RESUMEN
Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
Asunto(s)
Azepinas/farmacología , Encéfalo/patología , Proteínas de Ciclo Celular/metabolismo , Interneuronas/patología , Proteína 2 de Unión a Metil-CpG/fisiología , Síndrome de Rett/patología , Factores de Transcripción/metabolismo , Transcriptoma/efectos de los fármacos , Triazoles/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Femenino , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Factores de Transcripción/genéticaRESUMEN
Temporal lobe epilepsy (TLE) is characterized by recurrent spontaneous seizures and behavioral comorbidities. Reduced hippocampal theta oscillations and hyperexcitability that contribute to cognitive deficits and spontaneous seizures are present beyond the sclerotic hippocampus in TLE. However, the mechanisms underlying compromised network oscillations and hyperexcitability observed in circuits remote from the sclerotic hippocampus are largely unknown. Cholecystokinin (CCK)-expressing basket cells (CCKBCs) critically participate in hippocampal theta rhythmogenesis, and regulate neuronal excitability. Thus, we examined whether CCKBCs were vulnerable in nonsclerotic regions of the ventral hippocampus remote from dorsal sclerotic hippocampus using the intrahippocampal kainate (IHK) mouse model of TLE, targeting unilateral dorsal hippocampus. We found a decrease in the number of CCK+ interneurons in ipsilateral ventral CA1 regions from epileptic mice compared to those from sham controls. We also found that the number of boutons from CCK+ interneurons was reduced in the stratum pyramidale, but not in other CA1 layers, of ipsilateral hippocampus in epileptic mice, suggesting that CCKBCs are vulnerable. Electrical recordings showed that synaptic connectivity and strength from surviving CCKBCs to CA1 pyramidal cells (PCs) were similar between epileptic mice and sham controls. In agreement with reduced CCKBC number in TLE, electrical recordings revealed a significant reduction in amplitude and frequency of IPSCs in CA1 PCs evoked by carbachol (commonly used to excite CCK+ interneurons) in ventral CA1 regions from epileptic mice versus sham controls. These findings suggest that loss of CCKBCs beyond the hippocampal lesion may contribute to hyperexcitability and compromised network oscillations in TLE.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Colecistoquinina/biosíntesis , Epilepsia del Lóbulo Temporal/metabolismo , Neuronas GABAérgicas/metabolismo , Interneuronas/metabolismo , Ácido Kaínico/toxicidad , Animales , Región CA1 Hipocampal/efectos de los fármacos , Colecistoquinina/genética , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/genética , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Expresión Génica , Interneuronas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Gamma network oscillations in the brain are fast rhythmic network oscillations in the gamma frequency range (~30-100â¯Hz), playing key roles in the hippocampus for learning, memory, and spatial processing. There is evidence indicating that GABAergic interneurons, including parvalbumin-expressing basket cells (PVBCs), contribute to cortical gamma oscillations through synaptic interactions with excitatory cells. However, the molecular, cellular, and circuit underpinnings underlying generation and maintenance of cortical gamma oscillations are largely elusive. Recent studies demonstrated that intrinsic and synaptic properties of GABAergic interneurons and excitatory cells are regulated by a slowly inactivating or non-inactivating sodium current (i.e., persistent sodium current, INaP), suggesting that INaP is involved in gamma oscillations. Here, we tested whether INaP plays a role in hippocampal gamma oscillations using pharmacological, optogenetic, and electrophysiological approaches. We found that INaP blockers, phenytoin (40⯵M and 100⯵M) and riluzole (10⯵M), reduced gamma oscillations induced by optogenetic stimulation of CaMKII-expressing cells in CA1 networks. Whole-cell patch-clamp recordings further demonstrated that phenytoin (100⯵M) reduced INaP and firing frequencies in both PVBCs and pyramidal cells without altering threshold and amplitude of action potentials, but increased rheobase in both cell types. These results suggest that INaP in pyramidal cells and PVBCs is required for hippocampal gamma oscillations, supporting a pyramidal-interneuron network gamma model. Phenytoin-mediated modulation of hippocampal gamma oscillations may be a mechanism underlying its anticonvulsant efficacy, as well as its contribution to cognitive impairments in epilepsy patients.
Asunto(s)
Región CA1 Hipocampal/fisiología , Neuronas GABAérgicas/fisiología , Ritmo Gamma/fisiología , Interneuronas/fisiología , Células Piramidales/fisiología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/metabolismo , Ritmo Gamma/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Ratones , Optogenética , Parvalbúminas/metabolismo , Técnicas de Placa-Clamp , Fenitoína/farmacología , Células Piramidales/efectos de los fármacos , Riluzol/farmacología , Sodio/metabolismoRESUMEN
GABAergic interneurons in the hippocampus are critically involved in almost all hippocampal circuit functions including coordinated network activity. Somatostatin-expressing oriens-lacunosum moleculare (O-LM) interneurons are a major subtype of dendritically projecting interneurons in hippocampal subregions (e.g., CA1), and express group I metabotropic glutamate receptors (mGluRs), specifically mGluR1 and mGluR5. Group I mGluRs are thought to regulate hippocampal circuit functions partially through GABAergic interneurons. Previous studies suggest that a group I/II mGluR agonist produces slow supra-threshold membrane oscillations (<0.1â¯Hz), which are associated with high-frequency action potential (AP) discharges in O-LM interneurons. However, the properties and underlying mechanisms of these slow oscillations remain largely unknown. We performed whole-cell patch-clamp recordings from mouse interneurons in the stratum oriens/alveus (O/A interneurons) including CA1 O-LM interneurons. Our study revealed that the selective mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced slow membrane oscillations (<0.1â¯Hz), which were associated with gamma frequency APs followed by AP-free perithreshold gamma oscillations. The selective mGluR1 antagonist (S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) reduced the slow oscillations, and the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) partially blocked them. Blockade of nonselective cation-conducting transient receptor potential channels, L-type Ca2+ channels, or ryanodine receptors all abolished the slow oscillations, suggesting the involvement of multiple mechanisms. Our findings suggest that group I mGluR activation in O/A interneurons may play an important role in coordinated network activity, and O/A interneuron vulnerability to excitotoxicity, in disease states like seizures, is at least in part due to an excessive rise in intracellular Ca2+.
Asunto(s)
Ondas Encefálicas/fisiología , Hipocampo/metabolismo , Interneuronas/metabolismo , Potenciales de la Membrana/fisiología , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Ondas Encefálicas/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Hipocampo/efectos de los fármacos , Interneuronas/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Ratones Endogámicos C57BL , Periodicidad , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Convulsiones/metabolismo , Técnicas de Cultivo de Tejidos , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
PURPOSE: Treatment of de-epithelialized human corneas with riboflavin (RF) + long-wavelength ultraviolet light (UVA; RFUVA) increases corneal stroma tensile strength significantly. RFUVA treatment retards the progression of keratoconus, perhaps by cross-linking of collagen molecules, but exact molecular mechanisms remain unknown. Research described here tested possible chemical mechanisms of cross-linking. METHODS: Corneas of rabbits and spiny dogfish sharks were de-epithelialized mechanically, subjected to various chemical pretreatments, exposed to RFUVA, and then subjected to destructive tensile stress measurements. Tensile strength was quantified with a digital force gauge to measure degree of tissue cross-linking. RESULTS: For both rabbit and shark corneas, RFUVA treatment causes significant cross-linking by mechanism(s) that can be blocked by the presence of sodium azide. Conversely, such cross-linking is greatly enhanced in the presence of deuterium oxide (D(2)O), even when RF is present at only one tenth the currently used clinical concentrations. Blocking carbonyl groups preexisting in the stroma with 2,4-dinitrophenylhydrazide or hydroxylamine blocks essentially all corneal cross-linking. In contrast, blocking free amine groups preexisting in the stroma with acetic anhydride or ethyl acetimidate does not affect RFUVA corneal cross-linking. When both carbonyl groups are blocked and singlet oxygen is quenched, no RFUVA cross-linking occurs, indicating the absence of other cross-linking mechanisms. CONCLUSIONS: RFUVA catalyzes cross-linking reactions that require production of singlet oxygen ((1)O(2)), whose half-life is extended by D(2)O. Carbonyl-based cross-linking reactions dominate in the corneal stroma, but other possible reaction schemes are proposed. The use of D(2)O as solution media for RF would enable concentration decreases or significant strength enhancement in treated corneas.