Cell-specific NFIA upregulation promotes epileptogenesis by TRPV4-mediated astrocyte reactivity.

BACKGROUND: The astrocytes in the central nervous system (CNS) exhibit morphological and functional diversity in brain region-specific pattern. Functional alterations of reactive astrocytes are commonly present in human temporal lobe epilepsy (TLE) cases, meanwhile the neuroinflammation mediated by reactive astrocytes may advance the development of hippocampal epilepsy in animal models. Nuclear factor I-A (NFIA) may regulate astrocyte diversity in the adult brain. However, whether NFIA endows the astrocytes with regional specificity to be involved in epileptogenesis remains elusive. METHODS: Here, we utilize an interference RNA targeting NFIA to explore the characteristics of NFIA expression and its role in astrocyte reactivity in a 4-aminopyridine (4-AP)-induced seizure model in vivo and in vitro. Combined with the employment of a HA-tagged plasmid overexpressing NFIA, we further investigate the precise mechanisms how NIFA facilitates epileptogenesis. RESULTS: 4-AP-induced NFIA upregulation in hippocampal region is astrocyte-specific, and primarily promotes detrimental actions of reactive astrocyte. In line with this phenomenon, both NFIA and vanilloid transient receptor potential 4 (TRPV4) are upregulated in hippocampal astrocytes in human samples from the TLE surgical patients and mouse samples with intraperitoneal 4-AP. NFIA directly regulates mouse astrocytic TRPV4 expression while the quantity and the functional activity of TRPV4 are required for 4-AP-induced astrocyte reactivity and release of proinflammatory cytokines in the charge of NFIA upregulation. NFIA deficiency efficiently inhibits 4-AP-induced TRPV4 upregulation, weakens astrocytic calcium activity and specific astrocyte reactivity, thereby mitigating aberrant neuronal discharges and neuronal damage, and suppressing epileptic seizure. CONCLUSIONS: Our results uncover the critical role of NFIA in astrocyte reactivity and illustrate how epileptogenic brain injury initiates cell-specific signaling pathway to dictate the astrocyte responses.

Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4) Mitigates Seizures.

Astrocytes are critical regulators of the immune/inflammatory response in several human central nervous system (CNS) diseases. Emerging evidence suggests that dysfunctional astrocytes are crucial players in seizures. The objective of this study was to investigate the role of transient receptor potential vanilloid 4 (TRPV4) in 4-aminopyridine (4-AP)-induced seizures and the underlying mechanism. We also provide evidence for the role of Yes-associated protein (YAP) in seizures. 4-AP was administered to mice or primary cultured astrocytes. YAP-specific small interfering RNA (siRNA) was administered to primary cultured astrocytes. Mouse brain tissue and surgical specimens from epileptic patient brains were examined, and the results showed that TRPV4 was upregulated, while astrocytes were activated and polarized to the A1 phenotype. The levels of glial fibrillary acidic protein (GFAP), cytokine production, YAP, signal transducer activator of transcription 3 (STAT3), intracellular Ca2+([Ca2+]i) and the third component of complement (C3) were increased in 4-AP-induced mice and astrocytes. Perturbations in the immune microenvironment in the brain were balanced by TRPV4 inhibition or the manipulation of [Ca2+]i in astrocytes. Knocking down YAP with siRNA significantly inhibited 4-AP-induced pathological changes in astrocytes. Our study demonstrated that astrocytic TRPV4 activation promoted neuroinflammation through the TRPV4/Ca2+/YAP/STAT3 signaling pathway in mice with seizures. Astrocyte TRPV4 inhibition attenuated neuroinflammation, reduced neuronal injury, and improved neurobehavioral function. Targeting astrocytic TRPV4 activation may provide a promising therapeutic approach for managing epilepsy.

Potent half-sandwich Ru(â ¡) N

Herein a new series of organometallic half-sandwich Ru(Ⅱ) complexes bearing aryl-BIAN chelating ligands with various electron-withdrawing and electron-donating substituents have been developed as theranostic agents. All the complexes display much higher anti-proliferative potency than the clinical chemotherapeutic drug cisplatin towards seven cancer cell lines. The anti-proliferative efficacy of these complexes is correlated to their electron-withdrawing ability. Interestingly, complex Ru1 also potently suppresses cancer cell migration in vitro and effectively inhibit tumor growth in vivo in a CT26 colon cancer mouse xenograft model. Mechanisms of action studies display that Ru1 can favorably accumulate in lysosome and exerts anti-cancer potency by inducing a series of events related to lysosomal dysfunction in CT26 cells. Interestingly, inhibition of lysosomal enzymes leads to suppression of cytotoxicity and apoptosis induced by Ru1. Our results elucidate that complex Ru1 can elicit cytotoxicity through lysosome-mediated apoptosis in vitro and suppress tumor growth in vivo.

Functional glutamate transporters are expressed in the carotid chemoreceptor.

BACKGROUND: The carotid body (CB) plays a critical role in cyclic intermittent hypoxia (CIH)-induced chemosensitivity; however, the underlying mechanism remains uncertain. We have demonstrated the presence of multiple inotropic glutamate receptors (iGluRs) in CB, and that CIH exposure alters the level of some iGluRs in CB. This result implicates glutamatergic signaling in the CB response to hypoxia. The glutamatergic neurotransmission is not only dependent on glutamate and glutamate receptors, but is also dependent on glutamate transporters, including vesicular glutamate transporters (VGluTs) and excitatory amino acid transporters (EAATs). Here, we have further assessed the expression and distribution of VGluTs and EAATs in human and rat CB and the effect of CIH exposure on glutamate transporters expression. METHODS: The mRNA of VGluTs and EAATs in the human CB were detected by RT-PCR. The protein expression of VGluTs and EAATs in the human and rat CB were detected by Western blot. The distribution of VGluT3, EAAT2 and EAAT3 were observed by immunohistochemistry staining and immunofluorescence staining. Male Sprague-Dawley (SD) rats were exposed to CIH (FIO2 10-21%, 3 min/3 min for 8 h per day) for 2 weeks. The unpaired Student's t-test was performed. RESULTS: Here, we report on the presence of mRNAs for VGluT1-3 and EAAT1-3 in human CB, which is consistent with our previous results in rat CB. The proteins of VGluT1 and 3, EAAT2 and 3, but not VGluT2 and EAAT1, were detected with diverse levels in human and rat CB. Immunostaining showed that VGluT3, the major type of VGluTs in CB, was co-localized with tyrosine hydroxylase (TH) in type I cells. EAAT2 and EAAT3 were distributed not only in type I cells, but also in glial fibrillary acidic protein (GFAP) positive type II cells. Moreover, we found that exposure of SD rats to CIH enhanced the protein level of EAAT3 as well as TH, but attenuated the levels of VGluT3 and EAAT2 in CB. CONCLUSIONS: Our study suggests that glutamate transporters are expressed in the CB, and that glutamate transporters may contribute to glutamatergic signaling-dependent carotid chemoreflex to CIH.

Expression of BACE1 in the Rat Carotid Body.

This study explored the expression of BACE1 (ß-amyloid precursor protein cleaving enzyme 1) in the rat carotid body and the effect of CIH (cyclic intermittent hypoxia) on the expression of BACE1. We found that BACE1 was expressed in the rat carotid body and located in the nerve endings and type II cells but not in type I cells. CIH reduced BACE1 level in the carotid body, and reoxygenation or ROS scavenger alleviated this reduction. Furthermore, we found that CIH augmented the mRNA level of PGC-1α but attenuated the mRNA level of BACE1 in the carotid body. Taken together, our results suggest that CIH promotes the production of ROS that upregulates the level of PGC-1α, which may in turn inhibits the transcription of BACE1, and that a reduction in the BACE1 level may be related to CIH-induced reversible and ROS-dependent carotid body plasticity. Our study provides a new candidate molecule for further study of the mechanism of carotid body plasticity.

[Effect of dopamine on the sensitivity of carotid body to hypoxia induced by acute intermittent hypoxia in rats].

Objective: To investigate the sensitivity of carotid body to hypoxia and the effect of dopamine on the sensitivity of carotid body to hypoxia after acute intermittent hypoxia stimulation in rats. Methods: The isolated carotid body-sinus nerve in rat was transferred to incubator, and then the isolated sinus nerve was inhaled into the recorded glass electrode for recording electrical signals. The baseline buffer was bubbled with 95% O 2 + 5% CO 2 mixture gas, and the hypoxic stress was treated with 5% O 2 + 5% CO 2 + 90% N2 mixture gas, hypoxic stimulation was given for 30 seconds, 95% O 2 + 5% CO 2 for 90 seconds, a total of 10 cycles. No less than 5 rats in each group. Results: In this experiment, the electrical activity of sinus nerve isolated from rats was enhanced by hypoxia stimulation after acute intermittent hypoxia, but the response of sinus nerve to hypoxia was inhibited by dopamine. Before acute intermittent hypoxic stress, dopamine also inhibited the firing activity of sinus nerve, but after acute intermittent hypoxic cycle, the inhibition of dopamine on the firing activity of sinus nerve was strengthened. Conclusion: Acute intermittent hypoxia enhances the response of sinus nerve isolated from rats to hypoxia, dopamine inhibits the enhancement of carotid body sensitivity to hypoxia induced by acute intermittent hypoxic.

Evaluation of (-)-epigallocatechin-3-gallate (EGCG)-induced cytotoxicity on astrocytes: A potential mechanism of calcium overloading-induced mitochondrial dysfunction.

(-)-epigallocatechin-3-gallate (EGCG), the main component of green tea, has long been explored in the treatment and/or prevention of central nervous system (CNS) disorders. However, EGCG has been recently shown to exhibit acute and subacute toxicity. Although a lot of work has been done, the mechanisms of EGCG-induced mitochondrial dysfunction has not been delineated in primary astrocyte. Here, the mitotoxic effect of EGCG on primary astrocytes was investigated by measuring Ca2+ overloading-induced mitochondrial dysfunction. As expected, EGCG dose-dependently inhibited astrocytes growth depending on Ca2+ overloading, especially at 50 µM EGCG group. It is interesting to note that Ca2+ influx from the extracellular space was responsible for an increase in the cytosolic Ca2+ level ([Ca2+]i) by opening voltage-gated calcium channels (VGCCs) and, consequently, mitochondrial Ca2+ ([Ca2+]m) overloaded via the mitochondrial Ca2+ uniporter (MCU). As a result, mitochondrial dysfunction was induced, including the opening of the mitochondrial permeability transition pore (mPTP), mitochondrial membrane depolarization, an increasing in reactive oxygen species (ROS), and cytochrosome c (cyt c) releasing. Therefore, more apoptotic cells were observed in 50 µM EGCG group than that of in 1 µM EGCG group. These findings suggested that a high dose of EGCG was toxic to astrocytes partly by targeting mitochondria via calcium pathway, which would extend our understanding of the toxicity of EGCG and the underlying mechanisms.

Adaptive output feedback tracking of nonlinear systems with uncertain nonsymmetric dead-zone input.

In this paper, the problem of adaptive practical tracking is investigated by output feedback for a class of uncertain nonlinear systems subject to nonsymmetric dead-zone input nonlinearity with parameters of dead-zone being unknown. Instead of constructing the inverse of dead-zone nonlinearity, an adaptive robust control scheme is developed by designing an output compensator including two dynamic gains based respectively on identification and non-identification mechanism. With the aid of dynamic high-gain scaling approach and Backstepping method, stability analysis of the closed-loop system is proceeded using non-separation principle, which shows that the proposed controller guarantees that all closed-loop signal is bounded while the output of system tracks a broad class of bounded reference trajectories by arbitrarily small error prescribed previously. Finally, two examples are given to illustrate our controller effective.

Novel half-sandwich iridium OˆC (carbene)-Complexes: In vitro and in vivo tumor growth suppression and pro-apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes.

Herein we present half-sandwich IrIII complexes [(η5-Cpxbiph)Ir(OˆC)Cl] containing OˆC(NHC)-chelating ligand as anticancer and antimetastasis agents. All the complexes displayed high potency in vitro against a wide range of cancer cells. In addition, Ir2 significantly curb tumor growth in a colon cancer mouse xenograft model in vivo. Further mechanism of action studies indicate that Ir2-initiated apoptosis occurs through ROS-mediated cross-talk between mitochondria and lysosomes.

Combined effects of olfactory dysfunction and chronic stress on anxiety- and depressive- like behaviors in mice.

There is a close relationship between olfactory dysfunction and depression, but the underlying mechanism remains unknown. Studies have shown that olfactory deprived animal experience a higher level of stress compared with controls. In the present study, we aimed to investigate whether olfactory deprived mice would be more vulnerable to develop cognitive and emotional impairments under chronic stresses. Mice were treated with intranasal zinc sulfate infusion which resulted in a complete but reversible loss of olfactory function, and then they were treated with either chronic restraint stress (CRS) or chronic unpredictable mild stress (CUMS) for three consecutive weeks. After that, anxiety- and depressive-like behavior, as well as spatial learning and memory were measured. We found that olfactory deficit induced depressive-like behavior and impaired spatial learning and memory in mice, and the olfactory scores were significantly correlated with depressive-like behavior or the spatial learning. After CRS, olfactory deprived mice showed less anxiety- and depressive- like behaviors and better olfactory recovery than non-stressed anosmia mice. In contrast, CUMS led to increased anxiety- and depressive-like behavior and deterred the olfactory recovery. These results indicated that transient olfactory deprivation induces emotional and cognitive impairment in mice, which could be modulated by chronic stresses with a stressor intensity dependent way.

AMPA Receptor-Dependent Glutamatergic Signaling is Present in the Carotid Chemoreceptor.

Exposure to both sustained and intermittent hypoxia for as little as a day produces sustained augmentation of carotid chemoreceptor sensitivity; however, the molecular basis for this chemoreflex plasticity remains uncertain. We previously reported that NMDA receptor-dependent glutamatergic signaling in rat carotid body played a role in altered hypoxic sensitivity after exposure to cyclic intermittent hypoxia (CIH). Here we found that mRNAs of multiple AMPA and Kainate glutamate receptors were expressed in rat carotid body. The AMPA receptor subunit GluR1 showed intense immunoreactivity in the carotid body, co-localizing with tyrosine hydroxylase in type I cells. Treatment of rat carotid body-derived primary cells with AMPA activated ERK1/2 in a time-dependent manner. Exposing Sprague-Dawley rats to CIH for 8 h/day for 3 weeks significantly enhanced the expression level of GluA1 mRNA as well as GluR1 protein in the carotid body. In addition, our results showed that multiple of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) were expressed in the rat carotid body, indicating that glutamate might be as a neurotransmitter stored, released and uptake in the carotid body. Finally, we found that mRNAs of GluA1, GluA2 and GluA3 as well as PSD-95-like membrane-associated granulate kinase family members, PSD-95, PSD-93, and SAP97, were expressed in human carotid body. Our data suggest AMPA receptor-dependent glutamatergic signaling is present in the carotid body and might be involved in the carotid chemoreceptor response to hypoxia.

Anxiety- and depressive-like behaviors in olfactory deficient Cnga2 knockout mice.

There is a close neuroanatomical connection between odor and emotional processing. Olfactory dysfunction is found in various neurodegenerative and neuropsychiatric disorders. Here, mice take the cyclic nucleotide gated channel 2 mutant gene (Cnga2), which is critical for olfactory sensory neurons to generate odor induced action potentials were used. The Cnga2 mice were congenitally anosmic. Adult mice were tested in a series behavioral paradigm such as open field, light/dark box, forced swim test and Y-maze. Our study found that Cnga2 mice showed increased anxiety- and depressive-like behaviors than their wide type siblings. However, Cnga2 mice showed no difference from the wide types when tested in the two-trial recognition Y-maze. The results indicate that innate olfactory deficiency might modulate emotional behaviors in mice.