RESUMEN
The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small-molecule inhibitors targeting key epigenetic enzymes in DRG neurons, we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3, thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.
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Ganglios Espinales/fisiología , Histona Desacetilasas/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Axones , Células Cultivadas , Modelos Animales de Enfermedad , Epigénesis Genética/efectos de los fármacos , Femenino , Masculino , Ratones , Regeneración Nerviosa , Fosforilación/efectos de los fármacos , Transducción de SeñalRESUMEN
BACKGROUND: Neural tissue has limited regenerative ability. To cope with that, in recent years a diverse set of novel tools has been used to tailor neurostimulation therapies and promote functional regeneration after axonal injuries. METHOD: In this report, we explore cell-specific methods to modulate neuronal activity, including opto- and chemogenetics to assess the effect of specific neuronal stimulation in the promotion of axonal regeneration after injury. RESULTS: Opto- and chemogenetic stimulations of neuronal activity elicited increased in vitro neurite outgrowth in both sensory and cortical neurons, as well as in vivo regeneration in the sciatic nerve, but not after spinal cord injury. Mechanistically, inhibitory substrates such as chondroitin sulfate proteoglycans block the activity induced increase in axonal growth. CONCLUSIONS: We found that genetic modulations of neuronal activity on both dorsal root ganglia and corticospinal motor neurons increase their axonal growth capacity but only on permissive environments.
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Neuronas , Traumatismos de la Médula Espinal , Axones/fisiología , Ganglios Espinales , Humanos , Regeneración Nerviosa , Neuronas/fisiología , Nervio Ciático/lesiones , Traumatismos de la Médula Espinal/terapiaRESUMEN
The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malinKO). Strikingly, blocking glycogen synthesis in astrocytes-thus impeding Lafora bodies accumulation in this cell type-prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malinKO model. Conversely, mice that over-accumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neurons.
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Astrocitos/metabolismo , Encéfalo/metabolismo , Glucógeno/metabolismo , Enfermedad de Lafora/metabolismo , Degeneración Nerviosa/metabolismo , Animales , Astrocitos/patología , Encéfalo/patología , Modelos Animales de Enfermedad , Glucógeno Sintasa/genética , Glucógeno Sintasa/metabolismo , Enfermedad de Lafora/genética , Enfermedad de Lafora/patología , Ratones , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Regeneration of injured central nervous system axons is highly restricted, causing neurological impairment. To date, although the lack of intrinsic regenerative potential is well described, a key regulatory molecular mechanism for the enhancement of both axonal regrowth and functional recovery after central nervous system injury remains elusive. While ubiquitin ligases coordinate neuronal morphogenesis and connectivity during development as well as after axonal injury, their role specifically in axonal regeneration is unknown. Following a bioinformatics network analysis combining ubiquitin ligases with previously defined axonal regenerative proteins, we found a triad composed of the ubiquitin ligases MDM4, MDM2 and the transcription factor p53 (encoded by TP53) as a putative central signalling complex restricting the regeneration program. Indeed, conditional deletion of MDM4 or pharmacological inhibition of MDM2/p53 interaction in the eye and spinal cord promote axonal regeneration and sprouting of the optic nerve after crush and of supraspinal tracts after spinal cord injury. The double conditional deletion of MDM4-p53 as well as MDM2 inhibition in p53-deficient mice blocks this regenerative phenotype, showing its dependence upon p53. Genome-wide gene expression analysis from ex vivo fluorescence-activated cell sorting in MDM4-deficient retinal ganglion cells identifies the downstream target IGF1R, whose activity and expression was found to be required for the regeneration elicited by MDM4 deletion. Importantly, we demonstrate that pharmacological enhancement of the MDM2/p53-IGF1R axis enhances axonal sprouting as well as functional recovery after spinal cord injury. Thus, our results show MDM4-MDM2/p53-IGF1R as an original regulatory mechanism for CNS regeneration and offer novel targets to enhance neurological recovery.media-1vid110.1093/brain/awv125_video_abstractawv125_video_abstract.
Asunto(s)
Regeneración Nerviosa/fisiología , Traumatismos del Nervio Óptico/metabolismo , Recuperación de la Función/fisiología , Transducción de Señal/fisiología , Traumatismos de la Médula Espinal/metabolismo , Animales , Axones/metabolismo , Axones/patología , Biología Computacional , Modelos Animales de Enfermedad , Citometría de Flujo , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Compresión Nerviosa , Traumatismos del Nervio Óptico/patología , Traumatismos del Nervio Óptico/fisiopatología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Transcriptoma , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: The administration of µ-opioid receptors (MOR) and δ-opioid receptors (DOR) as well as cannabinoid-2 receptor (CB2R) agonists attenuates neuropathic pain. We investigated if treatment with two carbon monoxide-releasing molecules (CORM-2 and CORM-3) or an inducible heme oxygenase inducer (cobalt protoporphyrin IX, CoPP) could modulate the local and systemic effects and expression of MOR, DOR, and CB2R during neuropathic pain. METHODS: In C57BL/6 mice, at 10 days after the chronic constriction of sciatic nerve, we evaluated the effects of the intraperitoneal administration of 10 mg/kg of CORM-2, CORM-3, or CoPP on the antiallodynic and antihyperalgesic actions of a locally or systemically administered MOR (morphine), DOR ([d-Pen(2),d-Pen(5)]-enkephalin) or CB2R ((2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone ) agonist. The effects of CORM-2 and CoPP treatments on the expression of MOR, DOR, CB2R, inducible and constitutive heme oxygenases, microglia activation marker (CD11b/c), and neuronal and inducible nitric oxide synthases were also assessed. RESULTS: Treatments with CO-RMs and CoPP reduced the mechanical and thermal hypersensitivity induced by sciatic nerve injury, increased the local, but not systemic, antinociceptive effects of morphine, and decreased those produced by DPDPE and JWH-015. Both CORM-2 and CoPP treatments enhanced MOR and inducible heme oxygenase expression, unaltered DOR and constitutive heme oxygenase expression, and decreased the overexpression of CB2R, CD11b/c, and neuronal and inducible nitric oxide synthases induced by sciatic nerve injury. CONCLUSIONS: This study shows that CO-RMs and CoPP treatments increase the local antinociceptive effects of morphine through enhancing MOR peripheral expression and inhibiting spinal microglial activation and overexpression of neuronal/inducible nitric oxide synthases.
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Monóxido de Carbono/uso terapéutico , Hemo-Oxigenasa 1/biosíntesis , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Compuestos Organometálicos/uso terapéutico , Receptores Opioides mu/efectos de los fármacos , Analgésicos Opioides/uso terapéutico , Animales , Monóxido de Carbono/administración & dosificación , Constricción Patológica/complicaciones , Encefalina D-Penicilamina (2,5)/uso terapéutico , Inducción Enzimática/efectos de los fármacos , Calor , Indoles/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Morfina/uso terapéutico , Dimensión del Dolor/efectos de los fármacos , Estimulación Física , Protoporfirinas/uso terapéutico , Receptor Cannabinoide CB2/agonistas , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides mu/biosíntesisRESUMEN
Regeneration after a peripheral nerve injury still remains a challenge, due to the limited regenerative potential of axons after injury. While the endocannabinoid system (ECS) has been widely studied for its neuroprotective and analgesic effects, its role in axonal regeneration and during the conditioning lesion remains unexplored. In this study, we observed that a peripheral nerve injury induces axonal regeneration through an increase in the endocannabinoid tone. We also enhanced the regenerative capacity of dorsal root ganglia (DRG) neurons through the inhibition of endocannabinoid degradative enzyme MAGL or a CB1R agonist. Our results suggest that the ECS, via CB1R and PI3K-pAkt pathway activation, plays an important role in promoting the intrinsic regenerative capacity of sensory neurons after injury.
RESUMEN
Peripheral inputs continuously shape brain function and can influence memory acquisition, but the underlying mechanisms have not been fully understood. Cannabinoid type-1 receptor (CB1R) is a well-recognized player in memory performance, and its systemic modulation significantly influences memory function. By assessing low arousal/non-emotional recognition memory in mice, we found a relevant role of peripheral CB1R in memory persistence. Indeed, the peripherally-restricted CB1R specific antagonist AM6545 showed significant mnemonic effects that were occluded in adrenalectomized mice, and after peripheral adrenergic blockade. AM6545 also transiently impaired contextual fear memory extinction. Vagus nerve chemogenetic inhibition reduced AM6545-induced mnemonic effect. Genetic CB1R deletion in dopamine ß-hydroxylase-expressing cells enhanced recognition memory persistence. These observations support a role of peripheral CB1R modulating adrenergic tone relevant for cognition. Furthermore, AM6545 acutely improved brain connectivity and enhanced extracellular hippocampal norepinephrine. In agreement, intra-hippocampal ß-adrenergic blockade prevented AM6545 mnemonic effects. Altogether, we disclose a novel CB1R-dependent peripheral mechanism with implications relevant for lengthening the duration of non-emotional memory.
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Norepinefrina , Receptor Cannabinoide CB1 , Animales , Ratones , Adrenérgicos/farmacología , Encéfalo , Hipocampo , Norepinefrina/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidoresRESUMEN
While chemokines were originally described for their ability to induce cell migration, many studies show how these proteins also take part in many other cell functions, acting as adaptable messengers in the communication between a diversity of cell types. In the nervous system, chemokines participate both in physiological and pathological processes, and while their expression is often described on glial and immune cells, growing evidence describes the expression of chemokines and their receptors in neurons, highlighting their potential in auto- and paracrine signalling. In this study we analysed the role of nociception in the neuronal chemokinome, and in turn their role in axonal growth. We found that stimulating TRPV1+ nociceptors induces a transient increase in CCL21. Interestingly we also found that CCL21 enhances neurite growth of large diameter proprioceptors in vitro. Consistent with this, we show that proprioceptors express the CCL21 receptor CCR7, and a CCR7 neutralizing antibody dose-dependently attenuates CCL21-induced neurite outgrowth. Mechanistically, we found that CCL21 binds locally to its receptor CCR7 at the growth cone, activating the downstream MEK-ERK pathway, that in turn activates N-WASP, triggering actin filament ramification in the growth cone, resulting in increased axonal growth.
Asunto(s)
Ganglios Espinales , Nocicepción , Movimiento Celular , Quimiocina CCL21/metabolismo , Ganglios Espinales/metabolismo , Sistema de Señalización de MAP Quinasas , Receptores CCR7/metabolismoRESUMEN
Lafora disease (LD) is a fatal childhood-onset dementia characterized by the extensive accumulation of glycogen aggregates-the so-called Lafora Bodies (LBs)-in several organs. The accumulation of LBs in the brain underlies the neurological phenotype of the disease. LBs are composed of abnormal glycogen and various associated proteins, including p62, an autophagy adaptor that participates in the aggregation and clearance of misfolded proteins. To study the role of p62 in the formation of LBs and its participation in the pathology of LD, we generated a mouse model of the disease (malinKO) lacking p62. Deletion of p62 prevented LB accumulation in skeletal muscle and cardiac tissue. In the brain, the absence of p62 altered LB morphology and increased susceptibility to epilepsy. These results demonstrate that p62 participates in the formation of LBs and suggest that the sequestration of abnormal glycogen into LBs is a protective mechanism through which it reduces the deleterious consequences of its accumulation in the brain.
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Enfermedad de Lafora , Animales , Modelos Animales de Enfermedad , Glucógeno/metabolismo , Cuerpos de Inclusión/metabolismo , Enfermedad de Lafora/genética , Ratones , Ratones Noqueados , Proteína Sequestosoma-1RESUMEN
Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone.
RESUMEN
BACKGROUND: The local administration of µ-opioid receptor (MOR) agonists attenuates neuropathic pain but the precise mechanism implicated in this effect is not completely elucidated. We investigated if nitric oxide synthesized by neuronal (NOS1) or inducible (NOS2) nitric oxide synthases could modulate the local antiallodynic effects of morphine through the peripheral nitric oxide-cGMP-protein kinase G (PKG)-ATP-sensitive K+ (KATP) channels signaling pathway activation and affect the dorsal root ganglia MOR expression during neuropathic pain. RESULTS: In wild type (WT) mice, the subplantar administration of morphine dose-dependently decreased the mechanical and thermal allodynia induced by the chronic constriction of the sciatic nerve (CCI), which effects were significantly diminished after their co-administration with different subanalgesic doses of a selective NOS1 (N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris(trifluoroacetate) salt; NANT), NOS2 (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) inhibitor or a KATP channel blocker (glibenclamide). The evaluation of the expression of MOR in the dorsal root ganglia from sham-operated and sciatic nerve-injured WT, NOS1 knockout (KO) and NOS2-KO mice at 21 days after surgery demonstrated that, although the basal mRNA and protein levels of MOR were similar between WT and both NOS-KO animals, nerve injury only decreased their expression in WT mice. CONCLUSIONS: These results suggest that the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway activation participates in the local antiallodynic effects of morphine after sciatic nerve injury and that nitric oxide, synthesized by NOS1 and NOS2, is implicated in the dorsal root ganglia down-regulation of MOR during neuropathic pain.
Asunto(s)
Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Hiperalgesia/tratamiento farmacológico , Morfina/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Receptores Opioides mu/metabolismo , Animales , Hiperalgesia/etiología , Ratones , Ratones Noqueados , Morfina/antagonistas & inhibidores , Morfina/farmacología , Naloxona/análogos & derivados , Naloxona/farmacología , Óxido Nítrico/metabolismo , Fragmentos de Péptidos/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/genética , Nervio Ciático/lesiones , Transducción de Señal/efectos de los fármacos , Somatostatina/farmacologíaRESUMEN
Central nervous system (CNS) injuries do not heal properly in contrast to normal tissue repair, in which functional recovery typically occurs. The reason for this dichotomy in wound repair is explained in part by macrophage and microglial malfunction, affecting both the extrinsic and intrinsic barriers to appropriate axonal regeneration. In normal healing tissue, macrophages promote the repair of injured tissue by regulating transitions through different phases of the healing response. In contrast, inflammation dominates the outcome of CNS injury, often leading to secondary damage. Therefore, an understanding of the molecular mechanisms underlying this dichotomy is critical to advance in neuronal repair therapies. Recent studies highlight the plasticity and complexity of macrophages and microglia beyond the classical view of the M1/M2 polarization paradigm. This plasticity represents an in vivo continuous spectrum of phenotypes with overlapping functions and markers. Moreover, macrophage and microglial plasticity affect many events essential for neuronal regeneration after injury, such as myelin and cell debris clearance, inflammation, release of cytokines, and trophic factors, affecting both intrinsic neuronal properties and extracellular matrix deposition. Until recently, this complexity was overlooked in the translation of therapies modulating these responses for the treatment of neuronal injuries. However, recent studies have shed important light on the underlying molecular mechanisms of this complexity and its transitions and effects on regenerative events. Here we review the complexity of macrophages and microglia after neuronal injury and their roles in regeneration, as well as the underlying molecular mechanisms, and we discuss current challenges and future opportunities for treatment.
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Macrófagos , Microglía , Sistema Nervioso Central , Humanos , Inflamación , NeuronasRESUMEN
Both delta-opioid receptor (DOPr) and cannabinoid-2 receptor (CB2R) agonists attenuate neuropathic pain, but the precise mechanism implicated in these effects is not completely elucidated. We investigated whether nitric oxide synthesized by neuronal (NOS1) or inducible (NOS2) nitric-oxide synthases could modulate DOPr and/or CB2R antiallodynic and antihyperalgesic effects through the peripheral nitric oxide-cGMP-protein kinase G (PKG) pathway activation and affect their expression during neuropathic pain. In wild-type (WT) mice at 21 days after chronic constriction of sciatic nerve, we evaluated the effects of [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE); (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH-015); and a NOS1 [N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris(trifluoroacetate) salt; NANT], NOS2 [l-N(6)-(1-iminoethyl)-lysine; l-NIL], l-guanylate cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ], or PKG [(Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs] inhibitor administered alone or combined. Expression of DOPr and CB2R mRNA in the spinal cord and dorsal root ganglia of naive and nerve-injured WT, NOS1-knockout (KO), and NOS2-KO mice, also was assessed. The subplantar administration of NANT, l-NIL, ODQ, or Rp-8-pCPT-cGMPs dose-dependently inhibited neuropathic pain and enhanced the local effects of DPDPE or JWH-015. Moreover, although the basal levels of DOPr and CB2R mRNA were similar between WT and NOS-KO animals, nerve injury only decreased (DOPr) or increased (CB2R) their expression in the dorsal root ganglia of WT and NOS2-KO mice, and not in NOS1-KO mice. Results suggest that inactivation of the nitric oxide-cGMP-PKG peripheral pathway triggered by NOS1 and NOS2 enhanced the peripheral actions of DOPr and CB2R agonists and that nitric oxide synthesized by NOS1 is implicated in the peripheral regulation of DOPr and CB2R gene transcription during neuropathic pain.
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Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Óxido Nítrico/fisiología , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/metabolismo , Receptor Cannabinoide CB2/biosíntesis , Receptores Opioides delta/biosíntesis , Animales , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Ganglios Espinales/metabolismo , Calor , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo II/genética , Dimensión del Dolor/efectos de los fármacos , Estimulación Física , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neuropatía Ciática/patología , Transducción de Señal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
Brain glycogen is mainly stored in astrocytes. However, recent studies both in vitro and in vivo indicate that glycogen also plays important roles in neurons. By conditional deletion of glycogen synthase (GYS1), we previously developed a mouse model entirely devoid of glycogen in the central nervous system (GYS1Nestin-KO). These mice displayed altered electrophysiological properties in the hippocampus and increased susceptibility to kainate-induced seizures. To understand which of these functions are related to astrocytic glycogen, in the present study, we generated a mouse model in which glycogen synthesis is eliminated specifically in astrocytes (GYS1Gfap-KO). Electrophysiological recordings of awake behaving mice revealed alterations in input/output curves and impaired long-term potentiation, similar, but to a lesser extent, to those obtained with GYS1Nestin-KO mice. Surprisingly, GYS1Gfap-KO mice displayed no change in susceptibility to kainate-induced seizures as determined by fEPSP recordings and video monitoring. These results confirm the importance of astrocytic glycogen in synaptic plasticity.
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Astrocitos/metabolismo , Glucógeno/metabolismo , Plasticidad Neuronal/fisiología , Convulsiones/fisiopatología , Animales , Susceptibilidad a Enfermedades , Fenómenos Electrofisiológicos , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/fisiopatología , Ácido Kaínico , Masculino , Ratones NoqueadosRESUMEN
Peripheral nerve injuries, including motor neuron axonal injury, often lead to functional impairments. Current therapies are mostly limited to surgical intervention after lesion, yet these interventions have limited success in restoring functionality. Current activity-based therapies after axonal injuries are based on trial-error approaches in which the details of the underlying cellular and molecular processes are largely unknown. Here we show the effects of the modulation of both neuronal and muscular activity with optogenetic approaches to assess the regenerative capacity of cultured motor neuron (MN) after lesion in a compartmentalized microfluidic-assisted axotomy device. With increased neuronal activity, we observed an increase in the ratio of regrowing axons after injury in our peripheral-injury model. Moreover, increasing muscular activity induces the liberation of leukemia inhibitory factor and glial cell line-derived neurotrophic factor in a paracrine fashion that in turn triggers axonal regrowth of lesioned MN in our 3D hydrogel cultures. The relevance of our findings as well as the novel approaches used in this study could be useful not only after axotomy events but also in diseases affecting MN survival.
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Axones/patología , Dispositivos Laboratorio en un Chip , Unión Neuromuscular/patología , Comunicación Paracrina , Animales , Axotomía , Diferenciación Celular , Línea Celular , Channelrhodopsins/metabolismo , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Luz , Ratones , Neuronas Motoras/patología , Fibras Musculares Esqueléticas/citología , Mioblastos/citología , Optogenética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Médula Espinal/patologíaRESUMEN
Cyclin-dependent-like kinase 5 (CDKL5) gene mutations lead to an X-linked disorder that is characterized by infantile epileptic encephalopathy, developmental delay, and hypotonia. However, we found that a substantial percentage of these patients also report a previously unrecognized anamnestic deficiency in pain perception. Consistent with a role in nociception, we found that CDKL5 is expressed selectively in nociceptive dorsal root ganglia (DRG) neurons in mice and in induced pluripotent stem cell (iPS)-derived human nociceptors. CDKL5-deficient mice display defective epidermal innervation, and conditional deletion of CDKL5 in DRG sensory neurons impairs nociception, phenocopying CDKL5 deficiency disorder in patients. Mechanistically, CDKL5 interacts with calcium/calmodulin-dependent protein kinase II α (CaMKIIα) to control outgrowth and transient receptor potential cation channel subfamily V member 1 (TRPV1)-dependent signaling, which are disrupted in both CDKL5 mutant murine DRG and human iPS-derived nociceptors. Together, these findings unveil a previously unrecognized role for CDKL5 in nociception, proposing an original regulatory mechanism for pain perception with implications for future therapeutics in CDKL5 deficiency disorder.
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Células Receptoras Sensoriales , Transducción de Señal , Animales , Ciclinas , Modelos Animales de Enfermedad , Humanos , Ratones , Dolor , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Adult postmitotic mammalian cells, including neurons and cardiomyocytes, have a limited capacity to regenerate after injury. Therefore, an understanding of the molecular mechanisms underlying their regenerative ability is critical to advance tissue repair therapies. Recent studies highlight how redox signalling via paracrine cell-to-cell communication may act as a central mechanism coupling tissue injury with regeneration. Post-injury redox paracrine signalling can act by diffusion to nearby cells, through mitochondria or within extracellular vesicles, affecting specific intracellular targets such as kinases, phosphatases, and transcription factors, which in turn trigger a regenerative response. Here, we review redox paracrine signalling mechanisms in postmitotic tissue regeneration and discuss current challenges and future directions.
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Mitosis , Miocitos Cardíacos/metabolismo , Neuronas/metabolismo , Comunicación Paracrina , Transducción de Señal , Animales , Vesículas Extracelulares/metabolismo , Humanos , Miocitos Cardíacos/citología , Neuronas/citología , Oxidación-ReducciónRESUMEN
Axonal injury results in regenerative success or failure, depending on whether the axon lies in the peripheral or the CNS, respectively. The present study addresses whether epigenetic signatures in dorsal root ganglia discriminate between regenerative and non-regenerative axonal injury. Chromatin immunoprecipitation for the histone 3 (H3) post-translational modifications H3K9ac, H3K27ac and H3K27me3; an assay for transposase-accessible chromatin; and RNA sequencing were performed in dorsal root ganglia after sciatic nerve or dorsal column axotomy. Distinct histone acetylation and chromatin accessibility signatures correlated with gene expression after peripheral, but not central, axonal injury. DNA-footprinting analyses revealed new transcriptional regulators associated with regenerative ability. Machine-learning algorithms inferred the direction of most of the gene expression changes. Neuronal conditional deletion of the chromatin remodeler CCCTC-binding factor impaired nerve regeneration, implicating chromatin organization in the regenerative competence. Altogether, the present study offers the first epigenomic map providing insight into the transcriptional response to injury and the differential regenerative ability of sensory neurons.
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Axones/fisiología , Epigenómica , Ganglios Espinales/fisiología , Regeneración Nerviosa/fisiología , Células Receptoras Sensoriales/fisiología , Acetilación , Algoritmos , Animales , Factor de Unión a CCCTC/genética , Cromatina/metabolismo , Femenino , Ganglios Espinales/lesiones , Expresión Génica , Histonas/metabolismo , Aprendizaje Automático , Masculino , Ratones , Ratones Transgénicos , Nervio Ciático/lesiones , Análisis de Secuencia de ARNRESUMEN
After a spinal cord injury, axons fail to regenerate in the adult mammalian central nervous system, leading to permanent deficits in sensory and motor functions. Increasing neuronal activity after an injury using electrical stimulation or rehabilitation can enhance neuronal plasticity and result in some degree of recovery; however, the underlying mechanisms remain poorly understood. We found that placing mice in an enriched environment before an injury enhanced the activity of proprioceptive dorsal root ganglion neurons, leading to a lasting increase in their regenerative potential. This effect was dependent on Creb-binding protein (Cbp)-mediated histone acetylation, which increased the expression of genes associated with the regenerative program. Intraperitoneal delivery of a small-molecule activator of Cbp at clinically relevant times promoted regeneration and sprouting of sensory and motor axons, as well as recovery of sensory and motor functions in both the mouse and rat model of spinal cord injury. Our findings showed that the increased regenerative capacity induced by enhancing neuronal activity is mediated by epigenetic reprogramming in rodent models of spinal cord injury. Understanding the mechanisms underlying activity-dependent neuronal plasticity led to the identification of potential molecular targets for improving recovery after spinal cord injury.