Circular RNAs: Diagnostic and Therapeutic Perspectives in CNS Diseases.

Circular RNAs (circRNAs) are a class of regulatory non-coding RNAs characterized by the presence of covalently closed ends. A growing body of evidence suggests that circRNAs play important roles in physiology and pathology. In particular, accumulating data on circRNA functions in various central nervous system (CNS) diseases and their correlations indicate that circRNAs are critical contributors to the onset and development of brain disorders. In this review, we focus on the regulatory and functional roles of circRNAs in CNS diseases, highlighting their diagnostic and therapeutic potential, with the aim of providing new insights into CNS diseases.

N6-methyladenosine as a Novel Regulator of Brain Physiology and Diseases.

N6-methyladenosine (m6A) is identified as the most widespread and abundant internal chemical modification of RNA in eukaryotes. A series of proteins including methyltransferases (also known as "writers"), demethylases (also known as "erasers"), and m6A-binding proteins (also known as "readers") were indicated to participate in the m6A methylation. m6A has emerged as a regulator of various cellular, developmental, and disease processes. Notably, there is highest abundance of m6A methylation in brain than in other organs, which indicates that m6A plays an essential role in brain functions. Here, we describe the general features, mechanisms, and functions of m6A in the brain, and discuss the emerging roles of m6A in brain physiology and diseases.

Precise Characterization of the Penumbra Revealed by MRI: A Modified Photothrombotic Stroke Model Study.

AIMS: To precisely characterize the penumbra by MRI based on a modified photothrombotic stroke mouse model. METHODS: The proximal middle cerebral artery was occluded by a convenient laser system in conjunction with an intravenous injection of Rose Bengal in mice. And the suture MCAO model was performed in seven mice as a comparison of the reproducibility. One hour after occlusion, the penumbra was defined in six random photothrombotic stroke mice by mismatch between perfusion-weighted imaging and the apparent diffusion coefficient map on a home-made workstation. After imaging, three random mice of them were chosen to perform the reperfusion surgery. And the other three mice were sacrificed to stain for several potential penumbra markers, such as c-fos and heart shock protein 90. In the remaining mice, the evolution of the lesions was detected on the apparent diffusion coefficient map, diffusion-weighted imaging and T2-weighted imaging at 1, 3, 6, 12 and 24 hours. After evaluating the neurological deficit scores, the brains were sectioned and stained by triphenyltetrazolium chloride and Nissl. RESULTS: The mice subjected to photothrombosis showed significant behavioral deficits. One hour after occlusion, the low perfusion areas on the perfusion-weighted imaging interlaced with the hypointense areas on the apparent diffusion coefficient map, demonstrating that the penumbra was located both surrounding and inside the lesions. This phenomenon was subsequently confirmed by the c-fos and heart shock protein 90 staining. The final T2-weighted images of the mice subjected to the reperfusion surgery were also consistent with the penumbra images at one hour. At early stages, the lesions were clearly identified on the apparent diffusion coefficient map; the volumes of the lesions on the diffusion-weighted imaging and T2-weighted imaging did not reach a maximum until 12 hours. The coefficient of variation (CV) of the final lesions in the photothrombotic stroke mice was 21.7% (0.08 of 0.37) on T2-weighted imaging and 27.8% (0.10 of 0.35) on triphenyltetrazolium chloride, representing a high reproducibility (n = 7). While the CV of the lesions in the MCAO stroke mice was only 70% (0.24 of 0.34, n = 4). CONCLUSIONS: This study has provided a precise imaging definition of the penumbra based on a reproducible photothrombotic stroke mouse model.

Cystamine improves functional recovery via axon remodeling and neuroprotection after stroke in mice.

AIMS: Stroke is a leading cause of disability. However, there is no pharmacological therapy available for promoting recovery. Although treatment of stroke with cystamine has gained increasing interest, the detailed mechanisms underlying this process remain elusive. Thus, our aim is to examine the effect of cystamine on the function recovery after stroke and investigate further cystamine mechanisms. METHODS: Adult male C57BL/6J mice were subjected to photothrombotic model of focal stroke or sham operation. Cystamine or saline was administered intraperitoneally at 24 h after stroke. Functional recovery was analyzed using behavioral tests; axon remodeling was analyzed using magnetic resonance diffusion tensor imaging (DTI) and histological assessment. ANA-12, an antagonist of tropomyosin-related kinase B (TrkB), was administrated to examine the mechanisms underlying the neuroprotection mediated by cystamine. RESULTS: Treatment with cystamine resulted in amelioration of impaired function with concomitant enhancement of axonal remodeling. Cystamine treatment significantly increased brain-derived neurotrophic factor (BDNF) levels and phosphorylation of TrkB in brain after stroke. Cystamine significantly enhanced neuronal progenitor cell proliferation, neuronal survival, and plasticity through BDNF/TrkB pathway. CONCLUSIONS: These data provide evidence to investigate the promising utility of cystamine for therapy of stroke in a variety of ways, acting principally through BDNF/TrkB pathway.

Opening of microglial K(ATP) channels inhibits rotenone-induced neuroinflammation.

As activated microglia (MG) is an early sign that often precedes and triggers neuronal death, inhibition of microglial activation and reduction of subsequent neurotoxicity may offer therapeutic benefit. The present study demonstrates that rat primary cultured MG expressed Kir6.1 and SUR2 subunits of K(ATP) channel, which was identical to that expressed in BV-2 microglial cell line. The classic K(ATP) channel opener pinacidil and selective mitochondrial K(ATP) (mito-K(ATP)) channel opener diazoxide prevented rotenone-induced microglial activation and production of pro-inflammatory factors (tumour necrosis factor[TNF]-alpha and prostaglandin E(2)[PGE(2)]). And the effects of pinacidil and diazoxide were reversed by mito-K(ATP) blocker 5-hydroxydecanoate (5-HD), indicating that mito-K(ATP) channels participate in the regulation of microglial activation. Moreover, the underlying mechanisms involved the stabilization of mitochodrial membrane potential and inhibition of p38/c-Jun-N-terminal kinase (JNK) activation in microglia. Furthermore, the in vivo study confirmed that diazoxide exhibited neuroprotective effects against rotenone along with the inhibition of microglial activation and neuroinflammation. Thus, microglial mito-K(ATP) channel might be a novel prospective target for the treatment of neuroinflammation-related degenerative disorders such as Parkinson's disease.

Iptakalim alleviates rotenone-induced degeneration of dopaminergic neurons through inhibiting microglia-mediated neuroinflammation.

Inhibition of microglia-mediated neuroinflammation has been regarded as a prospective strategy for treating neurodegenerative disorders, such as Parkinson's disease (PD). In the present study, we demonstrated that systematic administration with iptakalim (IPT), an adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) opener, could alleviate rotenone-induced degeneration of dopaminergic neurons in rat substantia nigra along with the downregulation of microglial activation and mRNA levels of tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2). In rat primary cultured microglia, pretreatment with IPT suppressed rotenone-induced microglial activation evidenced by inhibition of microglial amoeboid morphological alteration, declined expression of ED1 (a marker for activated microglia), and decreased production of TNF-alpha and prostaglandin E2 (PGE(2)). These inhibitory effects of IPT could be reversed by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (5-HD). Furthermore, pretreatment with IPT prevented rotenone-induced mitochondrial membrane potential loss and p38/c-jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) activation in microglia, which might in turn regulate microglial activation and subsequent production of TNF-alpha and PGE(2). These data strongly suggest that the K(ATP) opener IPT may be a novel and promising neuroprotective drug via inhibiting microglia-mediated neuroinflammation.

Activation of Group II/III metabotropic glutamate receptors attenuates LPS-induced astroglial neurotoxicity via promoting glutamate uptake.

Altered glial function that leads to oxidative stress and excitotoxicity may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. We report the pivotal role of astroglial Group II and III metabotropic glutamate receptors (mGluR) against neurotoxicity. Activation of Group II or III mGluR on astrocytes with selective agonists DCG-IV or L-AP4 respectively inhibited astroglial lipopolysaccharide (LPS)-conditioned medium induced apoptosis of primary cultured mesencephalic neurons. Specific Group II or III mGluR antagonists APICA or MSOP completely abolished the neuroprotective effects of DCG-IV and L-AP4. Morphologic analysis showed that DCG-IV or L-AP4 could also attenuate the astroglial neurotoxicity to dopaminergic neurons. Measurement of extracellular glutamate concentration and [(3)H]-glutamate uptake showed that the restoration of glutamate uptake capability in LPS-treated astrocytes might be involved in the neuroprotective effects of activating astroglial Group II or III mGluR. Furthermore, we found that the repression of astroglial uptake function could be revived by GSH, and both Group II and III mGluR agonists could recover the endogenous reduced glutathione (GSH) level in LPS-treated astrocytes. These results suggested that the possible mechanisms of neuroprotection by either Type II or Type III mGluR activation may involve restoration of endogenous GSH, in turn affording recovery of astroglial capability to take up glutamate.

Protective effects of iptakalim, a novel ATP-sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils.

AIM: To investigate the protective role of iptakalim, a novel ATP sensitive potassium channel opener, on global cerebral ischemia-evoked insult in gerbils and glutamate-induced PC12 cell injury. METHODS: Global cerebral ischemia was induced by occluding the bilateral common carotid arteries in gerbils for 5 min. The open field maze and T-maze were employed to investigate the experimental therapeutic value of iptakalim on ischemic brain insult (n=8). The pyramidal cells in the hippocampal CA1 regions were counted to assess the protective effects of iptakalim. Glutamate released from the gerbil hippocampus and PC12 cells were determined by HPLC. Intracellular calcium was measured by Fluo-3 AM with A Bio-Rad Radiance 2100TM confocal system in conjunction with a Nikon TE300 microscope. Astrocyte glutamate uptake measurements were determined by liquid scintillation counting. RESULTS: Iptakalim (0.5-4.0 mg/kg per day, ip) could reduce the high locomotor activity evoked by ischemia and improve global cerebral ischemia-induced working memory impairments. Histological studies revealed that iptakalim could increase the survival neuron in the hippocampus CA1 zone in a dose-dependent manner. Moreover, iptakalim could reverse ischemia-evoked increases of glutamate in the hippocampus of gerbils. In an in vitro study, iptakalim protected PC12 cells against glutamate-induced excitotoxicity, reduced the [Ca(2+)](i) increases, and enhanced the glutamate uptake activity of primary cultured astrocytes. CONCLUSIONS: Iptakalim plays a key role in preventing global cerebral ischemia-evoked insults in gerbils and glutamate-induced PC12 cell injury by anti-excitotoxicity. Iptakalim might be a promising novel candidate for the prevention and/or treatment of stroke.

Activation of mitochondrial ATP-sensitive potassium channels improves rotenone-related motor and neurochemical alterations in rats.

Our previous studies revealed that activation of mitochondrial ATP-sensitive potassium channels exerted protective effects on rotenone-treated rats and cultured cells. The aim of the present study is to examine the potential therapeutic effects of iptakalim, an ATP-sensitive potassium-channel opener, and diazoxide, a selective mitochondrial ATP-sensitive potassium-channel opener, on Parkinsonian symptoms in rats induced by rotenone. Rats were treated with rotenone (2.5 mg/kg s.c.) daily for 4 wk. This treatment caused a depletion of dopamine in the striatum and substantia nigra. Behaviourally, rotenone-infused rats exhibit Parkinsonian symptoms. Catalepsy was estimated by a 9-cm bar test. Treatment with L-dopa (10 mg/kg.d p.o.), iptakalim (0.75, 1.5, 3.0 mg/kg.d p.o.) and diazoxide (3.0 mg/kg.d p.o.) for 2 wk improved behavioural dysfunction and elevated dopamine contents in the striatum and substantia nigra of rotenone-treated rats. Studies also found that iptakalim and diazoxide could reduce the enzymic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. All neurorestorative effects by both iptakalim and diazoxide were abolished by 5-hydroxydecanoate, a selective mitochondrial ATP-sensitive potassium-channel blocker. Collectively, the data suggested that mitochondrial ATP-sensitive potassium channels play a key role in improving both Parkinsonian symptoms and neurochemistry alterations of rotenone model rats, and selective activation of mitochondrial ATP-sensitive potassium channels may provide a new therapeutic strategy for treatment of early Parkinson's disease.

The regulation of rotenone-induced inflammatory factor production by ATP-sensitive potassium channel expressed in BV-2 cells.

Our previous studies have demonstrated that activating ATP-sensitive potassium channel (K(ATP) channel), not only improved Parkinsonian behavior and neurochemical symptoms, but also reduced iNOS activity and mRNA levels in striatum and nigra of rotenone rat model of Parkinson's disease (PD). In this study, it was first shown that the subunits of K(ATP) channels are expressed in BV-2 cells, and then it was investigated whether K(ATP) channel was involved in regulating inflammatory factor production from BV-2 cells activated by rotenone. It was found that K(ATP) channel was expressed in BV-2 cell and formed by the combination of Kir 6.1 and SUR 2A/2B. K(ATP) channel openers (KCOs) including pinacidil, diazoxide and iptakalim (Ipt) exerted beneficial effects on rotenone-induced morphological alterations of BV-2 cells, decreased tumor necrosis factor alpha (TNF-alpha) production and the expression and activity of inducible isoform of nitric oxide synthase (iNOS). Either glibenclamide or 5-hydroxydecanoate acid (a selective mitochondrial K(ATP) channel blocker) could abolish the effects of KCOs, suggesting that K(ATP) channels, especially mitochondrial ATP-sensitive potassium channels (mitoK(ATP) channels), played a crucial role in preventing the activation of BV-2 cells, and subsequently the production of a variety of proinflammatory factors. Therefore, activation of K(ATP) channel might be a new therapeutic strategy for treating neuroinflammatory and neurodegenerative disorders.

ATP-sensitive potassium channel opener iptakalim protected against the cytotoxicity of MPP+ on SH-SY5Y cells by decreasing extracellular glutamate level.

Mounting evidence reveals that ATP-sensitive potassium (K(ATP)) channel openers (KCOs) exert significant neuroprotection in vivo and in vitro in several models of Parkinson's disease (PD). However, the mechanisms are not well understood. In this study, we demonstrated that SH-SY5Y cells expressed mRNA and proteins for Kir6.1, Kir6.2, SUR1 and SUR2 subunits of K(ATP) channels. Moreover, our results showed that 1-methyl-4-phenyl-pyridinium ion (MPP+) induced up-regulation of mRNA for the Kir6.2 subunit and down-regulation of SUR1. It was further found that pretreatment with iptakalim, a novel K(ATP) channel opener, could attenuate increased extracellular glutamate level and decreased cell survival in SH-SY5Y cell culture after exposure to MPP+. Trans-pyrrolidine-2, 4-dicarboxylic acid (t-PDC), a glutamate transporter inhibitor, partially blocked the effect of iptakalim decreasing extracellular glutamate level. Additionally, iptakalim prevented MPP+-induced inhibition of glutamate uptake in primary cultured astrocytes. The beneficial effects of iptakalim on glutamate uptake of astrocytes were abolished by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-HD. These results suggest (i) K(ATP) channel dysfunction may be involved in the mechanisms of MPP+-induced cytotoxicity and (ii) iptakalim may modulate glutamate transporters and subsequently alleviate the increase of extracellular glutamate levels induced by MPP+ through opening mitoK(ATP) channels, thereby protecting SH-SY5Y cells against MPP+-induced cytotoxicity.

6-Hydroxydopamine-induced glutathione alteration occurs via glutathione enzyme system in primary cultured astrocytes.

AIM: To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes. METHODS: Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay. The mRNA levels of gamma-glutamylcysteine synthetase (gammaGCS), gamma-glutamyltransferase (gammaGT), glutathione peroxidase (GPx), GR(glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR. gammaGT activity was determined using gammaGT assay kits. RESULTS: In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h. However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h. RT-PCR data showed that the mRNA levels of gammaGCS, the rate-limiting enzyme of gamma-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of gammaGT after treatment for 48 h, which induced a decrease in GSx levels, despite the up-regulation of gammaGCS after exposure to 6-OHDA for 48 h. CONCLUSION: The change in gammaGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h. GSx levels decreased because of increased gammaGT mRNA levels and gammaGT activity induced by 6-OHDA after treatment for 48 h.

Systematic administration of iptakalim, an ATP-sensitive potassium channel opener, prevents rotenone-induced motor and neurochemical alterations in rats.

Our previous studies revealed that iptakalim, a novel ATP-sensitive potassium channel opener, has a significant neuroprotective function against ischemia in vivo or rotenone-induced neurotoxicity in vitro. To investigate the potential pharmaceutical benefit of ATP-sensitive potassium channel openers on neurodegenerative diseases, we studied the effects of iptakalim and diazoxide, a selective mitochondrial ATP-sensitive potassium channel opener, on the rotenone-induced nigrostriatal degeneration in rats. Iptakalim (1.5 mg/kg/day, orally) or diazoxide (1.5 mg/kg/day, orally) alone was administered to rats for 3 days, and then for 4 weeks was used daily with an injection of rotenone (2.5 mg/kg/day, subcutaneously) 1 hr later each time. The results showed that rotenone-infused rats exhibited parkinsonian symptoms and had dopamine depletion in the striatum and substantia nigra. Pretreatment with iptakalim or diazoxide prevented rotenone-induced catalepsy and the reduction of striatum dopamine contents. Moreover, iptakalim and diazoxide reduced the enzymatic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. These neuroprotective effects of iptakalim and diazoxide were abolished by 5-hydroxydecanoate, a selective mitochondrial ATP-sensitive potassium channel blocker. In conclusion, our data suggested that mitochondrial ATP-sensitive potassium channels might play a key role in preventing both parkinsonian symptoms and neurochemistry alterations induced by rotenone in rats. The selective activation of mitochondrial ATP-sensitive potassium channels may provide a new therapeutic strategy for prevention and treatment of neurodegenerative disorders such as Parkinson's disease.

Enhancement of glutamate uptake mediates the neuroprotection exerted by activating group II or III metabotropic glutamate receptors on astrocytes.

We investigated whether the activation of astroglial group II and III metabotropic glutamate receptors (mGluRs) could exert neuroprotective effects and whether the neuroprotection was related to glutamate uptake. Our results showed that the activation of astroglial group II or III mGluRs exerted neuroprotection against 1-methyl-4-phenylpyridinium (MPP+) astroglial conditioned medium-induced neurotoxicity in midbrain neuron cultures. Furthermore, MPP+ decreased glutamate uptake of primary astrocytes and C6 glioma cells, which was recovered by activating group II or III mGluRs. Specific group II or III mGluRs antagonists completely abolished the neuroprotective effects and the enhancement of glutamate uptake of their respective agonists. Our results showed that the primary cultured rat astrocytes and C6 glioma cells expressed receptor proteins for group II mGluR2/3, group III mGluR4, mGluR6 and mGluR7. C6 glioma cells expressed mRNA for group II mGluR3, group III mGluR4, mGluR6, mGluR7 and mGluR8. In conclusion, we confirmed that the activation of astroglial mGluRs exerted neuroprotection, and demonstrated that the mechanism underlying this protective role was at least partially related to the enhancement of glutamate uptake.

Effects of iptakalim on rotenone-induced cytotoxicity and dopamine release from PC12 cells.

Parkinson's disease is characterized by an extensive loss of dopaminergic neurons in the substantia nigra pars compacta. The final common pathway in the demise of these cells may involve dopamine-dependent oxidative stress. Previous studies revealed a new neuronal protective role of ATP-sensitive potassium channel openers. But the exact mechanism is still unknown. In the present study, the neuroprotective effect of iptakalim, a novel ATP-sensitive potassium channel opener, was studied against rotenone-induced cytotoxicity in rat dopaminergic PC12 cells. Rotenone decreased cell viability significantly after 48 h exposure and induced dopamine release from PC12 cells concentration-dependently. Iptakalim significantly enhanced dopamine uptake and alleviated rotenone-induced PC12 cells death and reduced dopamine release induced by rotenone or GBR-12909, a classical dopamine transporter inhibitor. These results suggest that iptakalim may open mitochondrial K(ATP) channels to modulate dopamine transporter and reduce extracellular dopamine levels, thereby it protecting PC12 cells against rotenone-induced injury.

Inhibitory effects of 1-methyl-4-phenylpyridinium on glutamate uptake into cultured C6 glioma cells.

AIM: To investigate the effect of 1-methyl-4-phenylpyridinium (MPP+) on the glutamate uptake into cultured C6 glioma cells. METHODS: The glutamate uptake into C6 glioma cells was measured by radio-ligand binding assay method. The effect of MPP+ on the morphology of C6 glioma cells was observed under phase contrast microscopy; apoptosis of C6 glioma cells were measured by FITC-labeled Annexin V staining and flow cytometry. Cell viability was measured by MTT method. RESULTS: MPP+ inhibited glutamate uptake into C6 glioma cells. However, MPP+ failed to induce any morphological changes of C6 glioma cells, and exposure to MPP+ had no effect on the viability and the apoptotic percentage of C6 glioma cells. Incubation with 12-O-tetradecanoylphorbol -13-acetate (TPA), a protein kinase C activator, caused a significant increase in glutamate uptake and completely reversed MPP+-induced inhibitory effect on glutamate uptake. CONCLUSION: The present results indicate that glutamate transporters may have important pathogenetic implications in Parkinson disease. MPP(+)-induced inhibition of glutamate uptake was due to the dysfunction of glutamate transporters; TPA enhanced glutamate uptake and completely reversed the inhibitory effect of MPP+.

Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient focal cerebral ischemia in mice.

The present observations examined the hypothesis that the iNOS expression in the ischemic penumbra after a transient focal ischemic insult is involved in the recruitment of penumbra into infarction. The middle cerebral artery in mice was occluded for 2 h by an intraluminal filament and then recirculated. The measurement of iNOS activity, iNOS protein formation and NO concentration in the ischemic core and penumbra, and the determination of infarct volume were performed at 6, 12, 24 and 48 h after reperfusion. iNOS protein and iNOS enzymatic activity appeared at 6 h, peaked at 24 h, and declined at 48 h in the penumbra after reperfusion. iNOS protein was not detectable in contralateral area and in sham-operated brains. The time course of iNOS protein, enzymatic activity and NO concentration in the penumbra but not in the core matched the process of infarct maturation. Treatment with iNOS inhibitor aminoguanidine (100 mg.kg(-1), i.p.) at 6 and 12 h after reperfusion inhibited iNOS activity by 88.0 +/- 10.4% and reduced NO concentration by 48.5 +/- 8.3% in the penumbra, and lessened infarct size by 48.8 +/- 7.2%. The iNOS activity and NO level in the core were not affected by the administration of aminoguanidine. These results suggest that iNOS expression in the ischemic penumbra is involved in the recruitment of penumbra into infarction and thereby contributing to the enlargement of infarct.