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Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However, the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection, skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors, ligands, ion channels and metabolites to generate injury-specific neuroimmune interactomes, we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts.
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Nociceptores , Dolor , Animales , Ratones , Dolor/inmunología , Dolor/metabolismo , Nociceptores/metabolismo , Transcriptoma , Ratones Endogámicos C57BL , Inflamación/inmunología , Masculino , Macrófagos/inmunología , Macrófagos/metabolismo , Modelos Animales de Enfermedad , Trombospondina 1/metabolismo , Trombospondina 1/genética , Piel/inmunología , Piel/metabolismo , Piel/patología , Zimosan , Análisis de la Célula Individual , Neuroinmunomodulación , Perfilación de la Expresión Génica , Neutrófilos/inmunología , Neutrófilos/metabolismoRESUMEN
The reduced ability of the central nervous system to regenerate with increasing age limits functional recovery following demyelinating injury. Previous work has shown that myelin debris can overwhelm the metabolic capacity of microglia, thereby impeding tissue regeneration in aging, but the underlying mechanisms are unknown. In a model of demyelination, we found that a substantial number of genes that were not effectively activated in aged myeloid cells displayed epigenetic modifications associated with restricted chromatin accessibility. Ablation of two class I histone deacetylases in microglia was sufficient to restore the capacity of aged mice to remyelinate lesioned tissue. We used Bacillus Calmette-Guerin (BCG), a live-attenuated vaccine, to train the innate immune system and detected epigenetic reprogramming of brain-resident myeloid cells and functional restoration of myelin debris clearance and lesion recovery. Our results provide insight into aging-associated decline in myeloid function and how this decay can be prevented by innate immune reprogramming.
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Astrocytes are heterogeneous glial cells of the central nervous system1-3. However, the physiological relevance of astrocyte diversity for neural circuits and behaviour remains unclear. Here we show that a specific population of astrocytes in the central striatum expresses µ-crystallin (encoded by Crym in mice and CRYM in humans) that is associated with several human diseases, including neuropsychiatric disorders4-7. In adult mice, reducing the levels of µ-crystallin in striatal astrocytes through CRISPR-Cas9-mediated knockout of Crym resulted in perseverative behaviours, increased fast synaptic excitation in medium spiny neurons and dysfunctional excitatory-inhibitory synaptic balance. Increased perseveration stemmed from the loss of astrocyte-gated control of neurotransmitter release from presynaptic terminals of orbitofrontal cortex-striatum projections. We found that perseveration could be remedied using presynaptic inhibitory chemogenetics8, and that this treatment also corrected the synaptic deficits. Together, our findings reveal converging molecular, synaptic, circuit and behavioural mechanisms by which a molecularly defined and allocated population of striatal astrocytes gates perseveration phenotypes that accompany neuropsychiatric disorders9-12. Our data show that Crym-positive striatal astrocytes have key biological functions within the central nervous system, and uncover astrocyte-neuron interaction mechanisms that could be targeted in treatments for perseveration.
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Astrocitos , Cuerpo Estriado , Rumiación Cognitiva , Cristalinas mu , Animales , Humanos , Ratones , Astrocitos/metabolismo , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Edición Génica , Técnicas de Inactivación de Genes , Cristalinas mu/deficiencia , Cristalinas mu/genética , Cristalinas mu/metabolismo , Rumiación Cognitiva/fisiología , Transmisión Sináptica , Sistemas CRISPR-Cas , Neuronas Espinosas Medianas/metabolismo , Sinapsis/metabolismo , Corteza Prefrontal/citología , Corteza Prefrontal/metabolismo , Terminales Presinápticos/metabolismo , Inhibición NeuralRESUMEN
The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.
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Encéfalo , Perfilación de la Expresión Génica , Vías Nerviosas , Neuronas , Médula Espinal , Animales , Ratones , Hipotálamo , Neuronas/metabolismo , Neuropéptidos , Médula Espinal/citología , Médula Espinal/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Neurotransmisores , Mesencéfalo/citología , Formación Reticular/citología , Electrofisiología , Cerebelo/citología , Corteza Cerebral/citologíaRESUMEN
The incidence of Alzheimer's disease (AD), the leading cause of dementia, increases rapidly with age, but why age constitutes the main risk factor is still poorly understood. Brain ageing affects oligodendrocytes and the structural integrity of myelin sheaths1, the latter of which is associated with secondary neuroinflammation2,3. As oligodendrocytes support axonal energy metabolism and neuronal health4-7, we hypothesized that loss of myelin integrity could be an upstream risk factor for neuronal amyloid-ß (Aß) deposition, the central neuropathological hallmark of AD. Here we identify genetic pathways of myelin dysfunction and demyelinating injuries as potent drivers of amyloid deposition in mouse models of AD. Mechanistically, myelin dysfunction causes the accumulation of the Aß-producing machinery within axonal swellings and increases the cleavage of cortical amyloid precursor protein. Suprisingly, AD mice with dysfunctional myelin lack plaque-corralling microglia despite an overall increase in their numbers. Bulk and single-cell transcriptomics of AD mouse models with myelin defects show that there is a concomitant induction of highly similar but distinct disease-associated microglia signatures specific to myelin damage and amyloid plaques, respectively. Despite successful induction, amyloid disease-associated microglia (DAM) that usually clear amyloid plaques are apparently distracted to nearby myelin damage. Our data suggest a working model whereby age-dependent structural defects of myelin promote Aß plaque formation directly and indirectly and are therefore an upstream AD risk factor. Improving oligodendrocyte health and myelin integrity could be a promising target to delay development and slow progression of AD.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Vaina de Mielina , Placa Amiloide , Animales , Ratones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Modelos Animales de Enfermedad , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Placa Amiloide/genética , Placa Amiloide/metabolismo , Placa Amiloide/patología , Axones/metabolismo , Axones/patología , Microglía/metabolismo , Microglía/patología , Análisis de Expresión Génica de una Sola Célula , Factores de Riesgo , Progresión de la EnfermedadRESUMEN
Astrocytes respond to injury and disease in the central nervous system with reactive changes that influence the outcome of the disorder1-4. These changes include differentially expressed genes (DEGs) whose contextual diversity and regulation are poorly understood. Here we combined biological and informatic analyses, including RNA sequencing, protein detection, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and conditional gene deletion, to predict transcriptional regulators that differentially control more than 12,000 DEGs that are potentially associated with astrocyte reactivity across diverse central nervous system disorders in mice and humans. DEGs associated with astrocyte reactivity exhibited pronounced heterogeneity across disorders. Transcriptional regulators also exhibited disorder-specific differences, but a core group of 61 transcriptional regulators was identified as common across multiple disorders in both species. We show experimentally that DEG diversity is determined by combinatorial, context-specific interactions between transcriptional regulators. Notably, the same reactivity transcriptional regulators can regulate markedly different DEG cohorts in different disorders; changes in the access of transcriptional regulators to DNA-binding motifs differ markedly across disorders; and DEG changes can crucially require multiple reactivity transcriptional regulators. We show that, by modulating reactivity, transcriptional regulators can substantially alter disorder outcome, implicating them as therapeutic targets. We provide searchable resources of disorder-related reactive astrocyte DEGs and their predicted transcriptional regulators. Our findings show that transcriptional changes associated with astrocyte reactivity are highly heterogeneous and are customized from vast numbers of potential DEGs through context-specific combinatorial transcriptional-regulator interactions.
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Astrocitos , Enfermedades del Sistema Nervioso Central , Regulación de la Expresión Génica , Factores de Transcripción , Transcripción Genética , Animales , Astrocitos/metabolismo , Enfermedades del Sistema Nervioso Central/genética , Enfermedades del Sistema Nervioso Central/patología , Cromatina/genética , Cromatina/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Neuropsychiatric disorders classically lack defining brain pathologies, but recent work has demonstrated dysregulation at the molecular level, characterized by transcriptomic and epigenetic alterations1-3. In autism spectrum disorder (ASD), this molecular pathology involves the upregulation of microglial, astrocyte and neural-immune genes, the downregulation of synaptic genes, and attenuation of gene-expression gradients in cortex1,2,4-6. However, whether these changes are limited to cortical association regions or are more widespread remains unknown. To address this issue, we performed RNA-sequencing analysis of 725 brain samples spanning 11 cortical areas from 112 post-mortem samples from individuals with ASD and neurotypical controls. We find widespread transcriptomic changes across the cortex in ASD, exhibiting an anterior-to-posterior gradient, with the greatest differences in primary visual cortex, coincident with an attenuation of the typical transcriptomic differences between cortical regions. Single-nucleus RNA-sequencing and methylation profiling demonstrate that this robust molecular signature reflects changes in cell-type-specific gene expression, particularly affecting excitatory neurons and glia. Both rare and common ASD-associated genetic variation converge within a downregulated co-expression module involving synaptic signalling, and common variation alone is enriched within a module of upregulated protein chaperone genes. These results highlight widespread molecular changes across the cerebral cortex in ASD, extending beyond association cortex to broadly involve primary sensory regions.
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Trastorno del Espectro Autista , Corteza Cerebral , Variación Genética , Transcriptoma , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Neuronas/metabolismo , ARN/análisis , ARN/genética , Transcriptoma/genética , Autopsia , Análisis de Secuencia de ARN , Corteza Visual Primaria/metabolismo , Neuroglía/metabolismoRESUMEN
Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.
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Cromatina/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Genoma Humano , Neurogénesis/genética , Regiones Promotoras Genéticas , Adulto , Línea Celular , Cerebro/citología , Cerebro/crecimiento & desarrollo , Cerebro/metabolismo , Cromatina/ultraestructura , Mapeo Cromosómico , Feto , Histonas/genética , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas del Tejido Nervioso/clasificación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/citología , Neuronas/metabolismo , Lóbulo Temporal/citología , Lóbulo Temporal/crecimiento & desarrollo , Lóbulo Temporal/metabolismo , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.
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Proliferación Celular/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regeneración Nerviosa/genética , Células-Madre Neurales/citología , Neuronas/metabolismo , Neuronas/patología , Transcripción Genética , Animales , Axones/patología , Axones/fisiología , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Proteína Huntingtina/genética , Ratones , Células-Madre Neurales/trasplante , Plasticidad Neuronal , Neuronas/citología , Neuronas/trasplante , Biosíntesis de Proteínas , Tractos Piramidales/citología , Tractos Piramidales/metabolismo , Tractos Piramidales/patología , RNA-Seq , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , TranscriptomaRESUMEN
Spinal cord injury in mammals is thought to trigger scar formation with little regeneration of axons1-4. Here we show that a crush injury to the spinal cord in neonatal mice leads to scar-free healing that permits the growth of long projecting axons through the lesion. Depletion of microglia in neonatal mice disrupts this healing process and stalls the regrowth of axons, suggesting that microglia are critical for orchestrating the injury response. Using single-cell RNA sequencing and functional analyses, we find that neonatal microglia are transiently activated and have at least two key roles in scar-free healing. First, they transiently secrete fibronectin and its binding proteins to form bridges of extracellular matrix that ligate the severed ends of the spinal cord. Second, neonatal-but not adult-microglia express several extracellular and intracellular peptidase inhibitors, as well as other molecules that are involved in resolving inflammation. We transplanted either neonatal microglia or adult microglia treated with peptidase inhibitors into spinal cord lesions of adult mice, and found that both types of microglia significantly improved healing and axon regrowth. Together, our results reveal the cellular and molecular basis of the nearly complete recovery of neonatal mice after spinal cord injury, and suggest strategies that could be used to facilitate scar-free healing in the adult mammalian nervous system.
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Microglía/fisiología , Traumatismos de la Médula Espinal/terapia , Regeneración de la Medula Espinal , Médula Espinal/citología , Médula Espinal/fisiología , Animales , Animales Recién Nacidos , Axones/efectos de los fármacos , Axones/fisiología , Cicatriz , Fibronectinas/metabolismo , Homeostasis , Ratones , Microglía/efectos de los fármacos , Inhibidores de Proteasas/farmacología , RNA-Seq , Análisis de la Célula Individual , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Regeneración de la Medula Espinal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.
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Nucléolo Celular/metabolismo , Ganglios Espinales/metabolismo , Cinesinas/metabolismo , Neuronas/metabolismo , Fosfoproteínas/química , Proteínas de Unión al ARN/química , Nervio Ciático/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Axonal/genética , Línea Celular Tumoral , Nucléolo Celular/ultraestructura , Ganglios Espinales/citología , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Neuronas/citología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultivo Primario de Células , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Nervio Ciático/citología , NucleolinaRESUMEN
Severe psychiatric illnesses, for instance schizophrenia, and affective diseases or autism spectrum disorders, have been associated with cognitive impairment and perturbed excitatory-inhibitory balance in the brain. Effects in juvenile mice can elucidate how erythropoietin (EPO) might aid in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, conceivably explaining mitigation of neuropsychiatric diseases. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing ~12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.
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Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1-3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factors that are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity2,5-9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesion cores of non-neural tissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.
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Axones/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Animales , Astrocitos/patología , Cicatriz/patología , Electrofisiología , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Hidrogeles , Laminina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuroglía/metabolismo , Proteoglicanos/metabolismo , Ratas , Ratas Endogámicas Lew , Recuperación de la Función , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/rehabilitación , Regeneración de la Medula Espinal , Células del Estroma/patologíaRESUMEN
Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.
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MicroARNs/genética , Tauopatías/genética , Proteínas tau/genética , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Corteza Cerebelosa/metabolismo , Corteza Cerebelosa/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , Ratones , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Oligodendroglía/metabolismo , RNA-Seq , Tauopatías/metabolismo , Tauopatías/patologíaRESUMEN
The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic Ccd42 gene is alternatively spliced to generate mRNAs with two different 3' untranslated regions (UTRs) that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CaaX and CCaX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during peripheral nervous system (PNS) axon regeneration. Functional studies indicate that prenyl-Cdc42 increases axon length in a manner that requires axonal targeting of its mRNA, which, in turn, needs an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, palmitoyl-Cdc42 has no effect on axon growth but selectively increases dendrite length. Together, these data show that alternative splicing of the Cdc42 gene product generates an axon growth promoting, locally synthesized prenyl-Cdc42 protein. This article has an associated First Person interview with one of the co-first authors of the paper.
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Axones , Isoformas de ARN , Axones/metabolismo , Lipoilación , Regeneración Nerviosa , Isoformas de ARN/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Vitamin A has diverse biological functions and is essential for human survival at every point from embryogenesis to adulthood. Vitamin A and its derivatives have been used to treat human diseases including vision diseases, skin diseases, and cancer. Both insufficient and excessive vitamin A uptake are detrimental, but how its transport is regulated is poorly understood. STRA6 is a multitransmembrane domain cell-surface receptor and mediates vitamin A uptake from plasma retinol binding protein (RBP). STRA6 can mediate both cellular vitamin A influx and efflux, but what regulates these opposing activities is unknown. To answer this question, we purified and identified STRA6-associated proteins in a native mammalian cell type that takes up vitamin A through STRA6 using mass spectrometry. We found that the major protein repeatedly identified as STRA6-associated protein is calmodulin, consistent with the cryogenic electron microscopy (cryo-EM) study of zebrafish STRA6 associated with calmodulin. Using radioactivity-based, high-performance liquid chromatography (HPLC)-based and real-time fluorescence techniques, we found that calmodulin profoundly affects STRA6's vitamin A transport activity. Increased calcium/calmodulin promotes cellular vitamin A efflux and suppresses vitamin A influx through STRA6. Further mechanistic studies revealed that calmodulin enhances the binding of apo-RBP to STRA6, and this enhancement is much more pronounced for apo-RBP than holo-RBP. This study revealed that calmodulin regulates STRA6's vitamin A influx or efflux activity by modulating its preferential interaction with apo-RBP or holo-RBP. This molecular mechanism of regulating vitamin A transport may point to new directions to treat human diseases associated with insufficient or excessive vitamin A uptake.
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Transporte Biológico/genética , Calmodulina/genética , Proteínas de la Membrana/genética , Proteínas Plasmáticas de Unión al Retinol/genética , Vitamina A/metabolismo , Animales , Apoproteínas/genética , Apoproteínas/metabolismo , Calcio/metabolismo , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , Microscopía por Crioelectrón , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica/genética , Receptores de Superficie Celular/genética , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Vitamina A/genética , Pez Cebra/genéticaRESUMEN
BACKGROUND: Vascular dementia (VaD) is the accumulation of vascular lesions in the subcortical white matter of the brain. These lesions progress and there is no direct medical therapy. AIMS: To determine the specific cellular responses in VaD so as to provide molecular targets for therapeutic development. MATERIALS AND METHODS: Single-nucleus transcriptome analysis was performed in human periventricular white matter (PVWM) samples of VaD and normal control (NC) subjects. RESULTS: Differential analysis shows that cell type-specific transcriptomic changes in VaD are associated with the disruption of specific biological processes, including angiogenesis, immune activation, axonal injury and myelination. Each cell type in the neurovascular unit within white matter has a specific alteration in gene expression in VaD. In a central cell type for this disease, subcluster analysis of endothelial cells (EC) indicates that VaD contains a disease-associated EC subcluster that expresses genes associated with programmed cell death and a response to protein folding. Two other subpopulations of EC in VaD express molecular systems associated with regenerative processes in angiogenesis, and in axonal sprouting and oligodendrocyte progenitor cell maturation. CONCLUSION: This comprehensive molecular profiling of brain samples from patients with VaD reveals previously unknown molecular changes in cells of the neurovascular niche, and an attempt at regeneration in injured white matter.
Asunto(s)
Demencia Vascular , Sustancia Blanca , Encéfalo/metabolismo , Demencia Vascular/genética , Demencia Vascular/patología , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Sustancia Blanca/metabolismo , Sustancia Blanca/patologíaRESUMEN
Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing astrocyte scar formation, attenuating scar-forming astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that astrocytes and non-astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, astrocyte scar formation aids rather than prevents central nervous system axon regeneration.
Asunto(s)
Astrocitos/patología , Axones/fisiología , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiología , Cicatriz/patología , Modelos Biológicos , Regeneración Nerviosa , Animales , Sistema Nervioso Central/citología , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Cicatriz/prevención & control , Femenino , Genómica , Ratones , Reproducibilidad de los Resultados , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patologíaRESUMEN
Subcortical white matter stroke is a common stroke subtype. White matter stroke stimulates adjacent oligodendrocyte progenitor cells (OPCs) to divide and migrate to the lesion, but stroke OPCs have only a limited differentiation into mature oligodendrocytes. To understand the molecular systems that are active in OPC responses in white matter stroke, OPCs were virally labeled and laser-captured in the region of partial damage adjacent to the infarct in male mice. RNAseq indicates two distinct OPC transcriptomes associated with the proliferative and limited-regeneration phases of OPCs after stroke. Molecular pathways related to nuclear receptor activation, ECM turnover, and lipid biosynthesis are activated during proliferative OPC phases after stroke; inflammatory and growth factor signaling is activated in the later stage of limited OPC differentiation. Within ECM proteins, Matrilin-2 is induced early after stroke and then rapidly downregulated. Prediction of upstream regulators of the OPC stroke transcriptome identifies several candidate molecules, including Inhibin A-a negative regulator of Matrilin-2. Inhibin A is induced in reactive astrocytes after stroke, including in humans. In functional assays, Matrilin-2 induces OPC differentiation, and Inhibin A inhibits OPC Matrilin-2 expression and inhibits OPC differentiation. In vivo, Matrilin-2 promotes motor recovery after white matter stroke, and promotes OPC differentiation and ultrastructural evidence of remyelination. These studies show that white matter stroke induces an initial proliferative and reparative response in OPCs, but this is blocked by a local cellular niche where reactive astrocytes secrete Inhibin A, downregulating Matrilin-2 and blocking myelin repair and recovery.SIGNIFICANCE STATEMENT Stroke in the cerebral white matter of the brain is common. The biology of damage and recovery in this stroke subtype are not well defined. These studies use cell-specific RNA sequencing and gain-of-function studies to show that white matter stroke induces a glial signaling niche, present in both humans and mice, between reactive astrocytes and oligodendrocyte progenitor cells. Astrocyte secretion of Inhibin A and downregulation of oligodendrocyte precursor production of Matrilin-2 limit OPC differentiation, tissue repair, and recovery in this disease.