocking sulfur-sulfhydrylation with DTT eliminated GYY4137-induced activation of AMPK and CaMKKß. DTT and preventing AMPK activation in ARC with Compound C and Ara-A could both attenuate the orexigenic effect of GYY4137. These findings suggest that H2S enhances appetite through protein sulfur-sulfhydrylation and the activation of AMPK and NPY function in ARC.

Sinomenine exerts anticonvulsant profile and neuroprotective activity in pentylenetetrazole kindled rats: involvement of inhibition of NLRP1 inflammasome.

BACKGROUND: Epilepsy is a common neurological disorder and is not well controlled by available antiepileptic drugs (AEDs). Inflammation is considered to be a critical factor in the pathophysiology of epilepsy. Sinomenine (SN), a bioactive alkaloid with anti-inflammatory effect, exerts neuroprotective activity in many nervous system diseases. However, little is known about the effect of SN on epilepsy. METHODS: The chronic epilepsy model was established by pentylenetetrazole (PTZ) kindling. Morris water maze (MWM) was used to test spatial learning and memory ability. H.E. staining and Hoechst 33258 staining were used to evaluate hippocampal neuronal damage. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: SN (20, 40, and 80 mg/kg) dose-dependently disrupts the kindling acquisition process, which decreases the seizure scores and the incidence of fully kindling. SN also increases the latency of seizure and decreases the duration of seizure in fully kindled rats. In addition, different doses of SN block the hippocampal neuronal damage and minimize the impairment of spatial learning and memory in PTZ kindled rats. Finally, PTZ kindling increases the expression of NLRP1 inflammasome complexes and the levels of inflammatory cytokines IL-1ß, IL-18, IL-6, and TNF-α, which are all attenuated by SN in a dose- dependent manner. CONCLUSIONS: SN exerts anticonvulsant and neuroprotective activity in PTZ kindling model of epilepsy. Disrupting the kindling acquisition, which inhibits NLRP1 inflammasome-mediated inflammatory process, might be involved in its effects.

Acid-sensing ion channels contribute to the effect of extracellular acidosis on proliferation and migration of A549 cells.

Acid-sensing ion channels, a proton-gated cation channel, can be activated by low extracellular pH and involved in pathogenesis of some tumors such as glioma and breast cancer. However, the role of acid-sensing ion channels in the growth of lung cancer cell is unclear. In this study, we investigated the expression of acid-sensing ion channels in human lung cancer cell line A549 and their possible role in proliferation and migration of A549 cells. The results show that acid-sensing ion channel 1, acid-sensing ion channel 2, and acid-sensing ion channel 3 are expressed in A549 cells at the messenger RNA and protein levels, and acid-sensing ion channel-like currents were elicited by extracellular acid stimuli. Moreover, we found that acidic extracellular medium or overexpressing acid-sensing ion channel 1a promotes proliferation and migration of A549 cells. In addition psalmotoxin 1, a specific acid-sensing ion channel 1a inhibitor, or acid-sensing ion channel 1a knockdown can abolish the effect of acid stimuli on A549 cells. In addition, acid-sensing ion channels mediate increase of [Ca2+]i induced by low extracellular pH in A549 cells. All these results indicate that acid-sensing ion channel-calcium signal mediate lung cancer cell proliferation and migration induced by extracellular acidosis, and acid-sensing ion channels may serve as a prognostic marker and a therapeutic target for lung cancer.

Cystathionine-ß-synthase-derived hydrogen sulfide is required for amygdalar long-term potentiation and cued fear memory in rats.

Hydrogen sulfide (H2S) is an endogenous gaseous molecule that functions as a neuromodulator in the brain. We previously reported that H2S regulated amygdalar synaptic plasticity and cued fear memory in rats. However, whether endogenous H2S is required for amygdalar long-term potentiation (LTP) induction and cued fear memory formation remains unclear. Here, we show that cystathionine-ß-synthase (CBS), the predominant H2S-producing enzyme in the brain, was highly expressed in the amygdala of rats. Suppressing CBS activity by inhibitor prevented activity-triggered generation of H2S in the lateral amygdala (LA) region. Incubating brain slices with CBS inhibitor significantly prevented the induction of NMDA receptors (NMDARs)-dependent LTP in the thalamo-LA pathway, and intra-LA infusion of CBS inhibitor impaired cued fear memory in rats. Notably, treatment with H2S donor, but not CBS activator, significantly reversed the impairments of LTP and fear memory caused by CBS inhibition. Mechanismly, inhibition of CBS activity led to a reduction in NMDAR-mediated synaptic response in the thalamo-LA pathway, and treatment with H2S donor restored the function of NMDARs. Collectively, these results indicate that CBS-derived H2S is required for amygdalar synaptic plasticity and cued fear memory in rats, and the effects of endogenous H2S might involve the regulation of NMDAR function.

Acid-sensing ion channel 1a contributes to the effect of extracellular acidosis on NLRP1 inflammasome activation in cortical neurons.

BACKGROUND: Acid-sensing ion channels (ASICs) are cation channels which were activated by extracellular acidosis and involved in various physiological and pathological processes in the nervous system. Inflammasome is a key component of the innate immune response in host against harmful and irritable stimuli. As the first discovered molecular platform, NLRP1 (nucleotide-binding oligomerization domain (NOD)-like receptor protein 1) inflammasome is expressed in neurons and implicated in many nervous system diseases such as brain injury, nociception and epilepsy. However, little is known about the effect of ASICs on NLRP1 inflammasome activation under acidosis. METHODS: The expression of inflammasome complex protein (NLRP1, ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) and caspase-1), inflammatory cytokines (IL-1ß and IL-18), and apoptosis-related protein (Bax, Bcl-2, and activated caspase-3) was detected by Western blot. Large-conductance Ca(2+) and voltage-activated K(+) (BK) channel currents were recorded by whole-cell patch-clamp technology. Measurement of [K(+)] i was performed by fluorescent ion imaging system. Co-expression of ASICs and BK channels was determined by dual immunofluorescence. Cell viability was assessed by MTT and LDH kit. RESULTS: ASICs and BK channels were co-expressed in primary cultured cortical neurons. Extracellular acidosis increased the expression of NLRP1, ASC, caspase-1, IL-1ß, and IL-18. Further mechanistic studies revealed that acidosis-induced ASIC1a activation results in the increase of BK channel currents, with the subsequent K(+) efflux and a low concentration of intracellular K(+), which activated NLRP1 inflammasome. Furthermore, these effects of acidosis could be blocked by specific ASIC1a inhibitor PcTX1 and BK channel inhibitor IbTX. The data also demonstrated neutralization of NLRP1-protected cortical neurons against injury induced by extracellular acidosis. CONCLUSIONS: Our data showed that NLRP1 inflammasome could be activated by extracellular acidosis though ASIC-BK channel K(+) signal pathway and was involved in extracellular acidosis-induced cortical neuronal injury.

Orexin-A activates hypothalamic AMP-activated protein kinase signaling through a Ca²âº-dependent mechanism involving voltage-gated L-type calcium channel.

Hypothalamic AMP-activated protein kinase (AMPK) and orexins/hypocretins are both involved in the control of feeding behavior, but little is known about the interaction between these two signaling systems. Here, we demonstrated that orexin-A elicited significant activation of AMPK in the arcuate nucleus (ARC) of the hypothalamus by elevating cytosolic free Ca²âº involving extracellular calcium influx. Electrophysiological results revealed that orexin-A increased the L-type calcium current via the orexin receptor-phospholipase C-protein kinase C signaling pathway in ARC neurons that produce neuropeptide Y, an important downstream effector of orexin-A's orexigenic effect. Furthermore, the L-type calcium channel inhibitor nifedipine attenuated orexin-A-induced AMPK activation in vitro and in vivo. We found that inhibition of AMPK by either compound C (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine) or the ATP-mimetic 9-ß-D-arabinofuranoside prevented the appetite-stimulating effect of orexin-A. This action can be mimicked by nifedipine, the blocker of the L-type calcium channel. Our results indicated that orexin-A activates hypothalamic AMPK signaling through a Ca²âº-dependent mechanism involving the voltage-gated L-type calcium channel, which may serve as a potential target for regulating feeding behavior.

Hyperoside protects cortical neurons from oxygen-glucose deprivation-reperfusion induced injury via nitric oxide signal pathway.

Hyperoside is a flavonoid compound and widely used in clinic to relieve pain and improve cardiovascular functions. However, the effects of hyperoside on ischemic neurons and the molecular mechanisms remain unclear. Here, we used an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD-R) to investigate the protective effects of hyperoside on ischemic neuron injury and further explore the possible related mechanisms. Our results demonstrated that hyperoside protected cultured cortical neurons from OGD-R injury, it also relieved glutamate-induced neuronal injury and NMDA-induced [Ca(2+)](i) elevation. As for the mechanisms, hyperoside firstly attenuated the phosphorylation of CaMKII caused by OGD-R lesions. Meanwhile, hyperoside lessened iNOS expression induced by OGD-R via inhibition of NF-κB activation. Furthermore, ameliorating of ERK, JNK and Bcl-2 family-related apoptotic signaling pathways were also involved in the neuroprotection of hyperoside. Taken together, these studies revealed that hyperoside had protective effects on neuronal ischemia-reperfusion impairment, which was related to the regulation of nitric oxide signaling pathway.

Acid-sensing ion channels contribute to the increase in vesicular release from SH-SY5Y cells stimulated by extracellular protons.

Acid-sensing ion channels (ASICs) have been reported to play a role in the neuronal dopamine pathway, but the exact role in neurotransmitter release remains elusive. Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line, which can release monoamine neurotransmitters. In this study, the expression of ASICs was identified in SH-SY5Y cells to further explore the role of ASICs in vesicular release stimulated by acid. We gathered evidence that ASICs could be detected in SH-SY5Y cells. In whole cell patch-clamp recording, a rapid decrease in extracellular pH evoked inward currents, which were reversibly inhibited by 100 µM amiloride. The currents were pH dependent, with a pH of half-maximal activation (pH(0.5)) of 6.01 ± 0.04. Furthermore, in calcium imaging and FM 1-43 dye labeling, it was shown that extracellular protons increased intracellular calcium levels and vesicular release in SH-SY5Y cells, which was attenuated by PcTx1 and amiloride. Interestingly, N-type calcium channel blockers inhibited the vesicular release induced by acidification. In conclusion, ASICs are functionally expressed in SH-SY5Y cells and involved in vesicular release stimulated by acidification. N-type calcium channels may be involved in the increase in vesicular release induced by acid. Our results provide a preliminary study on ASICs in SH-SY5Y cells and neurotransmitter release, which helps to further investigate the relationship between ASICs and dopaminergic neurons.

Sinomenine protects against ischaemic brain injury: involvement of co-inhibition of acid-sensing ion channel 1a and L-type calcium channels.

BACKGROUND AND PURPOSE: Sinomenine (SN), a bioactive alkaloid, has been utilized clinically to treat rheumatoid arthritis in China. Our preliminary experiments indicated that it could protect PC12 cells from oxygen-glucose deprivation-reperfusion (OGD-R), we thus investigated the possible effects of SN on cerebral ischaemia and the related mechanism. EXPERIMENTAL APPROACH: Middle cerebral artery occlusion in rats was used as an animal model of ischaemic stroke in vivo. The mechanisms of the effects of SN were investigated in vitro using whole-cell patch-clamp recording, calcium imaging in PC12 cells and rat cortical neurons subjected to OGD-R. KEY RESULTS: Pretreatment with SN (10 and 30 mg·kg(-1) , i.p.) significantly decreased brain infarction and the overactivation of calcium-mediated events in rats subjected to 2 h ischaemia followed by 24 h reperfusion. Extracellular application of SN inhibited the currents mediated by acid-sensing ion channel 1a and L-type voltage-gated calcium channels, in the rat cultured neurons, in a concentration-dependent manner. These inhibitory effects contribute to the neuroprotection of SN against OGD-R and extracellular acidosis-induced cytotoxicity. More importantly, administration of SN (30 mg·kg(-1) , i.p.) at 1 and 2 h after cerebral ischaemia also decreased brain infarction and improved functional recovery. CONCLUSION AND IMPLICATIONS: SN exerts potent protective effects against ischaemic brain injury when administered before ischaemia or even after the injury. The inhibitory effects of SN on acid-sensing ion channel 1a and L-type calcium channels are involved in this neuroprotection.

Acid-sensing ion channels contribute to the effect of acidosis on the function of dendritic cells.

As an H(+)-gated subgroup of the degenerin/epithelial Na(+) channel family, acid-sensing ion channels (ASICs) were reported to be involved in various physiological and pathological processes in neurons. However, little is known about the role of ASICs in the function of dendritic cells (DCs). In this study, we investigated the expression of ASICs in mouse bone marrow-derived DCs and their possible role in the function of DCs. We found that ASIC1, ASIC2, and ASIC3 are expressed in DCs at the mRNA and protein levels, and extracellular acid can evoke ASIC-like currents in DCs. We also demonstrated that acidosis upregulated the expression of CD11c, MHC class II, CD80, and CD86 and enhanced the Ag-presenting ability of DCs via ASICs. Moreover, the effect of acidosis on DCs can be abolished by the nonsteroidal anti-inflammatory drugs ibuprofen and diclofenac. These results suggest that ASICs are involved in the acidosis-mediated effect on DC function.

Tanshinone IIA attenuates neuronal damage and the impairment of long-term potentiation induced by hydrogen peroxide.

AIM OF THE STUDY: Tanshinone IIA (Tan IIA) is one of the key components of Salvia miltiorrhiza Bunge that has been widely used for various cardiovascular and cerebrovascular disorders in Asian countries. Many studies have reported that Tan IIA has antioxidative properties, but whether Tan IIA can rescue neurons from oxidative insult has never been reported. The present study was undertaken to evaluate the possible neuroprotective effects of Tan IIA on hydrogen peroxide (H(2)O(2))-induced oxidative stress in rats. MATERIALS AND METHODS: H(2)O(2)-induced cytotoxicity was evaluated by the cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and flow cytometry with PI staining. Calcium imaging experiments were carried out to measure intracellular free calcium concentration. Western blotting was used to determine the expression of Bax and Bcl-2 protein. Electrophysiological studies in hippocampal slices were performed to investigate the effect of Tan IIA on synaptic function and cognitive impairment caused by H(2)O(2). RESULTS: It was found that pretreatment with Tan IIA protected primary rat cortical neurons against H(2)O(2)-induced cytotoxicity. Furthermore, Tan IIA markedly reduced the elevation of [Ca(2+)](i) evoked by H(2)O(2). Western blot analysis indicated that pretreatment with Tan IIA prevented the increase in Bax/Bcl-2 ratio induced by H(2)O(2). In addition, preincubation of Tan IIA 20 min prior to H(2)O(2) exposure could reverse H(2)O(2)-induced hippocampal LTP impairment, but without significant alteration in basal synaptic transmission and LTP induction. CONCLUSIONS: These findings demonstrate that Tan IIA might serve as a novel promising therapeutic agent for oxidative stress injury in neurodegenerative diseases.

Disruption of PICK1 attenuates the function of ASICs and PKC regulation of ASICs.

Acid-sensing ion channels (ASICs) extensively exist in both central and peripheral neuronal systems and contribute to many physiological and pathological processes. The protein that interacts with C kinase 1 (PICK1) was cloned as one of the proteins interacting with protein kinase C (PKC) and colocalized with ASIC1 and ASIC2. Here, we used PICK1 knockout (PICK1-KO) C57/BL6 mice together with the whole cell patch clamp, calcium imaging, RT-PCR, Western blot, and immunocytochemistry techniques to explore the possible change in ASICs and the regulatory effects of PKC on ASICs. The results showed that PICK1 played a key role in regulation of ASIC functions. In PICK1-KO mouse cortical neurons, both the amplitude of ASIC currents and elevation of [Ca(2+)](i) mediated by acid were decreased, which were attributable to the decreased expression of ASIC1a and ASIC2a proteins in the plasma membrane. PKC, a partner protein of PICK1, regulated ASIC functions via PICK1. The agonist and antagonist of PKC only altered ASIC currents and acid-induced increase in [Ca(2+)](i) in wild-type, but not in KO mice. In conclusion, our data provided the direct evidence from PICK1-KO mice that a novel target protein, PICK1, would regulate ASIC function and membrane expression in the brain. In addition, PICK1 played the bridge role between PKC and ASICs.

Reversal of aging-associated hippocampal synaptic plasticity deficits by reductants via regulation of thiol redox and NMDA receptor function.

Deficits in learning and memory accompanied by age-related neurodegenerative diseases are closely related to the impairment of synaptic plasticity. In this study, we investigated the role of thiol redox status in the modulation of the N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) in CA1 areas of hippocampal slices. Our results demonstrated that the impaired LTP induced by aging could be reversed by acute administration of reductants that can regulate thiol redox status directly, such as dithiothreitol or ß-mercaptoethanol, but not by classical anti-oxidants such as vitamin C or trolox. This repair was mediated by the recruitment of aging-related deficits in NMDAR function induced by these reductants and was mimicked by glutathione, which can restore the age-associated alterations in endogenous thiol redox status. Moreover, antioxidant prevented but failed to reverse H(2)O(2) -induced impairment of NMDAR-mediated synaptic plasticity. These results indicate that the restoring of thiol redox status may be a more effective strategy than the scavenging of oxidants in the treatment of pre-existing oxidative injury in learning and memory.