RESUMEN
GABAergic interneurons are key regulators of cortical circuit function. Among the dozens of reported transcriptionally distinct subtypes of cortical interneurons, neurogliaform cells (NGCs) are unique: they are recruited by long-range excitatory inputs, are a source of slow cortical inhibition and are able to modulate the activity of large neuronal populations. Despite their functional relevance, the developmental emergence and diversity of NGCs remains unclear. Here, by combining single-cell transcriptomics, genetic fate mapping, and electrophysiological and morphological characterization, we reveal that discrete molecular subtypes of NGCs, with distinctive anatomical and molecular profiles, populate the mouse neocortex. Furthermore, we show that NGC subtypes emerge gradually through development, as incipient discriminant molecular signatures are apparent in preoptic area (POA)-born NGC precursors. By identifying NGC developmentally conserved transcriptional programs, we report that the transcription factor Tox2 constitutes an identity hallmark across NGC subtypes. Using CRISPR-Cas9-mediated genetic loss of function, we show that Tox2 is essential for NGC development: POA-born cells lacking Tox2 fail to differentiate into NGCs. Together, these results reveal that NGCs are born from a spatially restricted pool of Tox2+ POA precursors, after which intra-type diverging molecular programs are gradually acquired post-mitotically and result in functionally and molecularly discrete NGC cortical subtypes.
Asunto(s)
Neocórtex , Neuronas , Ratones , Animales , Factores de Transcripción/genética , Interneuronas/fisiología , Movimiento CelularRESUMEN
Cortical expansion in primate brains relies on enlargement of germinal zones during a prolonged developmental period. Although most mammals have two cortical germinal zones, the ventricular zone (VZ) and subventricular zone (SVZ), gyrencephalic species display an additional germinal zone, the outer subventricular zone (oSVZ), which increases the number and diversity of neurons generated during corticogenesis. How the oSVZ emerged during evolution is poorly understood, but recent studies suggest a role for non-coding RNAs, which allow tight genetic program regulation during development. Here, using in vivo functional genetics, single-cell RNA sequencing, live imaging, and electrophysiology to assess progenitor and neuronal properties in mice, we identify two oSVZ-expressed microRNAs (miRNAs), miR-137 and miR-122, which regulate key cellular features of cortical expansion. miR-137 promotes basal progenitor self-replication and superficial layer neuron fate, whereas miR-122 decreases the pace of neuronal differentiation. These findings support a cell-type-specific role of miRNA-mediated gene expression in cortical expansion.
Asunto(s)
Diferenciación Celular/genética , MicroARNs/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , ARN no Traducido/metabolismo , Animales , Proliferación Celular/genética , Reprogramación Celular/genética , Hurones , Células HEK293 , Humanos , Ventrículos Laterales , Ratones , MicroARNs/genética , Mitosis/genética , Neurogénesis/genética , Neuronas/metabolismo , ARN no Traducido/genéticaRESUMEN
BACKGROUND & OBJECTIVE: Diabetes and neurodegenerative diseases (ND) are progressive morbidities and represent a major public health burden. A growing body of evidence points towards the comorbidity of diabetes and NDs with a possible exacerbation of latter by former. Considering the high prevalence of both morbidities in aging world population, even a modest impact of diabetes on NDs could lead to significant public health implications. Several hypotheses and mechanistic evidence were proposed linking altered glucose metabolism to the risk of progressive dementia. Unregulated production of reactive oxygen species (ROS) and resultant oxidative stress (OS) are the common features of diabetes as well as NDs. CONCLUSION: This review explores the concept of altered glucose metabolic pathways leading to ROS increase and its possible link to NDs, with a special emphasis on Alzheimer's diseases (AD). We also discuss the detailed mechanistic link between hyperglycemia, ROS generation, and neurodegeneration to highlight potential therapeutic avenues for better prevention and treatment.
Asunto(s)
Diabetes Mellitus/epidemiología , Diabetes Mellitus/metabolismo , Enfermedades Neurodegenerativas/epidemiología , Enfermedades Neurodegenerativas/metabolismo , Estrés Oxidativo/fisiología , Progresión de la Enfermedad , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Endothelin-1 (ET-1) has been demonstrated to be a pro-nociceptive as well as an anti-nociceptive agent. However, underlying molecular mechanisms for these pain modulatory actions remain unclear. In the present study, we evaluated the ability of ET-1 to alter the nociceptor excitability using a patch clamp technique in acutely dissociated rat dorsal root ganglion (DRG) neurons. ET-1 produced an increase in threshold current to evoke an action potential (I threshold) and hyperpolarization of resting membrane potential (RMP) indicating decreased excitability of DRG neurons. I threshold increased from 0.25 ± 0.08 to 0.33 ± 0.07 nA and hyperpolarized RMP from -57.51 ± 1.70 to -67.41 ± 2.92 mV by ET-1 (100 nM). The hyperpolarizing effect of ET-1 appears to be orchestrated via modulation of membrane conductances, namely voltage-gated sodium current (I Na) and outward transient potassium current (I KT). ET-1, 30 and 100 nM, decreased the peak I Na by 41.3 ± 6.8 and 74 ± 15.2%, respectively. Additionally, ET-1 (100 nM) significantly potentiated the transient component (I KT) of the potassium currents. ET-1-induced effects were largely attenuated by BQ-788, a selective ETBR blocker. However, a selective ETAR blocker BQ-123 did not alter the effects of ET-1. A selective ETBR agonist, IRL-1620, mimicked the effect of ET-1 on I Na in a concentration-dependent manner (IC50 159.5 ± 92.6 µM). In conclusion, our results demonstrate that ET-1 hyperpolarizes nociceptors by blocking I Na and potentiating I KT through selective activation of ETBR, which may represent one of the underlying mechanisms for reported anti-nociceptive effects of ET-1.
Asunto(s)
Potenciales de Acción , Endotelina-1/farmacología , Ganglios Espinales/citología , Neuronas/metabolismo , Receptor de Endotelina B/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Antagonistas de los Receptores de la Endotelina B/farmacología , Activación del Canal Iónico/efectos de los fármacos , Neuronas/efectos de los fármacos , Nociceptores/metabolismo , Canales de Potasio/metabolismo , Ratas Sprague-Dawley , Canales de Sodio/metabolismoRESUMEN
Microsomal and soluble epoxide hydrolase (mEH and sEH) fulfill apparently distinct roles: Whereas mEH detoxifies xenobiotics, sEH hydrolyzes fatty acid (FA) signaling molecules and is thus implicated in a variety of physiological functions. These epoxy FAs comprise epoxyeicosatrienoic acids (EETs) and epoxy-octadecenoic acids (EpOMEs), which are formed by CYP epoxygenases from arachidonic acid (AA) and linoleic acid, respectively, and then are hydrolyzed to their respective diols, the so-called DHETs and DiHOMEs. Although EETs and EpOMEs are also substrates for mEH, its role in lipid signaling is considered minor due to lower abundance and activity relative to sEH. Surprisingly, we found that in plasma from mEH KO mice, hydrolysis rates for 8,9-EET and 9,10-EpOME were reduced by 50% compared to WT plasma. This strongly suggests that mEH contributes substantially to the turnover of these FA epoxides-despite kinetic parameters being in favor of sEH. Given the crucial role of liver in controlling plasma diol levels, we next studied the capacity of sEH and mEH KO liver microsomes to synthesize DHETs with varying concentrations of AA (1-30 µM) and NADPH. mEH-generated DHET levels were similar to the ones generated by sEH, when AA concentrations were low (1 µM) or epoxygenase activity was curbed by modulating NADPH. With increasing AA concentrations sEH became more dominant and with 30 µM AA produced twice the level of DHETs compared to mEH. Immunohistochemistry of C57BL/6 liver slices further revealed that mEH expression was more widespread than sEH expression. mEH immunoreactivity was detected in hepatocytes, Kupffer cells, endothelial cells, and bile duct epithelial cells, while sEH immunoreactivity was confined to hepatocytes and bile duct epithelial cells. Finally, transcriptome analysis of WT, mEH KO, and sEH KO liver was carried out to discern transcriptional changes associated with the loss of EH genes along the CYP-epoxygenase-EH axis. We found several prominent dysregulations occurring in a parallel manner in both KO livers: (a) gene expression of Ephx1 (encoding for mEH protein) was increased 1.35-fold in sEH KO, while expression of Ephx2 (encoding for sEH protein) was increased 1.4-fold in mEH KO liver; (b) Cyp2c genes, encoding for the predominant epoxygenases in mouse liver, were mostly dysregulated in the same manner in both sEH and mEH KO mice, showing that loss of either EH has a similar impact. Taken together, mEH appears to play a leading role in the hydrolysis of 8,9-EET and 9,10-EpOME and also contributes to the hydrolysis of other FA epoxides. It probably profits from its high affinity for FA epoxides under non-saturating conditions and its close physical proximity to CYP epoxygenases, and compensates its lower abundance by a more widespread expression, being the only EH present in several sEH-lacking cell types.
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Epóxido Hidrolasas/metabolismo , Metabolismo de los Lípidos/fisiología , Hígado/fisiología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Epóxido Hidrolasas/genética , Compuestos Epoxi/metabolismo , Expresión Génica , Inactivación Metabólica , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/metabolismo , Ácidos Oléicos/metabolismo , Oxilipinas/sangre , Oxilipinas/metabolismoRESUMEN
Recent studies suggest a role for the arachidonic acid-derived epoxyeicosatrienoic acids (EETs) in attenuating epileptic seizures. However, their effect on neurotransmission has never been investigated in detail. Here, we studied how 11,12- and 14,15 EET affect excitability and excitatory neurotransmission in mouse hippocampus. 11,12 EET (2 µM), but not 14,15 EET (2 µM), induced the opening of a hyperpolarizing K+ conductance in CA1 pyramidal cells. This action could be blocked by BaCl2, the G protein blocker GDPß-S and the GIRK1/4 blocker tertiapin Q and the channel was thus identified as a GIRK channel. The 11,12 EET-mediated opening of this channel significantly reduced excitability of CA1 pyramidal cells, which could not be blocked by the functional antagonist EEZE (10 µM). Furthermore, both 11,12 EET and 14,15 EET reduced glutamate release on CA1 pyramidal cells with 14,15 EET being the less potent regioisomer. In CA1 pyramidal cells, 11,12 EET reduced the amplitude of excitatory postsynaptic currents (EPSCs) by 20% and the slope of field excitatory postsynaptic potentials (fEPSPs) by 50%, presumably via a presynaptic mechanism. EEZE increased both EPSC amplitude and fEPSP slope by 40%, also via a presynaptic mechanism, but failed to block 11,12 EET-mediated reduction of EPSCs and fEPSPs. This strongly suggests the existence of distinct targets for 11,12 EET and EEZE in neurons. In summary, 11,12 EET substantially reduced excitation in CA1 pyramidal cells by inhibiting the release of glutamate and opening a GIRK channel. These findings might explain the therapeutic potential of EETs in reducing epileptiform activity.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Hipocampo/efectos de los fármacos , Neurotransmisores/farmacología , Transmisión Sináptica/efectos de los fármacos , Ácido 8,11,14-Eicosatrienoico/antagonistas & inhibidores , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Anticonvulsivantes/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Transmisión Sináptica/fisiología , Técnicas de Cultivo de TejidosRESUMEN
Microsomal epoxide hydrolase (mEH) is a detoxifying enzyme for xenobiotic compounds. Enzymatic activity of mEH can be greatly increased by a point mutation, leading to an E404D amino acid exchange in its catalytic triad. Surprisingly, this variant is not found in any vertebrate species, despite the obvious advantage of accelerated detoxification. We hypothesized that this evolutionary avoidance is due to the fact that the mEH plays a dualistic role in detoxification and control of endogenous vascular signaling molecules. To test this, we generated mEH E404D mice and assessed them for detoxification capacity and vascular dynamics. In liver microsomes from these mice, turnover of the xenobiotic compound phenanthrene-9,10-oxide was four times faster compared to WT liver microsomes, confirming accelerated detoxification. mEH E404D animals also showed faster metabolization of a specific class of endogenous eicosanoids, arachidonic acid-derived epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Significantly higher DHETs/EETs ratios were found in mEH E404D liver, urine, plasma, brain and cerebral endothelial cells compared to WT controls, suggesting a broad impact of the mEH mutant on endogenous EETs metabolism. Because EETs are strong vasodilators in cerebral vasculature, hemodynamics were assessed in mEH E404D and WT cerebral cortex and hippocampus using cerebral blood volume (CBV)-based functional magnetic resonance imaging (fMRI). Basal CBV0 levels were similar between mEH E404D and control mice in both brain areas. But vascular reactivity and vasodilation in response to the vasodilatory drug acetazolamide were reduced in mEH E404D forebrain compared to WT controls by factor 3 and 2.6, respectively. These results demonstrate a critical role for mEH E404D in vasodynamics and suggest that deregulation of endogenous signaling pathways is the undesirable gain of function associated with the E404D variant.