RESUMO
Although microglia are macrophages of the central nervous system, their involvement is not limited to immune functions. The roles of microglia during development in humans remain poorly understood due to limited access to fetal tissue. To understand how microglia can impact human neurodevelopment, the methyl-CpG binding protein 2 (MECP2) gene was knocked out in human microglia-like cells (MGLs). Disruption of the MECP2 in MGLs led to transcriptional and functional perturbations, including impaired phagocytosis. The co-culture of healthy MGLs with MECP2-knockout (KO) neurons rescued synaptogenesis defects, suggesting a microglial role in synapse formation. A targeted drug screening identified ADH-503, a CD11b agonist, restored phagocytosis and synapse formation in spheroid-MGL co-cultures, significantly improved disease progression, and increased survival in MeCP2-null mice. These results unveil a MECP2-specific regulation of human microglial phagocytosis and identify a novel therapeutic treatment for MECP2-related conditions.
Assuntos
Proteína 2 de Ligação a Metil-CpG , Microglia , Transtornos do Neurodesenvolvimento , Fagocitose , Microglia/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Animais , Camundongos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Técnicas de Cocultura , Modelos Animais de Doenças , Camundongos Knockout , Sinapses/metabolismo , Neurônios/metabolismoRESUMO
Hydrogen sulfide (H2S) formed by cystathionine-γ-lyase (CSE) enhances the activity of Cav3.2â¯T-type Ca2+ channels, contributing to the bladder pain accompanying hemorrhagic cystitis caused by systemic administration of cyclophosphamide (CPA) in mice. Given clinical and fundamental evidence for the involvement of the substance P/NK1 receptor systems in bladder pain syndrome (BPS)/interstitial cystitis (IC), we created an intravesical substance P-induced bladder pain model in mice and analyzed the possible involvement of the CSE/Cav3.2 pathway. Bladder pain/cystitis was induced by i.p. CPA or intravesical substance P in female mice. Bladder pain was evaluated by counting nociceptive behavior and by detecting referred hyperalgesia in the lower abdomen and hindpaw. The isolated bladder tissue was weighed to estimate bladder swelling and subjected to histological observation and Western blotting. Intravesical substance P caused profound referred hyperalgesia accompanied by little bladder swelling or edema 6-24â¯h after the administration, in contrast to i.p. CPA-induced nociceptive behavior/referred hyperalgesia with remarkable bladder swelling/edema and urothelial damage. The bladder pain and/or cystitis symptoms caused by substance P or CPA were prevented by the NK1 receptor antagonist. CSE in the bladder was upregulated by substance P or CPA, and the NK1 antagonist prevented the CPA-induced CSE upregulation. A CSE inhibitor, a T-type Ca2+ channel blocker and gene silencing of Cav3.2 abolished the intravesical substance P-induced referred hyperalgesia. The intravesical substance P-induced pain in mice is useful as a model for nonulcerative BPS, and involves the activation of the NK1 receptor/CSE/H2S/Cav3.2 cascade.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Cistationina gama-Liase/metabolismo , Dor/etiologia , Transdução de Sinais/efeitos dos fármacos , Substância P/toxicidade , Doenças da Bexiga Urinária/induzido quimicamente , Doenças da Bexiga Urinária/complicações , Animais , Benzimidazóis/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/genética , Ciclofosfamida/toxicidade , Ciclopropanos/farmacologia , Cistationina gama-Liase/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Imunossupressores/toxicidade , Camundongos , Naftalenos/farmacologia , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Dor/induzido quimicamente , Medição da Dor , RNA Mensageiro/metabolismo , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Medula Espinal/patologia , Doenças da Bexiga Urinária/patologia , Urotélio/efeitos dos fármacos , Urotélio/patologiaRESUMO
We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2) in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL), caused delayed mechanical hypersensitivity in the lower abdomen, namely 'referred hyperalgesia', 6-24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca2+ channels.
Assuntos
Canais de Cálcio Tipo T/fisiologia , Dinoprostona/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Dor Nociceptiva/induzido quimicamente , Dor Nociceptiva/genética , Oligopeptídeos/farmacologia , Receptor PAR-2/metabolismo , Receptor PAR-2/fisiologia , Canais de Cátion TRPV/fisiologia , Bexiga Urinária , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Hiperalgesia/prevenção & controle , Indometacina , Camundongos Endogâmicos , Dor Nociceptiva/prevenção & controle , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismoRESUMO
Cav3.2 T-type Ca2+ channel activity is suppressed by zinc that binds to the extracellular histidine-191 of Cav3.2, and enhanced by H2S that interacts with zinc. Cav3.2 in nociceptors is upregulated in an activity-dependent manner. The enhanced Cav3.2 activity by H2S formed by the upregulated cystathionine-γ-lyase (CSE) is involved in the cyclophosphamide (CPA)-induced cystitis-related bladder pain in mice. We thus asked if zinc deficiency affects the cystitis-related bladder pain in mice by altering Cav3.2 function and/or expression. Dietary zinc deficiency for 2 weeks greatly decreased zinc concentrations in the plasma but not bladder tissue, and enhanced the bladder pain/referred hyperalgesia (BP/RH) following CPA at 200mg/kg, a subeffective dose, but not 400mg/kg, a maximal dose, an effect abolished by pharmacological blockade or gene silencing of Cav3.2. Acute zinc deficiency caused by systemic N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN), a zinc chelator, mimicked the dietary zinc deficiency-induced Cav3.2-dependent promotion of BP/RH following CPA at 200mg/kg. CPA at 400mg/kg alone or TPEN plus CPA at 200mg/kg caused Cav3.2 overexpression accompanied by upregulation of Egr-1 and USP5, known to promote transcriptional expression and reduce proteasomal degradation of Cav3.2, respectively, in the dorsal root ganglia (DRG). The CSE inhibitor, ß-cyano-l-alanine, prevented the BP/RH and upregulation of Cav3.2, Egr-1 and USP5 in DRG following TPEN plus CPA at 200mg/kg. Together, zinc deficiency promotes bladder pain accompanying CPA-induced cystitis by enhancing function and expression of Cav3.2 in nociceptors, suggesting a novel therapeutic avenue for treatment of bladder pain, such as zinc supplementation.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Cistite/metabolismo , Dor/metabolismo , Zinco/deficiência , Animais , Quelantes/farmacologia , Ciclofosfamida , Cistationina gama-Liase/metabolismo , Cistite/induzido quimicamente , Dieta , Modelos Animais de Doenças , Etilaminas , Gânglios Espinais/metabolismo , Camundongos , Oligodesoxirribonucleotídeos/farmacologia , Piridinas , Bexiga Urinária/metabolismo , Zinco/sangue , Zinco/metabolismoRESUMO
Hydrogen sulfide (H2 S) is generated from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE), and promotes nociception by targeting multiple molecules such as Cav 3.2 T-type Ca2+ channels. Bladder pain accompanying cyclophosphamide (CPA)-induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H2 S/Cav 3.2 pathway. Therefore, we investigated whether NF-κB, as an upstream signal of the CSE/H2 S system, contributes to bladder pain in mice with CPA-induced cystitis. Bladder pain-like nociceptive behaviour was observed in CPA-treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. ß-Cyano-l-alanine, a reversible selective CSE inhibitor, prevented CPA-induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF-κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor. PDTC and curcumin, which inhibits NF-κB signals, abolished CPA-induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA-induced activation of NF-κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H2 S/Cav 3.2 signaling. Therefore, NF-κB-inhibiting compounds including curcumin may be useful for the treatment of cystitis-related bladder pain.
Assuntos
Cistationina gama-Liase/metabolismo , Cistite/complicações , Sulfeto de Hidrogênio/metabolismo , NF-kappa B/metabolismo , Dor/metabolismo , Regulação para Cima , Bexiga Urinária/metabolismo , Alanina/química , Alanina/farmacologia , Animais , Canais de Cálcio Tipo T/metabolismo , Curcumina/farmacologia , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Camundongos , Dor/complicações , Dor/enzimologia , Dor/patologia , Prolina/análogos & derivados , Prolina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiocarbamatos/farmacologia , Regulação para Cima/efeitos dos fármacos , Bexiga Urinária/patologiaRESUMO
T-type Ca channels (T channels), particularly Cav3.2 among the 3 isoforms, play a role in neuropathic and visceral pain. We thus characterized the effects of RQ-00311651 (RQ), a novel T-channel blocker, in HEK293 cells transfected with human Cav3.1 or Cav3.2 by electrophysiological and fluorescent Ca signaling assays, and also evaluated the antiallodynic/antihyperalgesic activity of RQ in somatic, visceral, and neuropathic pain models in rodents. RQ-00311651 strongly suppressed T currents when tested at holding potentials of -65 â¼ -60 mV, but not -80 mV, in the Cav3.1- or Cav3.2-expressing cells. RQ-00311651 also inhibited high K-induced Ca signaling in those cells. In mice, RQ, administered intraperitoneally (i.p.) at 5 to 20 mg/kg or orally at 20 to 40 mg/kg, significantly suppressed the somatic hyperalgesia and visceral pain-like nociceptive behavior/referred hyperalgesia caused by intraplantar and intracolonic administration of NaHS or Na2S, H2S donors, respectively, which involve the enhanced activity of Cav3.2 channels. RQ-00311651, given i.p. at 5 to 20 mg/kg, exhibited antiallodynic or antihyperalgesic activity in rats with spinal nerve injury-induced neuropathy or in rats and mice with paclitaxel-induced neuropathy. Oral and i.p. RQ at 10 to 20 mg/kg also suppressed the visceral nociceptive behavior and/or referred hyperalgesia accompanying cerulein-induced acute pancreatitis and cyclophosphamide-induced cystitis in mice. The analgesic and antihyperalgesic/antiallodynic doses of oral and i.p. RQ did not significantly affect the locomotor activity and motor coordination. Together, RQ is considered a state-dependent blocker of Cav3.1/Cav3.2 T channels and may serve as an orally available analgesic for treatment of neuropathic and inflammatory pain including distinct visceral pain with minimum central side effects.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Nociceptividade/efeitos dos fármacos , Dor Visceral/tratamento farmacológico , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Hiperalgesia/induzido quimicamente , Masculino , Camundongos , Neuralgia/induzido quimicamente , Paclitaxel , Ratos , Ratos Wistar , Dor Visceral/induzido quimicamenteRESUMO
BACKGROUND AND PURPOSE: Hydrogen sulfide (H(2) S), generated by enzymes such as cystathionine-γ-lyase (CSE) from L-cysteine, facilitates pain signals by activating the Ca(v) 3.2 T-type Ca(2+) channels. Here, we assessed the involvement of the CSE/H(2) S/Ca(v) 3.2 pathway in cystitis-related bladder pain. EXPERIMENTAL APPROACH: Cystitis was induced by i.p. administration of cyclophosphamide in mice. Bladder pain-like nociceptive behaviour was observed and referred hyperalgesia was evaluated using von Frey filaments. Phosphorylation of ERK in the spinal dorsal horn was determined immunohistochemically following intravesical administration of NaHS, an H(2) S donor. KEY RESULTS: Cyclophosphamide caused cystitis-related symptoms including increased bladder weight, accompanied by nociceptive changes (bladder pain-like nociceptive behaviour and referred hyperalgesia). Pretreatment with DL-propargylglycine, an inhibitor of CSE, abolished the nociceptive changes and partly prevented the increased bladder weight. CSE protein in the bladder was markedly up-regulated during development of cystitis. Mibefradil or NNC 55-0396, blockers of T-type Ca(2+) channels, administered after the symptoms of cystitis appeared, reversed the nociceptive changes. Further, silencing of Ca(v) 3.2 protein by repeated intrathecal administration of mouse Ca(v) 3.2-targeting antisense oligodeoxynucleotides also significantly attenuated the nociceptive changes, but not the increased bladder weight. Finally, the number of cells staining positive for phospho-ERK was increased in the superficial layer of the L6 spinal cord after intravesical administration of NaHS, an effect inhibited by NNC 55-0396. CONCLUSION AND IMPLICATIONS: Endogenous H(2) S, generated by up-regulated CSE, caused bladder pain and referred hyperalgesia through the activation of Ca(v) 3.2 channels, one of the T-type Ca(2+) channels, in mice with cyclophosphamide-induced cystitis.