Interaction of nNOS with PSD-95 negatively controls regenerative repair after stroke.

Stroke is a major public health concern. The lack of effective therapies heightens the need for new therapeutic targets. Mammalian brain has the ability to rewire itself to restore lost functionalities. Promoting regenerative repair, including neurogenesis and dendritic remodeling, may offer a new therapeutic strategy for the treatment of stroke. Here, we report that interaction of neuronal nitric oxide synthase (nNOS) with the protein postsynaptic density-95 (PSD-95) negatively controls regenerative repair after stroke in rats. Dissociating nNOS-PSD-95 coupling in neurons promotes neuronal differentiation of neural stem cells (NSCs), facilitates the migration of newborn cells into the injured area, and enhances neurite growth of newborn neurons and dendritic spine formation of mature neurons in the ischemic brain of rats. More importantly, blocking nNOS-PSD-95 binding during the recovery stage improves stroke outcome via the promotion of regenerative repair in rats. Histone deacetylase 2 in NSCs may mediate the role of nNOS-PSD-95 association. Thus, nNOS-PSD-95 can serve as a target for regenerative repair after stroke.

Delayed Administration of Tat-HA-NR2B9c Promotes Recovery After Stroke in Rats.

BACKGROUND AND PURPOSE: Previous studies reported that Tat-NR2B9c, a peptide disrupting the N-methyl-d-aspartate receptor-postsynaptic density protein-95 interaction, reduced ischemic damage in the acute phase after stroke. However, its effect in the subacute phase is unknown. The aim of this study is to determine whether disrupting the N-methyl-d-aspartate receptor-postsynaptic density protein-95 interaction in the subacute phase promotes recovery after stroke. METHODS: Studies were performed on Sprague-Dawley rats or nNOS(-/-) mice, and experimental ischemic stroke was induced by middle cerebral artery occlusion. Animals were treated with drugs starting at day 4 after ischemia. Sensorimotor functions and spatial learning and memory ability were assessed after drug treatment. Then, rats were euthanized for morphological observation and biochemical tests. RESULTS: Disrupting the N-methyl-d-aspartate receptor-postsynaptic density protein-95 interaction with Tat-HA-NR2B9c significantly ameliorated the ischemia-induced impairments of spatial memory and sensorimotor functions in rats during subacute stage but did not improve stroke outcome in nNOS(-/-) mice. Consistent with the functional recovery, Tat-HA-NR2B9c substantially increased neurogenesis in the dentate gyrus and dendritic spine density of mature neurons in the motor cortex of rats, meanwhile, reversed the ischemia-induced formation of S-nitrosylation-cyclin-dependent kinase 5 and increased cyclin-dependent kinase 5 activity in ipsilateral hippocampus. However, directly blocking N-methyl-d-aspartate receptors with MK-801 or Ro 25-6981 did not show the beneficial effects above. CONCLUSIONS: Dissociating N-methyl-d-aspartate receptor-postsynaptic density protein-95 coupling by Tat-HA-NR2B9c in the subacute phase after stroke promotes functional recovery, probably because of that it increases neurogenesis and dendritic spine density of mature neurons via regulating cyclin-dependent kinase 5 in the ischemic brain.

Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide.

To design a peptide disrupting the interaction between N-methyl-d-aspartate receptors-2B (NR2B) and postsynaptic density protein-95 (PSD-95), a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide contained a fragment of the cell membrane transduction domain of the human immunodeficiency virus type1 (HIV-1) Tat, a influenza virus hemagglutinin (HA) epitope-tag, and the C-terminal 9 amino acids of NR2B (NR2B9c). We named the chimeric peptide Tat-HA-NR2B9c. The expression plasmid contained a gene fragment encoding the Tat-HA-NR2B9c was ligated to the C-terminal fragment of l-asparaginase (AnsB-C) via a unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in inclusion body in Escherichia coli under isopropyl ß-d-1-thiogalactopyranoside (IPTG) and purified by washing with 2M urea, solubilizing in 4M urea, and then ethanol precipitation. The target chimeric peptide Tat-HA-NR2B9c was released from the fusion partner following acid hydrolysis and purified by isoelectric point precipitation and ultrafiltration. SDS-PAGE analysis and MALDI-TOF-MS analysis showed that the purified Tat-HA-NR2B9c was highly homogeneous. Furthermore, we investigated the effects of Tat-HA-NR2B9c on ischemia-induced cerebral injury in the rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion, and found that the peptide reduced infarct size and improved neurological functions.

Tat-HA-NR2B9c attenuate oxaliplatin-induced neuropathic pain.

Oxaliplatin is a commonly used chemotherapy drug, which can produce acute and chronic peripheral neurotoxicity. Currently, there is no good therapeutic drug in clinic. Excessive stimulation of N-methyl-d-aspartate receptors (NMDARs) is crucial for the transmission of pain signals. However, directly inhibiting NMDARs can cause severe side effects because they have key physiological functions in the Central nervous system (CNS). Several years ago, we prepared a polypeptide Tat-HA-NR2B9c which can disturb NMDARs-postsynaptic density protein-95 (PSD-95) interaction. In this study, we studied whether Tat-HA-NR2B9c could be an effective treatment for oxaliplatin-induced neuropathic pain. To conform it, a rat model of oxaliplatin-induced neuropathic was established, and analgesic effect of Tat-HA-NR2B9c was studied. Here, we show that oxaliplatin induces the interaction of NMDARs with PSD-95. Uncoupling the complex by Tat-HA-NR2B9c has potent analgesic effect in oxaliplatin-induced cold hyperalgesia and mechanical allodynia without suppressing general behavioral. Tat-HA-NR2B9c neither inhibits NMDARs function nor impacts antitumor activity of oxaliplatin. Thus, this new drug may serve as a treatment for oxaliplatin-induced neuropathic pain, perhaps without major side effects.

Chimeric Peptide Tat-HA-NR2B9c Improves Regenerative Repair after Transient Global Ischemia.

Transient global ischemia (TGI) is a major public health problem, and it heightens the need of effective treatments. The present study was undertaken to investigate whether recombinant polypeptide Tat-HA-NR2B9c improves spatial learning and memory deficits in rats after TGI. Rats were subjected to 20-min ischemia induced by four-vessel occlusion (4-VO) method and daily injected with Tat-HA-NR2B9c (1.12 mg/kg) for 1 week. Tat-HA-NR2B9c increased CREB activity, upregulated B-cell lymphoma-2 (Bcl-2) expression after treated for 24 h. There was a significant increase in dendrite spine density in hippocampal CA1 region and BrdU-positive cells and BrdU/NeuN-positive cells in the dentate gyrus after Tat-HA-NR2B9c treatment, compared with ischemia group at postischemic day 28. Inhibition of the CREB activation by recombinant lentivirus, LV-CREB133-GFP, abolished the upregulation effects of Tat-HA-NR2B9c on Bcl-2 expression. Moreover, Tat-HA-NR2B9c improved the impaired spatial learning and memory ability in Morris water maze. These results suggest that Tat-HA-NR2B9c substantially ameliorated the TGI-induced loss of dendrite spine in hippocampal CA1, increased neurogenesis in dentate gyrus, and significantly improved cognitive abilities by the CREB pathway in rats after transient global cerebral ischemia. It may be served as a treatment for TGI.

Hippocampal TERT Regulates Spatial Memory Formation through Modulation of Neural Development.

The molecular mechanism of memory formation remains a mystery. Here, we show that TERT, the catalytic subunit of telomerase, gene knockout (Tert-/-) causes extremely poor ability in spatial memory formation. Knockdown of TERT in the dentate gyrus of adult hippocampus impairs spatial memory processes, while overexpression facilitates it. We find that TERT plays a critical role in neural development including dendritic development and neuritogenesis of hippocampal newborn neurons. A monosynaptic pseudotyped rabies virus retrograde tracing method shows that TERT is required for neural circuit integration of hippocampal newborn neurons. Interestingly, TERT regulated neural development and spatial memory formation in a reverse transcription activity-independent manner. Using X-ray irradiation, we find that hippocampal newborn neurons mediate the modulation of spatial memory processes by TERT. These observations reveal an important function of TERT through a non-canonical pathway and encourage the development of a TERT-based strategy to treat neurological disease-associated memory impairment.

L-Tetrahydropalmatine alleviates mechanical hyperalgesia in models of chronic inflammatory and neuropathic pain in mice.

Chronic pain is categorized as inflammatory and neuropathic, and there are common mechanisms underlying the generation of each pain state. Such pain is difficult to treat and the treatment at present is inadequate. Corydalis yanhusuo is a traditional Chinese medicine with demonstrated analgesic efficacy in humans. The potential antihyperalgesic effect of its active component is L-tetrahydropalmatine (L-THP). L-THP has been used for the treatment of headache and other mild pain. However, little is known about its analgesic effect on chronic pain and its mechanism. Here, we report that L-THP exerts remarkable antihyperalgesic effects on neuropathic and inflammatory pain in animal models. Neuropathic hypersensitivity was induced by segmental spinal nerve ligation and inflammatory hypersensitivity was induced by an intraplantar injection of complete Freund's adjuvant. To determine the receptor mechanism underlying the antihyperalgesic actions of L-THP, we used SCH23390, an antagonist of a dopamine D1 receptor, in an attempt to block the antihyperalgesic effects of L-THP. We found that L-THP (1-4 mg/kg, i.p.) produced a dose-dependent antihyperalgesic effect in spinal nerve ligation and complete Freund's adjuvant models. The antihyperalgesic effects of L-THP were abolished by a dopamine D1 receptor antagonist SCH23390 (0.02 mg/kg). Furthermore, L-THP (4 mg/kg, i.p.) did not influence motor function. These findings suggest that L-THP may ameliorate mechanical hyperalgesia by enhancing dopamine D1 receptor-mediated dopaminergic transmission.

(+)-Borneol attenuates oxaliplatin-induced neuropathic hyperalgesia in mice.

Common chemotherapeutic agents such as oxaliplatin often cause neuropathic pain during cancer treatment in patients. Such neuropathic pain is difficult to treat and responds poorly to common analgesics, which represents a clinical challenge. (+)-Borneol, a bicyclic monoterpene present in the essential oil of plants, is used for analgesia and anesthesia in traditional Chinese medicine. Although borneol has an antinociceptive effect on acute pain models, little is known about its effect on chemotherapy-induced neuropathic pain and its mechanism. We found that (+)-borneol exerted remarkable antihyperalgesic effects in a mouse model of oxaliplatin-induced neuropathic pain. In addition, (+)-borneol blocked the action of the transient receptor potential ankyrin 1 agonist in mechanical and cold stimulus tests. Repeated treatment with (+)-borneol did not lead to the development of antinociceptive tolerance and did not affect body weight and locomotor activity. (+)-Borneol showed robust analgesic efficacy in mice with neuropathic pain by blocking transient receptor potential ankyrin 1 in the spinal cord and may be a useful analgesic in the management of neuropathic pain.

[Synthesis and SERS characterization of silver nanocubes].

Silver nanocubes were synthesized by reducing silver nitrate with ethylene glycol in the presence of poly(vinyl pyrrolidone) based on the report by Xia's group. Silver nanocubes were immobilized on silicon wafers by self-assembly processes. SERS activity of silver nanocubes was detected by using pyridine and SCN- respectively as probe molecules. The preliminary results show that the Raman intensities of pyridine and SCN- adsorbed at silver nanocubes were enhanced considerably, indicating that silver nanocubes can be used as a good SERS substrate. On the other hand, SERS combined with the probe molecule method can be used to characterize the optical property of silver nanocubes.

The different roles of glucocorticoids in the hippocampus and hypothalamus in chronic stress-induced HPA axis hyperactivity.

Hypothalamus-pituitary-adrenal (HPA) hyperactivity is observed in many patients suffering from depression and the mechanism underling the dysfunction of HPA axis is not well understood. Chronic stress has a causal relationship with the hyperactivity of HPA axis. Stress induces the over-synthesis of glucocorticoids, which will arrive at all the body containing the brain. It is still complicated whether glucocorticoids account for chronic stress-induced HPA axis hyperactivity and in which part of the brain the glucocorticoids account for chronic stress-induced HPA axis hyperactivity. Here, we demonstrated that glucocorticoids were indispensable and sufficient for chronic stress-induced hyperactivity of HPA axis. Although acute glucocorticoids elevation in the hippocampus and hypothalamus exerted a negative regulation of HPA axis, we found that chronic glucocorticoids elevation in the hippocampus but not in the hypothalamus accounted for chronic stress-induced hyperactivity of HPA axis. Chronic glucocorticoids exposure in the hypothalamus still exerted a negative regulation of HPA axis activity. More importantly, we found mineralocorticoid receptor (MR) - neuronal nitric oxide synthesis enzyme (nNOS) - nitric oxide (NO) pathway mediated the different roles of glucocorticoids in the hippocampus and hypothalamus in regulating HPA axis activity. This study suggests that the glucocorticoids in the hippocampus play an important role in the development of HPA axis hyperactivity and the glucocorticoids in the hypothalamus can't induce hyperactivity of HPA axis, revealing new insights into understanding the mechanism of depression.

CAPON-nNOS coupling can serve as a target for developing new anxiolytics.

Anxiety disorders are highly prevalent psychiatric diseases. There is need for a deeper understanding of anxiety control mechanisms in the mammalian brain and for development of new anxiolytic agents. Here we report that the coupling between neuronal nitric oxide synthase (nNOS) and its carboxy-terminal PDZ ligand (CAPON) can serve as a target for developing new anxiolytic agents. Augmenting nNOS-CAPON interaction in the hippocampus of mice by overexpressing full-length CAPON gave rise to anxiogenic-like behaviors, whereas dissociating CAPON from nNOS by overexpressing CAPON-125C or CAPON-20C (the C-terminal 125 or 20 amino acids of CAPON) or delivering Tat-CAPON-12C (a peptide comprising Tat and the 12 C-terminal amino acids of CAPON) in the hippocampus of mice produced anxiolytic-like effects. Mice subjected to chronic mild stress (CMS) displayed a substantial increase in nNOS-CAPON coupling in the hippocampus and a consequent anxiogenic-like phenotype. Disrupting nNOS-CAPON coupling reversed the CMS-induced anxiogenic-like behaviors. Moreover, small-molecule blockers of nNOS-CAPON binding rapidly produced anxiolytic-like effects. Dexamethasone-induced ras protein 1 (Dexras1)-extracellular signal-regulated kinase (ERK) signaling was involved in the behavioral effects of nNOS-CAPON association. Thus, nNOS-CAPON association contributes to the modulation of anxiety-related behaviors via regulating Dexras1-ERK signaling and can serve as a target for developing potential anxiolytics.