Gabrb2-knockout mice displayed schizophrenia-like and comorbid phenotypes with interneuron-astrocyte-microglia dysregulation.

Intronic polymorphisms of the GABAA receptor ß2 subunit gene (GABRB2) under adaptive evolution were associated with schizophrenia and reduced expression, especially of the long isoform which differs in electrophysiological properties from the short isoform. The present study was directed to examining the gene dosage effects of Gabrb2 in knockout mice of both heterozygous (HT) and homozygous (KO) genotypes with respect to possible schizophrenia-like and comorbid phenotypes. The KO mice, and HT mice to a lesser extent, were found to display prepulse inhibition (PPI) deficit, locomotor hyperactivity, stereotypy, sociability impairments, spatial-working and spatial-reference memory deficits, reduced depression and anxiety, and accelerated pentylenetetrazol (PTZ)-induced seizure. In addition, the KO mice were highly susceptible to audiogenic epilepsy. Some of the behavioral phenotypes showed evidence of imprinting, gender effect and amelioration by the antipsychotic risperidone, and the audiogenic epilepsy was inhibited by the antiepileptic diazepam. GABAergic parvalbumin (PV)-positive interneuron dystrophy, astrocyte dystrophy, and extensive microglia activation were observed in the frontotemporal corticolimbic regions, and reduction of newborn neurons was observed in the hippocampus by immunohistochemical staining. The neuroinflammation indicated by microglial activation was accompanied by elevated brain levels of oxidative stress marker malondialdehyde (MDA) and the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). These extensive schizophrenia-like and comorbid phenotypes brought about by Gabrb2 knockout, in conjunction with our previous findings on GABRB2 association with schizophrenia, support a pivotal role of GABRB2 in schizophrenia etiology.

Neuron-Derived ADAM10 Production Stimulates Peripheral Nerve Injury-Induced Neuropathic Pain by Cleavage of E-Cadherin in Satellite Glial Cells.

BACKGROUND: Increasing evidence suggests the potential involvement of metalloproteinase family proteins in the pathogenesis of neuropathic pain, although the underlying mechanisms remain elusive. METHODS: Using the spinal nerve ligation model, we investigated whether ADAM10 proteins participate in pain regulation. By implementing invitro methods, we produced a purified culture of satellite glial cells to study the underlying mechanisms of ADAM10 in regulating neuropathic pain. RESULTS: Results showed that the ADAM10 protein was expressed in calcitonin gene-related peptide (CGRP)-containing neurons of the dorsal root ganglia, and expression was upregulated following spinal nerve ligation surgery invivo. Intrathecal administration of GI254023X, an ADAM10 selective inhibitor, to the rats one to three days after spinal nerve ligation surgery attenuated the spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia. Intrathecal injection of ADAM10 recombinant protein simulated pain behavior in normal rats to a similar extent as those treated by spinal nerve ligation surgery. These results raised a question about the relative contribution of ADAM10 in pain regulation. Further results showed that ADAM10 might act by cleaving E-cadherin, which is mainly expressed in satellite glial cells. GI254023X reversed spinal nerve ligation-induced downregulation of E-cadherin and activation of cyclooxygenase 2 after spinal nerve ligation. ß-catenin, which creates a complex with E-cadherin in the membranes of satellite glial cells, was also downregulated by spinal nerve ligation surgery in satellite glial cells. Finally, knockdown expression of ß-catenin by lentiviral infection in purified satellite glial cells increased expression of inducible nitric oxide synthase and cyclooxygenase 2. CONCLUSION: Our findings indicate that neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleaving E-cadherin in satellite glial cells.

Effects of antidepressants on P2X7 receptors.

Antidepressants including paroxetine, fluoxetine and desipramine are commonly used for treating depression. P2×7 receptors are member of the P2X family. Recent studies indicate that these receptors may constitute a novel potential target for the treatment of depression. In the present study, we examined the action of these antidepressants on cloned rat P2×7 receptors that were stably expressed in human embryonic kidney (HEK) 293 cells by using the whole-cell patch-clamp technique, and found that paroxetine at a dose of 10µM could significantly reduce the inward currents evoked by the P2×7 receptors agonist BzATP by pre-incubation for 6-12 but not by acute application (10µM) or pre-incubation for 2-6h at a dose of 1µM, 3µM or 10µM paroxetine. Neither fluoxetine nor desipramine had significant effects on currents evoked by BzATP either applied acutely or by pre-incubation at various concentrations. These results suggest that the sensitivity of rat P2×7 receptors to antidepressants is different, which may represent an unknown mechanism by which these drugs exert their therapeutic effects and side effects.

Pirt reduces bladder overactivity by inhibiting purinergic receptor P2X3.

Pirt is a transmembrane protein predominantly expressed in peripheral neurons. However, the physiological and pathological roles of Pirt in hollow viscus are largely unknown. Here we show that Pirt deficiency in mice causes bladder overactivity. The density of α,ß-meATP-induced currents is significantly reinforced in Pirt-deficient dorsal root ganglion (DRG) neurons. Pirt and P2X3 receptor co-localize in bladder nerve fibres and heterologous Pirt expression significantly reduces P2X3-mediated currents. Pirt interacts with P2X3 through the N-terminal 14 amino-acid residues. TAT-conjugated Pirt(N14) peptide (Pirt(N14)) is sufficient to inhibit P2X3 activation in bladder DRG neurons and to alleviate bladder overactivity in Pirt(-/-) mice. Pirt expression is decreased in the bladder of cyclophosphamide (CYP)-treated mice, a commonly used model of bladder overactivity. Importantly, Pirt(N14) administration reduces the frequency of bladder voiding and restores the voided volume of CYP-treated mice. Therefore, our results demonstrate that Pirt is an endogenous regulator of P2X3 in bladder function.

Involvement of MAPK ERK activation in upregulation of water channel protein aquaporin 1 in a mouse model of Bell's palsy.

The aim of this study is to immunolocalize the aquaporin 1 water channel protein (AQP1) in Schwann cells of idiopathic facial nerve and explore its possible role during the development of facial palsy induced by herpes simplex virus type 1 (HSV-1). HSV-1 was inoculated into the surface of posterior auricle of mouse to establish a paralyzed animal model. In HSV-1-induced facial palsy mice, protein levels of AQP1 significantly increased on the 9th to 16th day after inoculation of HSV-1. The upregulation of AQP1 was closely related to the intratemporal facial nerve edema in facial nerve canal, which was also consistent with the symptom of facial palsy in mice. In a hypoxia model of Schwann cells in vitro, we found that U0126, an ERK antagonist, inhibited not only morphological changes of cultures Schwann cells but also upregulation of both AQP1 and phosphorylated ERK. Combined with increased phosphorylated ERK in HSV-1-induced facial palsy mice, we inferred that ERK MAPK pathway might also be involved in increased AQP1 in mouse model of Bell's palsy. Although the precise mechanism needs to be further explored, our findings suggest that AQP1 in Schwann cells of intratemporal facial nerve is involved in the evolution of facial palsy induced by HSV-1 and may play an important role in the pathogenesis of this disease. AQP1 might be a potential target, and the ERK antagonist U0126 could be a new drug for the treatment of HSV-1-induced Bell's palsy in an early stage.

Molecular hydrogen suppresses reactive astrogliosis related to oxidative injury during spinal cord injury in rats.

AIMS: Spinal cord injury (SCI) can induce excessive astrocyte activation. Hydrogen has been deemed as a novel antioxidant. We investigated whether molecular hydrogen could act as an antiastrogliosis agent during SCI and oxidative injury in experimental rats and cultured astrocytes. METHODS: Hydrogen-rich saline (HS, 8 mL/kg, i.p.) was injected every 12 h after SCI in rats. The expression of STAT3, p-STAT3, and glial fibrillary acidic protein (GFAP); the release of IL-1ß, IL-6, and TNF-α; and astrogliosis, along with the BBB score, were evaluated. Culturing astrocytes with hydrogen-rich medium, the intracellular reactive oxygen species (ROS), astrogliosis, and the release of proinflammatory cytokines were assessed after H2O2-induced injury. RESULTS: In the HS group, the expression of STAT3, p-STAT3, and GFAP and the proinflammatory cytokines were decreased in local spinal cord on postoperation day (POD) 3; on PODs 7 and 14, reactive astrogliosis was suppressed, and the locomotor function was also improved. Furthermore, hydrogen-rich medium attenuated the intracellular production of ROS (especially HO•), astrogliosis, and the secretion of proinflammatory cytokines in astrocytes 12 h after H2O2-induced injury. CONCLUSIONS: Molecular hydrogen could suppress reactive astrogliosis after contusive SCI and reduce the release of proinflammatory cytokines produced by active astrocytes related to oxidative injury. Thus, molecular hydrogen is potential to be a neuroprotective agent.

K(V)7/KCNQ channels are functionally expressed in oligodendrocyte progenitor cells.

BACKGROUND: K(V)7/KCNQ channels are widely expressed in neurons and they have multiple important functions, including control of excitability, spike afterpotentials, adaptation, and theta resonance. Mutations in KCNQ genes have been demonstrated to associate with human neurological pathologies. However, little is known about whether K(V)7/KCNQ channels are expressed in oligodendrocyte lineage cells (OLCs) and what their functions in OLCs. METHODS AND FINDINGS: In this study, we characterized K(V)7/KCNQ channels expression in rat primary cultured OLCs by RT-PCR, immunostaining and electrophysiology. KCNQ2-5 mRNAs existed in all three developmental stages of rat primary cultured OLCs. K(V)7/KCNQ proteins were also detected in oligodendrocyte progenitor cells (OPCs, early developmental stages of OLCs) of rat primary cultures and cortex slices. Voltage-clamp recording revealed that the I(M) antagonist XE991 significantly reduced K(V)7/KCNQ channel current (I(K(Q))) in OPCs but not in differentiated oligodendrocytes. In addition, inhibition of K(V)7/KCNQ channels promoted OPCs motility in vitro. CONCLUSIONS: These findings showed that K(V)7/KCNQ channels were functionally expressed in rat primary cultured OLCs and might play an important role in OPCs functioning in physiological or pathological conditions.

Astroglial P2X7 receptor current density increased following long-term exposure to rotenone.

The role of the interaction between neurons and glial cells in the pathogenesis of neurodegenerative diseases is gaining more attention. Neuroinflammation participates in the progressive nature of diverse neurologic diseases including Parkinson's disease, Alzheimer's disease and multiple sclerosis. Activated microglia release neurotoxic molecules, which take part in the neuroinflammatory responses. Astrocytes are also key players in these responses. Reactive astrocytes secrete inflammatory factors, including tumor necrosis factor-α (TNF-α). This secretion can be regulated by extracellular ATP mediated through P2X7 receptors. However, whether the activity of astrocytic P2X7 receptors changes in Parkinson's disease and whether these changes would influence the secretion of inflammatory factors in astrocytes are still unclear. In our study, through immunocytochemistry, whole-cell patch clamp and ELISA assay, we found that P2X7 receptors were expressed in midbrain astrocytes, and that, rotenone, a Parkinson's disease model used at a low concentration (2-20 nM) for 48 h increased the P2X7 receptor current density and thereby inhibited the secretion of TNF-α. Our research suggests that rotenone can regulate cytokine secretion of astrocytes through elevated P2X7 channel current density and, in turn, take part in the neuroinflammatory process in the rotenone Parkinson's disease model.

[The correlation of asymmetrical dimethylarginine level and oxidative stress to the onset of Alzheimer's disease].

This study is to investigate the influence and mechanism of action of asymmetrical dimethylarginine (ADMA) and the induced oxidative stress level on Alzheimer's disease (AD) incidence. ADMA concentration, nitric oxide, Abeta(40)/Abeta(42) ratio, inducible NO synthase (iNOS) activity and the concentrations of the induced free radicals including malondialdehyde (MDA), 3-nitrotyrosine (3-NT) and peroxynitrite (ONOO-) in the cerebrospinal fluid (CSF) from 34 neurologically normal controls and 37 AD patients were quantitatively determined and statistically compared. The results showed that the ADMA concentration significantly decreased in AD patients, and it showed negative correlation with the NO, iNOS activity, and showed positive correlation with MMSE score. ADMA concentration was negatively correlated with Abeta(40)/Abeta(42) ratio (P<0.01) with the observation that Abeta(40)/Abeta(42) ratio increased while ADMA level decreased in CSF in AD patients. The concentration levels of MDA, 3-NT and ROS significantly increased compared with the control with all the P values less than 0.05. These findings suggested that the ADMA disorder and the oxidative damage effect of the induced free radicals in CSF of AD patients are an important mechanism of AD incidence, and their joint regulation may provide new idea for the prevention and clinical treatment of AD.

Polygalasaponin G promotes neurite outgrowth of cultured neuron on myelin.

Myelin contains many axonal outgrowth inhibitory components which contribute to regeneration failure after neuronal injury in the mammalian central nervous system (CNS). In an attempt to develop small molecular agents to promote axonal outgrowth, we screened a compound library purified from traditional Chinese herbs, and found a small molecular compound polygalasaponin G (PS-G), extracted from Polygala japonica, which has a potent neurotrophic activity on PC12 cells and cultured cortical neurons. We reported, to our knowledge for the first time, that PS-G could promote neurite outgrowth of neurons cultured on the myelin substrates and inhibit the activation of RhoA. Thus, our results could represent a therapeutic approach to improve axon regeneration after CNS injuries.

Expression of P2X purinoceptors in PC12 phaeochromocytoma cells.

1. The PC12 cell line, which was cloned from a rat adrenal phaeochromocytoma, is a useful model system. It expresses neuronal properties after treatment with nerve growth factor (NGF). The nervous system-specific P2X receptor subtype P2X(2) was initially cloned from PC12 cells, but little is known about the expression of other subtypes of P2X receptors in PC12 cells. The aim of the present study was to investigate whether PC12 cells express the other P2X receptors when exposed to NGF. 2. Reverse transcription-polymerase chain reaction at the mRNA level and immunocytochemisty at the protein level showed that, among the seven P2X purinoceptor subtypes, only P2X(2) was found to be expressed in undifferentiated PC12 phaeochromocytoma cells, but all seven P2X purinoceptor subtypes were expressed in differentiated PC12 cells treated with 50 microg/mL NGF. 3. Electrophysiological recordings indicated that ATP (30 micromol/L) but not alpha,beta-methylene ATP (alpha,beta-meATP; 30 micromol/L) evoked an inward current in undifferentiated PC12 cells, but both alpha,beta-meATP and ATP evoked inward currents in differentiated PC12 cells. The results indicate that the NGF-induced P2X receptors expressed in PC12 cells are functional channels. 4. The present study suggests that the NGF-induced neuronal phenotype of PC12 cells may be a model for the study of P2X heteromeric receptors.