RESUMO
Mammalian neurons lose the ability to regenerate their central nervous system axons as they mature during embryonic or early postnatal development. Neuronal maturation requires a transformation from a situation in which neuronal components grow and assemble to one in which these components are fixed and involved in the machinery for effective information transmission and computation. To regenerate after injury, neurons need to overcome this fixed state to reactivate their growth programme. A variety of intracellular processes involved in initiating or sustaining neuronal maturation, including the regulation of gene expression, cytoskeletal restructuring and shifts in intracellular trafficking, have been shown to prevent axon regeneration. Understanding these processes will contribute to the identification of targets to promote repair after injury or disease.
RESUMO
Axons in the adult mammalian central nervous system fail to regenerate after spinal cord injury. Neurons lose their capacity to regenerate during development, but the intracellular processes underlying this loss are unclear. We found that critical components of the presynaptic active zone prevent axon regeneration in adult mice. Transcriptomic analysis combined with live-cell imaging revealed that adult primary sensory neurons downregulate molecular constituents of the synapse as they acquire the ability to rapidly grow their axons. Pharmacogenetic reduction of neuronal excitability stimulated axon regeneration after adult spinal cord injury. Genetic gain- and loss-of-function experiments uncovered that essential synaptic vesicle priming proteins of the presynaptic active zone, but not clostridial-toxin-sensitive VAMP-family SNARE proteins, inhibit axon regeneration. Systemic administration of Baclofen reduced voltage-dependent Ca2+ influx in primary sensory neurons and promoted their regeneration after spinal cord injury. These findings indicate that functional presynaptic active zones constitute a major barrier to axon regeneration.
Assuntos
Axônios , Traumatismos da Medula Espinal , Animais , Axônios/metabolismo , Sistema Nervoso Central/metabolismo , Mamíferos , Camundongos , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
An inhibitory extracellular milieu and neuron-intrinsic processes prevent axons from regenerating in the adult central nervous system (CNS). Here we show how the two aspects are interwoven. Genetic loss-of-function experiments determine that the small GTPase RhoA relays extracellular inhibitory signals to the cytoskeleton by adapting mechanisms set in place during neuronal polarization. In response to extracellular inhibitors, neuronal RhoA restricts axon regeneration by activating myosin II to compact actin and, thereby, restrain microtubule protrusion. However, astrocytic RhoA restricts injury-induced astrogliosis through myosin II independent of microtubules by activating Yes-activated protein (YAP) signaling. Cell-type-specific deletion in spinal-cord-injured mice shows that neuronal RhoA activation prevents axon regeneration, whereas astrocytic RhoA is beneficial for regenerating axons. These data demonstrate how extracellular inhibitors regulate axon regeneration, shed light on the capacity of reactive astrocytes to be growth inhibitory after CNS injury, and reveal cell-specific RhoA targeting as a promising therapeutic avenue.
Assuntos
Actinas , Axônios , Doenças do Sistema Nervoso Central , Regeneração Nervosa , Proteína rhoA de Ligação ao GTP , Actinas/metabolismo , Animais , Astrócitos/metabolismo , Axônios/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/patologia , Camundongos , Regeneração Nervosa/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
In this issue of Neuron, Wang et al. demonstrate that both cell-intrinsic and -extrinsic factors restrict the myelination of newly regenerated axons. Pharmalogical targeting of GPR17 signaling in oligodendrocyte precursor cells (OPCs) and microglial inhibition of oligodendrocyte maturation together promote robust myelination of regenerated axons after CNS injury.
Assuntos
Bainha de Mielina , Oligodendroglia , Axônios , Microglia , NeurôniosRESUMO
Oligodendrocyte progenitor cells (OPCs) are the most proliferative and dispersed population of progenitor cells in the adult central nervous system, which allows these cells to rapidly respond to damage. Oligodendrocytes and myelin are lost after traumatic spinal cord injury (SCI), compromising efficient conduction and, potentially, the long-term health of axons. In response, OPCs proliferate and then differentiate into new oligodendrocytes and Schwann cells to remyelinate axons. This culminates in highly efficient remyelination following experimental SCI in which nearly all intact demyelinated axons are remyelinated in rodent models. However, myelin regeneration comprises only one role of OPCs following SCI. OPCs contribute to scar formation after SCI and restrict the regeneration of injured axons. Moreover, OPCs alter their gene expression following demyelination, express cytokines and perpetuate the immune response. Here, we review the functional contribution of myelin regeneration and other recently uncovered roles of OPCs and their progeny to repair following SCI.
Assuntos
Células Precursoras de Oligodendrócitos/citologia , Oligodendroglia/metabolismo , Remielinização/fisiologia , Traumatismos da Medula Espinal/patologia , Animais , Humanos , Bainha de Mielina/metabolismo , Células de Schwann/metabolismo , Traumatismos da Medula Espinal/metabolismoRESUMO
The specification of an axon and its subsequent outgrowth are key steps during neuronal polarization, a prerequisite to wire the brain. The Rho-guanosine triphosphatase (GTPase) RhoA is believed to be a central player in these processes. However, its physiological role has remained undefined. Here, genetic loss- and gain-of-function experiments combined with time-lapse microscopy, cell culture, and in vivo analysis show that RhoA is not involved in axon specification but confines the initiation of neuronal polarization and axon outgrowth during development. Biochemical analysis and super-resolution microscopy together with molecular and pharmacological manipulations reveal that RhoA restrains axon growth by activating myosin-II-mediated actin arc formation in the growth cone to prevent microtubules from protruding toward the leading edge. Through this mechanism, RhoA regulates the duration of axon growth and pause phases, thus controlling the tightly timed extension of developing axons. Thereby, this work unravels physiologically relevant players coordinating actin-microtubule interactions during axon growth.
Assuntos
Axônios/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Axônios/fisiologia , Encéfalo/embriologia , Encéfalo/metabolismo , Polaridade Celular/fisiologia , Feminino , Mutação com Ganho de Função/genética , Cones de Crescimento/metabolismo , Mutação com Perda de Função/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Injured axons fail to regenerate in the adult CNS, which contrasts with their vigorous growth during embryonic development. We explored the potential of re-initiating axon extension after injury by reactivating the molecular mechanisms that drive morphogenetic transformation of neurons during development. Genetic loss- and gain-of-function experiments followed by time-lapse microscopy, in vivo imaging, and whole-mount analysis show that axon regeneration is fueled by elevated actin turnover. Actin depolymerizing factor (ADF)/cofilin controls actin turnover to sustain axon regeneration after spinal cord injury through its actin-severing activity. This pinpoints ADF/cofilin as a key regulator of axon growth competence, irrespective of developmental stage. These findings reveal the central role of actin dynamics regulation in this process and elucidate a core mechanism underlying axon growth after CNS trauma. Thereby, neurons maintain the capacity to stimulate developmental programs during adult life, expanding their potential for plasticity. Thus, actin turnover is a key process for future regenerative interventions.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Cofilina 1/genética , Cofilina 2/genética , Destrina/genética , Cones de Crescimento/patologia , Regeneração Nervosa/genética , Traumatismos da Medula Espinal/genética , Animais , Axônios/patologia , Cofilina 1/metabolismo , Cofilina 2/metabolismo , Destrina/metabolismo , Cones de Crescimento/metabolismo , Microscopia Intravital , Camundongos , Microscopia Confocal , Neurônios/metabolismo , Neurônios/patologia , Ratos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Imagem com Lapso de TempoRESUMO
Here we present a protocol for analyses of axon regeneration and density in unsectioned adult mouse spinal cord. This includes methods for injury and tracing of dorsal column sensory and corticospinal axons; clearing and staining of unsectioned spinal cord; visualization of axon degeneration and regeneration in cleared and uncleared specimens using two-photon microscopy; and either manual or semi-automatic analysis of axon density and regeneration in 3D space using Imaris and ImageJ software. This protocol can be used to elucidate the molecular and cellular mechanisms underlying nervous system degeneration and regeneration and to establish the therapeutic efficacy of candidate neuroregenerative treatments. Because tissue sectioning is not required, this protocol enables unambiguous evaluation of regeneration and greatly accelerates the speed at which analyses can be conducted. Surgical procedures take <30 min per mouse, with a wait period of 2 weeks between axonal injury and tracing and 2-8 weeks between tracing and tissue processing. Clearing and immunolabeling take ~1-2 weeks, depending on the size of the sample. Imaging and analysis can be performed in 1 d. All these procedures can be accomplished by a competent graduate student or experienced technician.
Assuntos
Axônios/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Traumatismos da Medula Espinal/diagnóstico por imagem , Medula Espinal/ultraestrutura , Animais , Axônios/metabolismo , Feminino , Furanos , Masculino , Cloreto de Metileno , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/fisiologia , Éteres Fenílicos , Software , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/reabilitaçãoRESUMO
Axons in the adult mammalian brain and spinal cord fail to regenerate upon lesion. In vivo imaging serves as a tool to investigate the immediate response of axons to injury and how the same injured axons behave over time. Here, we describe the dynamic changes that injured sensory axons undergo and methods of imaging them in vivo. First, we explain how sensory axons in the dorsal column of the adult mouse spinal cord respond to axotomy. Then, we highlight practical considerations for implementing two-photon based in vivo imaging of these axons. Finally, we describe future directions for this technique, including the possibility of in vivo imaging of subcellular dynamics within the axon.
Assuntos
Axônios/ultraestrutura , Sistema Nervoso Central/diagnóstico por imagem , Sistema Nervoso Central/lesões , Células Receptoras Sensoriais/ultraestrutura , Animais , Humanos , Regeneração NervosaRESUMO
Remyelination occurs after spinal cord injury (SCI) but its functional relevance is unclear. We assessed the necessity of myelin regulatory factor (Myrf) in remyelination after contusive SCI by deleting the gene from platelet-derived growth factor receptor alpha positive (PDGFRα-positive) oligodendrocyte progenitor cells (OPCs) in mice prior to SCI. While OPC proliferation and density are not altered by Myrf inducible knockout after SCI, the accumulation of new oligodendrocytes is largely prevented. This greatly inhibits myelin regeneration, resulting in a 44% reduction in myelinated axons at the lesion epicenter. However, spontaneous locomotor recovery after SCI is not altered by remyelination failure. In controls with functional MYRF, locomotor recovery precedes the onset of most oligodendrocyte myelin regeneration. Collectively, these data demonstrate that MYRF expression in PDGFRα-positive cell derived oligodendrocytes is indispensable for myelin regeneration following contusive SCI but that oligodendrocyte remyelination is not required for spontaneous recovery of stepping.
Assuntos
Oligodendroglia/metabolismo , Remielinização/fisiologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Comportamento Animal , Diferenciação Celular , Proliferação de Células , Modelos Animais de Doenças , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Regeneração Nervosa/fisiologia , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS.
Assuntos
Envelhecimento/patologia , Axônios/patologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Regeneração Nervosa , Animais , Mamíferos/crescimento & desenvolvimento , Modelos BiológicosRESUMO
Spinal cord injury can lead to severe motor, sensory and autonomic dysfunction. Currently, there is no effective treatment for the injured spinal cord. The transplantation of Schwann cells, neural stem cells or progenitor cells, olfactory ensheathing cells, oligodendrocyte precursor cells and mesenchymal stem cells has been investigated as potential therapies for spinal cord injury. However, little is known about the mechanisms through which these individual cell types promote repair and functional improvements. The five most commonly proposed mechanisms include neuroprotection, immunomodulation, axon regeneration, neuronal relay formation and myelin regeneration. A better understanding of the mechanisms whereby these cells promote functional improvements, as well as an appreciation of the obstacles in implementing these therapies and effectively modeling spinal cord injury, will be important to make cell transplantation a viable clinical option and may lead to the development of more targeted therapies.
Assuntos
Transplante de Células , Regeneração Nervosa/fisiologia , Células-Tronco Neurais/citologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Transplante de Células/métodos , HumanosRESUMO
Neuroprotection refers to the attenuation of pathophysiological processes triggered by acute injury to minimize secondary damage. The development of neuroprotective treatments represents a major goal in the field of spinal cord injury (SCI) research. In this review, we discuss the strengths and limitations of the methodologies employed to assess secondary damage and neuroprotection in preclinical models of traumatic SCI. We also discuss modelling issues and how new tools might be exploited to study secondary damage and neuroprotection.
Assuntos
Neuroproteção , Fármacos Neuroprotetores/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Potenciais de Ação , Animais , Modelos Animais de Doenças , Neurônios/patologia , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Motor cortical plasticity contributes to spontaneous recovery after incomplete spinal cord injury (SCI), but the pathways underlying this remain poorly understood. We performed optogenetic mapping of motor cortex in channelrhodopsin-2 expressing mice to assess the capacity of the cortex to re-establish motor output longitudinally after a C3/C4 dorsal column SCI that bilaterally ablated the dorsal corticospinal tract (CST) containing â¼96% of corticospinal fibers but spared â¼3% of CST fibers that project via the dorsolateral funiculus. Optogenetic mapping revealed extensive early deficits, but eventual reestablishment of motor cortical output maps to the limbs at the same latency as preoperatively by 4 weeks after injury. Analysis of skilled locomotion on the horizontal ladder revealed early deficits followed by partial spontaneous recovery by 6 weeks after injury. To dissociate between the contributions of injured dorsal projecting versus spared dorsolateral projecting corticospinal neurons, we established a transient silencing approach to inactivate spared dorsolaterally projecting corticospinal neurons specifically by injecting adeno-associated virus (AAV)-expressing Cre-dependent DREADD (designer receptor exclusively activated by designer drug) receptor hM4Di in sensorimotor cortex and AAV-expressing Cre in C7/C8 dorsolateral funiculus. Transient silencing uninjured dorsolaterally projecting corticospinal neurons via activation of the inhibitory DREADD receptor hM4Di abrogated spontaneous recovery and resulted in a greater change in skilled locomotion than in control uninjured mice using the same silencing approach. These data demonstrate the pivotal role of a minor dorsolateral corticospinal pathway in mediating spontaneous recovery after SCI and support a focus on spared corticospinal neurons as a target for therapy. SIGNIFICANCE STATEMENT: Spontaneous recovery can occur after incomplete spinal cord injury (SCI), but the pathways underlying this remain poorly understood. We performed optogenetic mapping of motor cortex after a cervical SCI that interrupts most corticospinal transmission but results in partial recovery on a horizontal ladder task of sensorimotor function. We demonstrate that the motor cortex can reestablish output to the limbs longitudinally. To dissociate the roles of injured and uninjured corticospinal neurons in mediating recovery, we transiently silenced the minor dorsolateral corticospinal pathway spared by our injury. This abrogated spontaneous recovery and resulted in a greater change in skilled locomotion than in uninjured mice using the same approach. Therefore, uninjured corticospinal neurons substantiate remarkable motor cortical plasticity and partial recovery after SCI.
Assuntos
Córtex Motor/patologia , Tratos Piramidais/patologia , Traumatismos da Medula Espinal/patologia , Animais , Mapeamento Encefálico , Vias Eferentes/crescimento & desenvolvimento , Vias Eferentes/patologia , Imuno-Histoquímica , Locomoção , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Optogenética , Recuperação de Função Fisiológica , Córtex Sensório-Motor/patologiaRESUMO
How aging impacts axon regeneration after CNS injury is not known. We assessed the impact of age on axon regeneration induced by Pten deletion in corticospinal and rubrospinal neurons, two neuronal populations with distinct innate regenerative abilities. As in young mice, Pten deletion in older mice remains effective in preventing axotomy-induced decline in neuron-intrinsic growth state, as assessed by mTOR activity, neuronal soma size, and axonal growth proximal to a spinal cord injury. However, axonal regeneration distal to injury is greatly diminished, accompanied by increased expression of astroglial and inflammatory markers at the injury site. Thus, the mammalian CNS undergoes an age-dependent decline in axon regeneration, as revealed when neuron-intrinsic growth state is elevated. These results have important implications for developing strategies to promote axonal repair after CNS injuries or diseases, which increasingly affect middle-aged to aging populations.
Assuntos
Envelhecimento/patologia , Axônios/patologia , Sistema Nervoso Central/patologia , Mamíferos/crescimento & desenvolvimento , Regeneração Nervosa , Animais , Astrócitos/patologia , Axotomia , Biomarcadores/metabolismo , Regulação para Baixo , Deleção de Genes , Inflamação/patologia , Camundongos , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/metabolismo , Tratos Piramidais/patologia , Tratos Piramidais/fisiopatologia , Transdução de Sinais , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Coral reefs are among the most productive and diverse marine ecosystems on the Earth. They are also particularly sensitive to changing energetic requirements by different trophic levels. Microbialization specifically refers to the increase in the energetic metabolic demands of microbes relative to macrobes and is significantly correlated with increasing human influence on coral reefs. In this study, metabolic theory of ecology is used to quantify the relative contributions of two broad bacterioplankton groups, autotrophs and heterotrophs, to energy flux on 27 Pacific coral reef ecosystems experiencing human impact to varying degrees. The effective activation energy required for photosynthesis is lower than the average energy of activation for the biochemical reactions of the Krebs cycle, and changes in the proportional abundance of these two groups can greatly affect rates of energy and materials cycling. We show that reef-water communities with a higher proportional abundance of microbial autotrophs expend more metabolic energy per gram of microbial biomass. Increased energy and materials flux through fast energy channels (i.e. water-column associated microbial autotrophs) may dampen the detrimental effects of increased heterotrophic loads (e.g. coral disease) on coral reef systems experiencing anthropogenic disturbance.
Assuntos
Antozoários/metabolismo , Antozoários/microbiologia , Bactérias/metabolismo , Recifes de Corais , Fitoplâncton/metabolismo , Animais , Biomassa , Ecossistema , Metabolismo Energético , Humanos , Microbiologia da ÁguaRESUMO
Bacterial meningitis is a serious infection of the CNS that results when blood-borne bacteria are able to cross the blood-brain barrier (BBB). Group B Streptococcus (GBS) is the leading cause of neonatal meningitis; however, the molecular mechanisms that regulate bacterial BBB disruption and penetration are not well understood. Here, we found that infection of human brain microvascular endothelial cells (hBMECs) with GBS and other meningeal pathogens results in the induction of host transcriptional repressor Snail1, which impedes expression of tight junction genes. Moreover, GBS infection also induced Snail1 expression in murine and zebrafish models. Tight junction components ZO-1, claudin 5, and occludin were decreased at both the transcript and protein levels in hBMECs following GBS infection, and this repression was dependent on Snail1 induction. Bacteria-independent Snail1 expression was sufficient to facilitate tight junction disruption, promoting BBB permeability to allow bacterial passage. GBS induction of Snail1 expression was dependent on the ERK1/2/MAPK signaling cascade and bacterial cell wall components. Finally, overexpression of a dominant-negative Snail1 homolog in zebrafish elevated transcription of tight junction protein-encoding genes and increased zebrafish survival in response to GBS challenge. Taken together, our data support a Snail1-dependent mechanism of BBB disruption and penetration by meningeal pathogens.
Assuntos
Barreira Hematoencefálica/metabolismo , Sistema de Sinalização das MAP Quinases , Infecções Estreptocócicas/metabolismo , Streptococcus agalactiae , Junções Íntimas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Barreira Hematoencefálica/microbiologia , Barreira Hematoencefálica/patologia , Células Cultivadas , Claudina-5/genética , Claudina-5/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fatores de Transcrição da Família Snail , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/patologia , Junções Íntimas/genética , Junções Íntimas/patologia , Fatores de Transcrição/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis.