RESUMO
Müller cell gliosis is a common response in many retinal pathological conditions. We previously demonstrated that downregulation of Kir channels contributes to Müller cell gliosis in a rat chronic ocular hypertension (COH) model. Here, the possible involvement of outward K+ currents in Müller cell gliosis was investigated. Outward K+ current densities in Müller cells isolated from COH rats, as compared with those in normal rats, showed a significant increase, which was mainly contributed by large-conductance Ca2+ -activated K+ (BKCa ) channels. The involvement of BKCa channels in Müller cell gliosis is suggested by the fact that glial fibrillary acidic protein (GFAP) levels were augmented in COH retinas when these channels were suppressed by intravitreal injections of iberiotoxin. In COH retinas an increase in dopamine (DA) D1 receptor (D1R) expression in Müller cells was revealed by both immunohistochemistry and Western blotting. Moreover, protein levels of tyrosine hydroxylase were also increased, and consistent to this, retinal DA contents were elevated. SKF81297, a selective D1R agonist, enhanced BKCa currents of normal Müller cells through intracellular cAMP-PKA signaling pathway. Furthermore, GFAP levels were increased by the D1R antagonist SCH23390 injected intravitreally through eliminating the BKCa current upregulation in COH retinas, but partially reduced by SKF81297. All these results strongly suggest that the DA-D1R system may be activated to a stronger extent in COH rat retinas, thus increasing BKCa currents of Müller cells. The upregulation of BKCa channels may antagonize the Kir channel inhibition-induced depolarization of Müller cells, thereby attenuating the gliosis of these cells.
Assuntos
Células Ependimogliais/metabolismo , Gliose/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Hipertensão Ocular/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Modelos Animais de Doenças , Células Ependimogliais/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Potenciais da Membrana/fisiologia , Hipertensão Ocular/patologia , Ratos Sprague-Dawley , Receptores de Dopamina D1/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo , Corpo Vítreo/metabolismo , Corpo Vítreo/patologiaRESUMO
Endocannabinoid receptor system is extensively expressed in the vertebrate retina. There are two types of cannabinoid receptors, CB1 and CB2. Activation of these two receptors by endocannabinoids N-arachidonoylethanolamide (anandamine, AEA) and 2-arachidonyl glycerol (2-AG) regulates multiple neuronal and glial ion channels, thus getting involved in retinal visual information processing. In this review, incorporating our results, we discuss the modulation of cannabinoid CB1 and CB2 receptors on retinal neuronal and glial ion channels and retinal synaptic transmission.
Assuntos
Canais Iônicos/fisiologia , Receptores de Canabinoides/fisiologia , Retina/fisiologia , Transmissão Sináptica/fisiologia , Animais , Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Glicerídeos/farmacologia , Humanos , Alcamidas Poli-Insaturadas/farmacologiaRESUMO
Retinal ganglion cell apoptotic death is the main pathological characteristic of glaucoma, which is the leading cause of irreversible blindness. Disruption of Ca2+ homeostasis plays an important role in glaucoma. Voltage-gated Ca2+ channel blockers have been shown to improve vision in patients with glaucoma. However, whether and how voltage-gated Ca2+ channels are involved in retinal ganglion cell apoptotic death are largely unknown. In this study, we found that total Ca2+ current densities in retinal ganglion cells were reduced in a rat model of chronic ocular hypertension experimental glaucoma, as determined by whole-cell patch-clamp electrophysiological recordings. Further analysis showed that L-type Ca2+ currents were downregulated while T-type Ca2+ currents were upregulated at the later stage of glaucoma. Western blot assay and immunofluorescence experiments confirmed that expression of the CaV1.2 subunit of L-type Ca2+ channels was reduced and expression of the CaV3.3 subunit of T-type Ca2+ channels was increased in retinas of the chronic ocular hypertension model. Soluble tumor necrosis factor-α, an important inflammatory factor, inhibited the L-type Ca2+ current of isolated retinal ganglion cells from control rats and enhanced the T-type Ca2+ current. These changes were blocked by the tumor necrosis factor-α inhibitor XPro1595, indicating that both types of Ca2+ currents may be mediated by soluble tumor necrosis factor-α. The intracellular mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and nuclear factor kappa-B signaling pathway mediate the effects of tumor necrosis factor-α. TUNEL assays revealed that mibefradil, a T-type calcium channel blocker, reduced the number of apoptotic retinal ganglion cells in the rat model of chronic ocular hypertension. These results suggest that T-type Ca2+ channels are involved in disrupted Ca2+ homeostasis and apoptosis of retinal ganglion cells in glaucoma, and application of T-type Ca2+ channel blockers, especially a specific CaV3.3 blocker, may be a potential strategy for the treatment of glaucoma.
RESUMO
Direct conversion of readily available non-neural cells from patients into induced neurons holds great promise for neurological disease modeling and cell-based therapy. Olfactory ensheathing cells (OECs) is a unique population of glia in olfactory nervous system. Based on the regeneration-promoting properties and the relative clinical accessibility, OECs are attracting increasing attention from neuroscientists as potential therapeutic agents for use in neural repair. Here, we report that OECs can be directly, rapidly and efficiently reprogrammed into neuronal cells by the single transcription factor Neurogenin 2 (NGN2). These induced cells exhibit typical neuronal morphologies, express multiple neuron-specific markers, produce action potentials, and form functional synapses. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of OECs is effectively reprogrammed towards that of neuronal lineage. Importantly, these OEC-derived induced neurons survive and mature after transplantation into adult mouse spinal cords. Taken together, our study provides a direct and efficient strategy to quickly obtain neuronal cells from adult OECs, suggestive of promising potential for personalized disease modeling and cell replacement-mediated therapeutic approaches to neurological disorders.
Assuntos
Regeneração Nervosa/fisiologia , Bulbo Olfatório/fisiopatologia , Células Cultivadas , Humanos , NeurôniosRESUMO
Group I metabotropic glutamate receptor (mGluR I) activation exerts a slow postsynaptic excitatory effect in the CNS. Here, the issues of whether and how this receptor is involved in regulating retinal ganglion cell (RGC) excitability were investigated in retinal slices using patch-clamp techniques. Under physiological conditions, RGCs displayed spontaneous firing. Extracellular application of LY367385 (10 µM)/MPEP (10 µM), selective mGluR1 and mGluR5 antagonists, respectively, significantly reduced the firing frequency, suggesting that glutamate endogenously released from bipolar cells constantly modulates RGC firing. DHPG (10 µM), an mGluR I agonist, significantly increased the firing and caused depolarization of the cells, which were reversed by LY367385, but not by MPEP, suggesting the involvement of the mGluR1 subtype. Intracellular Ca2+-dependent PI-PLC/PKC and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways mediated the DHPG-induced effects. In the presence of cocktail synaptic blockers (CNQX, D-AP5, bicuculline, and strychnine), which terminated the spontaneous firing in both ON and OFF RGCs, DHPG still induced depolarization and triggered the cells to fire. The DHPG-induced depolarization could not be blocked by TTX. In contrast, Ba2+, an inwardly rectifying potassium channel (Kir) blocker, and Cs+ and ZD7288, hyperpolarization-activated cation channel (I h) blockers, mimicked the effect of DHPG. Furthermore, in the presence of Ba2+/ZD7288, DHPG did not show further effects. Moreover, Kir and I h currents could be recorded in RGCs, and extracellular application of DHPG indeed suppressed these currents. Our results suggest that activation of mGluR I regulates the excitability of rat RGCs by inhibiting Kir and I h.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Células Ganglionares da Retina/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Benzoatos/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Masculino , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Proteína Quinase C/metabolismo , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismoRESUMO
Endocannabinoid signaling system is involved in regulating multiple neuronal functions in the central nervous system by activating G-protein coupled cannabinoid CB1 and CB2 receptors (CB1Rs and CB2Rs). Growing evidence has shown that CB1Rs and CB2Rs are extensively expressed in retinal ganglion cells (RGCs). Here, modulation of L- and T-types Ca2+ channels by activating CB1Rs and CB2Rs in RGCs was investigated. Triple immunofluorescent staining showed that L-type subunit CaV1.2 was co-localized with T-type subunits (CaV3.1, CaV3.2 and CaV3.3) in rat RGCs. In acutely isolated rat RGCs, the CB1R agonist WIN55212-2 suppressed both peak and steady-state Ca2+ currents in a dose-dependent manner, with IC50 being 9.6 µM and 8.4 µM, respectively. It was further shown that activation of CB1Rs by WIN55212-2 or ACEA, another CB1R agonist, significantly suppressed both L- and T-type Ca2+ currents, and shifted inactivation curve of T-type one toward hyperpolarization direction. While the effect on L-type Ca2+ channels was mediated by intracellular cAMP/protein kinase A (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways, only CaMKII signaling pathway was involved in the effect on T-type Ca2+ channels. Furthermore, CB65 and HU308, two specific CB2R agonists, significantly suppressed T-type Ca2+ channels, which was mediated by intracellular cAMP/PKA and CaMKII signaling pathways, but had no effect on L-type channels. These results imply that endogenous cannabinoids may modulate the excitability and the output of RGCs by differentially suppressing the activity of L- and T-type Ca2+ channels through activation of CB1Rs and CB2Rs. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".
Assuntos
Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo T/fisiologia , Cicloexanos/farmacologia , Morfolinas/farmacologia , Quinolinas/farmacologia , Receptor CB1 de Canabinoide/fisiologia , Receptor CB2 de Canabinoide/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Ácidos Araquidônicos/farmacologia , Benzoxazinas/farmacologia , Canabinoides/farmacologia , Relação Dose-Resposta a Droga , Masculino , Potenciais da Membrana/efeitos dos fármacos , Naftalenos/farmacologia , Ratos , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Transdução de Sinais/fisiologiaRESUMO
Spinal muscular atrophy (SMA) is a devastating motor neuron disease caused by mutations of the survival motor neuron 1 (SMN1) gene. SMN2, a paralogous gene to SMN1, can partially compensate for the loss of SMN1. On the basis of age at onset, highest motor function and SMN2 copy numbers, childhood-onset SMA can be divided into three types (SMA I-III). An inverse correlation was observed between SMN2 copies and the differential phenotypes of SMA. Interestingly, this correlation is not always absolute. Using SMA induced pluripotent stem cells (iPSCs), we found that the SMN was significantly decreased in both SMA III and SMA I iPSCs derived postmitotic motor neurons (pMNs) and γ-aminobutyric acid (GABA) neurons. Moreover, the significant differences of SMN expression level between SMA III (3 copies of SMN2) and SMA I (2 copies of SMN2) were observed only in pMNs culture, but not in GABA neurons or iPSCs. From these findings, we further discovered that the neurite outgrowth was suppressed in both SMA III and SMA I derived MNs. Meanwhile, the significant difference of neurite outgrowth between SMA III and SMA I group was also found in long-term cultures. However, significant hyperexcitability was showed only in SMA I derived mature MNs, but not in SMA III group. Above all, we propose that SMN protein is a major factor of phenotypic modifier. Our data may provide a new insight into recognition for differential phenotypes of SMA disease.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Fenótipo , Biomarcadores , Diferenciação Celular , Reprogramação Celular , Fenômenos Eletrofisiológicos , Feminino , Neurônios GABAérgicos/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Atrofia Muscular Espinal/genética , Mutação , Neuritos/metabolismo , Linhagem , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Somatic cells can be directly converted into functional neurons by ectopic expression of defined factors and/or microRNAs. Since the first report of conversion mouse embryonic fibroblasts into functional neurons, the postnatal mouse, and human fibroblasts, astroglia, hepatocytes, and pericyte-derived cells have been converted into functional dopaminergic and motor neurons both in vitro and in vivo. However, it is invasive to get all these materials. In the current study, we provide a noninvasive approach to obtain directly reprogrammed functional neurons by overexpression of the transcription factors Ascl1, Brn2, NeuroD, c-Myc, and Myt1l in human urine cells. These induced neuronal (iN) cells could express multiple neuron-specific proteins and generate action potentials. Moreover, urine cells from Wilson's disease (WD) patient could also be directly converted into neurons. In conclusion, generation of iN cells from nonneural lineages is a feasible and befitting approach for neurological disease modeling.
RESUMO
Dopamine plays an important role in regulating neuronal functions in the central nervous system by activating the specific G-protein coupled receptors. Both D1 and D2 dopamine receptors are extensively distributed in the retinal neurons. In the present study, we investigated the effects of D1 receptor signaling on outward K(+) currents in acutely isolated rat retinal ganglion cells (RGCs) by patch-clamp techniques. Extracellular application of SKF81297 (10 µM), a specific D1 receptor agonist, significantly and reversibly suppressed outward K(+) currents of the cells, which was reversed by SCH23390 (10 µM), a selective D1 receptor antagonist. We further showed that SKF81297 mainly suppressed the glybenclamide (Gb)- and 4-aminopyridine (4-AP)-sensitive K(+) current components, but did not show effect on the tetraethylammonium (TEA)-sensitive one. Both protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways were likely involved in the SKF81297-induced suppression of the K(+) currents since either Rp-cAMP (10 µM), a cAMP/PKA signaling inhibitor, or KN-93 (10 µM), a specific CaMKII inhibitor, eliminated the SKF81297 effect. In contrast, neither protein kinase C (PKC) nor mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway seemed likely to be involved because both the PKC inhibitor bisindolylmaleimide IV (Bis IV) (10 µM) and the MAPK/ERK1/2 inhibitor U0126 (10 µM) did not block the SKF81297-induced suppression of the K(+) currents. These results suggest that activation of D1 receptors suppresses the Gb- and 4-AP-sensitive K(+) current components in rat RGCs through the intracellular PKA and CaMKII signaling pathways, thus modulating the RGC excitability.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Potássio/fisiologia , Receptores de Dopamina D1/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Benzazepinas/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: To establish the validation method and criteria for counting bacteria and fungi in microbial limit test which is described in the Pharmacopeia of China (ChP) 2005. METHOD: According to the method set up for validation, the tested microorganisms with known counts were added to samples followed by the determination of the recovery. RESULT: With different preparing method for testing samples, the recoveries for the tested microorganisms in testing samples were found to be over 70%. CONCLUSION: Validation method for counting contaminated bacteria and fungi in drugs is recommended to follow the method established in this paper. The recovery for tested microorganisms should be not less than 70%.
Assuntos
Bactérias/isolamento & purificação , Contaminação de Medicamentos , Fungos/isolamento & purificação , Aspergillus niger/isolamento & purificação , Bacillus subtilis/isolamento & purificação , Candida albicans/isolamento & purificação , Contagem de Colônia Microbiana/métodos , Medicamentos de Ervas Chinesas/normas , Escherichia coli/isolamento & purificação , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Paroxysmal kinesigenic dyskinesia (PKD) is a monogenic movement disorder with autosomal dominant inheritance. We previously identified the proline-rich transmembrane protein 2 (PRRT2) as a causative gene of PKD. However, the pathogenesis of PKD remains largely unknown so far. In addition, applicable modeling tools to investigate the underlying mechanisms of PKD are still lacking. The combination of disease-specific human induced pluripotent stem cells (iPSCs) and directed cell differentiation offers an ideal platform for disease modeling. In this study, we generated two iPSC lines from the renal epithelial cells of one PKD patient with the hotspot c.649dupC mutation (PKD-iPSCs). These cell lines were positive for alkaline phosphatase Nanog, Tra-1-80, Tra-1-60, SSEA-3 and SSEA-4. Teratomas with three blastoderms including ectoderm, mesoderm, and endoderm were obtained two months after injection of PKD-iPSCs into NOD/SCID mice. The expression of PRRT2 mRNA was decreased in PKD-iPSCs compared with that of the control iPSCs. Furthermore, PKD-iPSCs possessed the differentiation potential of functional glutamatergic, dopaminergic and motor neurons in vitro. Electrophysiological examinations revealed that the current densities of fast activated and deactivated sodium channels as well as voltage gated potassium channels were not different between the neurons from PKD-iPSCs and control iPSCs. Thus, PKD-iPSCs are a feasible modeling tool to investigate the pathogenic mechanisms of PKD.