RESUMEN
Antibody delivery to the CNS remains a huge hurdle for the clinical application of antibodies targeting a CNS antigen. The blood-brain barrier and blood-CSF barrier restrict access of therapeutic antibodies to their CNS targets in a major way. The very high amounts of therapeutic antibodies that are administered systemically in recent clinical trials to reach CNS targets are barely viable cost-wise for broad, routine applications. Though global CNS delivery of antibodies can be achieved by intrathecal application, these procedures are invasive. A non-invasive method to bring antibodies into the CNS reliably and reproducibly remains an important unmet need in neurology. In the present study, we show that intranasal application of a mouse monoclonal antibody against the neurite growth-inhibiting and plasticity-restricting membrane protein Nogo-A leads to a rapid transfer of significant amounts of antibody to the brain and spinal cord in intact adult rats. Daily intranasal application for 2 wk of anti-Nogo-A antibody enhanced growth and compensatory sprouting of corticofugal projections and functional recovery in rats after large unilateral cortical strokes. These findings are a starting point for clinical translation for a less invasive route of application of therapeutic antibodies to CNS targets for many neurological indications.
Asunto(s)
Anticuerpos Monoclonales , Proteínas de la Mielina , Animales , Ratas , Encéfalo/metabolismo , Proteínas de la Mielina/metabolismo , Proteínas Nogo , Médula Espinal/metabolismo , Anticuerpos Monoclonales/administración & dosificación , Administración IntranasalRESUMEN
Dopaminergic signaling in the striatum, particularly at dopamine 2 receptors (D2R), has been a topic of active investigation in obesity research in the past decades. However, it still remains unclear whether variations in striatal D2Rs modulate the risk for obesity and if so in which direction. Human studies have yielded contradictory findings that likely reflect a complex nonlinear relationship, possibly involving a combination of causal effects and compensatory changes. Animal work indicates that although chronic obesogenic diets reduce striatal D2R function, striatal D2R down-regulation does not lead to obesity. In this study, we evaluated the consequences of striatal D2R up-regulation on body-weight gain susceptibility and energy balance in mice. We used a mouse model of D2R overexpression (D2R-OE) in which D2Rs were selectively up-regulated in striatal medium spiny neurons. We uncover a pathological mechanism by which striatal D2R-OE leads to reduced brown adipose tissue thermogenesis, reduced energy expenditure, and accelerated obesity despite reduced eating. We also show that D2R-OE restricted to development is sufficient to promote obesity and to induce energy-balance deficits. Together, our findings indicate that striatal D2R-OE during development persistently increases the propensity for obesity by reducing energy output in mice. This suggests that early alterations in the striatal dopamine system could represent a key predisposition factor toward obesity.
Asunto(s)
Cuerpo Estriado/metabolismo , Dieta/efectos adversos , Metabolismo Energético , Neostriado/metabolismo , Obesidad/etiología , Receptores de Dopamina D2/fisiología , Animales , Humanos , Masculino , Ratones , Ratones Transgénicos , Obesidad/patología , Aumento de PesoRESUMEN
Loss of bladder control is common after spinal cord injury (SCI) and no causal therapies are available. Here we investigated whether function-blocking antibodies against the nerve-fiber growth inhibitory protein Nogo-A applied to rats with severe SCI could prevent development of neurogenic lower urinary tract dysfunction. Bladder function of rats with SCI was repeatedly assessed by urodynamic examination in fully awake animals. Four weeks after SCI, detrusor sphincter dyssynergia had developed in all untreated or control antibody-infused animals. In contrast, 2 weeks of intrathecal anti-Nogo-A antibody treatment led to significantly reduced aberrant maximum detrusor pressure during voiding and a reduction of the abnormal EMG high-frequency activity in the external urethral sphincter. Anatomically, we found higher densities of fibers originating from the pontine micturition center in the lumbosacral gray matter in the anti-Nogo-A antibody-treated animals, as well as a reduced number of inhibitory interneurons in lamina X. These results suggest that anti-Nogo-A therapy could also have positive effects on bladder function clinically.SIGNIFICANCE STATEMENT After spinal cord injury, loss of bladder control is common. Detrusor sphincter dyssynergia is a potentially life-threatening consequence. Currently, only symptomatic treatment options are available. First causal treatment options are urgently needed in humans. In this work, we show that function-blocking antibodies against the nerve-fiber growth inhibitory protein Nogo-A applied to rats with severe spinal cord injury could prevent development of neurogenic lower urinary tract dysfunction, in particular detrusor sphincter dyssynergia. Anti-Nogo-A therapy has entered phase II clinical trial in humans and might therefore soon be the first causal treatment option for neurogenic lower urinary tract dysfunction.
Asunto(s)
Anticuerpos/farmacología , Proteínas Nogo/antagonistas & inhibidores , Traumatismos de la Médula Espinal/complicaciones , Vejiga Urinaria Neurogénica/etiología , Animales , Femenino , Ratas , Ratas Endogámicas LewRESUMEN
Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) in which multiple sites of blood-brain barrier (BBB) disruption, focal inflammation, demyelination and tissue destruction are the hallmarks. Here we show that sphingosine-1-phosphate receptor 2 (S1PR2) has a negative role in myelin repair as well as an important role in demyelination by modulating BBB permeability. In lysolecithin-induced demyelination of adult mouse spinal cord, S1PR2 inactivation by either the pharmacological inhibitor JTE-013 or S1PR2 gene knockout led to enhanced myelin repair as determined by higher numbers of differentiated oligodendrocytes and increased numbers of remyelinated axons at the lesion sites. S1PR2 inactivation in lysolecithin-induced demyelination of the optic chiasm, enhanced oligodendrogenesis and improved the behavioral outcome in an optokinetic reflex test. In order to see the effect of S1PR2 inactivation on demyelination, experimental autoimmune encephalitis (EAE) was induced by MOG-peptide. S1PR2 inhibition or knockout decreased the extent of demyelinated areas as well as the clinical disability in this EAE model. Both toxin induced and EAE models showed decreased BBB leakage and reduced numbers of Iba1+ macrophages following S1PR2 inactivation. Our results suggest that S1PR2 activity impairs remyelination and also enhances BBB leakage and demyelination. The former effect could be mediated by Nogo-A, as antagonism of this factor enhances remyelination and S1PR2 can act as a Nogo-A receptor.
Asunto(s)
Esclerosis Múltiple/fisiopatología , Remielinización , Receptores de Esfingosina-1-Fosfato/fisiología , Animales , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/fisiología , Esclerosis Múltiple/patología , Vaina de Mielina/ultraestructura , Receptores de Esfingosina-1-Fosfato/genética , Médula Espinal/patología , Médula Espinal/fisiopatologíaRESUMEN
Nogo-A has been well described as a myelin-associated inhibitor of neurite outgrowth and functional neuroregeneration after central nervous system (CNS) injury. Recently, a new role of Nogo-A has been identified as a negative regulator of synaptic plasticity in the uninjured adult CNS. Nogo-A is present in neurons and oligodendrocytes. However, it is yet unclear which of these two pools regulate synaptic plasticity. To address this question we used newly generated mouse lines in which Nogo-A is specifically knocked out in (1) oligodendrocytes (oligoNogo-A KO) or (2) neurons (neuroNogo-A KO). We show that both oligodendrocyte- and neuron-specific Nogo-A KO mice have enhanced dendritic branching and spine densities in layer 2/3 cortical pyramidal neurons. These effects are compartmentalized: neuronal Nogo-A affects proximal dendrites whereas oligodendrocytic Nogo-A affects distal regions. Finally, we used two-photon laser scanning microscopy to measure the spine turnover rate of adult mouse motor cortex layer 5 cells and find that both Nogo-A KO mouse lines show enhanced spine remodeling after 4 days. Our results suggest relevant control functions of glial as well as neuronal Nogo-A for synaptic plasticity and open new possibilities for more selective and targeted plasticity enhancing strategies.
Asunto(s)
Espinas Dendríticas/metabolismo , Corteza Motora/metabolismo , Plasticidad Neuronal/fisiología , Proteínas Nogo/metabolismo , Oligodendroglía/metabolismo , Animales , Ratones , Ratones Noqueados , Neuronas/metabolismoRESUMEN
AIMS: The search for novel drugs that enhance myelin repair in entities such as multiple sclerosis has top priority in neurological research, not least because remyelination can hinder further neurodegeneration in neuro-inflammatory conditions. Recently, several new compounds with the potential to boost remyelination have been identified using high-throughput in vitro screening methods. However, assessing their potential to enhance remyelination in vivo using plastic embedded semi-thin sections or electron microscopy, even though being the gold standard for assessing remyelination, is toxic, extremely time-consuming and expensive. METHODS: We screened available myelin dyes for a staining candidate which offers a faster and easier alternative to visualize remyelination in cryo-sections. RESULTS: We identified sudan black as a candidate with excellent myelin resolution and we show that our adapted sudan black staining can demonstrate myelin repair in rodent spinal cord cryosections as reliable as in semithin sections, but much faster, easier, less toxic and less expensive. Besides that, it can resolve the small myelinated axons in the corpus callosum. The staining can yet readily be combined with immunostainings which can be challenging in semithin sections. We validated the method in human spinal cord tissue as well as in experimental demyelination of the rat spinal cord by a lysolecithin time course experiment. As proof-of-principle, we demonstrate that sudan black is able to reliably detect the remyelination enhancing properties of benztropine. CONCLUSION: Our adapted sudan black staining can be used to rapidly and non-toxically screen for remyelinating therapies in demyelinating diseases.
Asunto(s)
Compuestos Azo , Naftalenos , Remielinización , Médula Espinal/patología , Coloración y Etiquetado/métodos , Animales , Enfermedades Desmielinizantes/patología , Femenino , Humanos , Ratas , Ratas Long-EvansRESUMEN
Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G13, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A(-/-) mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity.
Asunto(s)
Hipocampo/metabolismo , Corteza Motora/metabolismo , Proteínas de la Mielina/genética , Plasticidad Neuronal/genética , Receptores de Lisoesfingolípidos/genética , Animales , Proliferación Celular , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Regulación de la Expresión Génica , Hipocampo/citología , Potenciación a Largo Plazo , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Corteza Motora/citología , Proteínas de la Mielina/deficiencia , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Neuritas/metabolismo , Proteínas Nogo , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Sinapsis/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
We have generated a transgenic rat model using RNAi and used it to study the role of the membrane protein Nogo-A in synaptic plasticity and cognition. The membrane protein Nogo-A is expressed in CNS oligodendrocytes and subpopulations of neurons, and it is known to suppress neurite growth and regeneration. The constitutively expressed polymerase II-driven transgene was composed of a microRNA-targeting Nogo-A placed into an intron preceding the coding sequence for EGFP, thus quantitatively labeling cells according to intracellular microRNA expression. The transgenic microRNA in vivo efficiently reduced the concentration of Nogo-A mRNA and protein preferentially in neurons. The resulting significant increase in long-term potentiation in both hippocampus and motor cortex indicates a repressor function of Nogo-A in synaptic plasticity. The transgenic rats exhibited prominent schizophrenia-like behavioral phenotypes, such as perseveration, disrupted prepulse inhibition, and strong withdrawal from social interactions. This fast and efficient microRNA-mediated knockdown provides a way to silence gene expression in vivo in transgenic rats and shows a role of Nogo-A in regulating higher cognitive brain functions.
Asunto(s)
Cognición/fisiología , Regulación de la Expresión Génica/fisiología , MicroARNs/farmacología , Proteínas de la Mielina/metabolismo , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Potenciación a Largo Plazo/fisiología , Proteínas Nogo , Interferencia de ARN , Ratas , Ratas Transgénicas , Transgenes/genéticaRESUMEN
Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A-specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti-Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.
Asunto(s)
Encéfalo/irrigación sanguínea , Encéfalo/crecimiento & desarrollo , Células Endoteliales/metabolismo , Proteínas de la Mielina/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Encéfalo/citología , Células Cultivadas , Circulación Cerebrovascular/fisiología , Células Endoteliales/citología , Ratones , Ratones Noqueados , Proteínas de la Mielina/genética , Proteínas NogoRESUMEN
Neuronal signal integration as well as synaptic transmission and plasticity highly depend on the morphology of dendrites and their spines. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. Nogo-A is also expressed by certain neurons, in particular during development, but its physiological function in this cell type is less well understood. We addressed this question in the cerebellum, where Nogo-A is transitorily highly expressed in the Purkinje cells (PCs) during early postnatal development. We used general genetic ablation (KO) as well as selective overexpression of Nogo-A in PCs to analyze its effect on dendritogenesis and on the formation of their main input synapses from parallel (PFs) and climbing fibers (CFs). PC dendritic trees were larger and more complex in Nogo-A KO mice and smaller than in wild-type in Nogo-A overexpressing PCs. Nogo-A KO resulted in premature soma-to-dendrite translocation of CFs and an enlargement of the CF territory in the molecular layer during development. Although spine density was not influenced by Nogo-A, the size of postsynaptic densities of PF-PC synapses was negatively correlated with the Nogo-A expression level. Electrophysiological studies revealed that Nogo-A negatively regulates the strength of synaptic transmission at the PF-PC synapse. Thus, Nogo-A appears as a negative regulator of PC input synapses, which orchestrates cerebellar connectivity through regulation of synapse morphology and the size of the PC dendritic tree.
Asunto(s)
Cerebelo/fisiología , Proteínas de la Mielina/fisiología , Animales , Cerebelo/citología , Dendritas/fisiología , Dendritas/ultraestructura , Fenómenos Electrofisiológicos , Proteínas Ligadas a GPI/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de la Mielina/deficiencia , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Neuronas/fisiología , Neuronas/ultraestructura , Proteínas Nogo , Receptor Nogo 1 , Células de Purkinje/fisiología , Células de Purkinje/ultraestructura , Receptores de Superficie Celular/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Anatomically incomplete spinal cord injuries are often followed by considerable functional recovery in patients and animal models, largely because of processes of neuronal plasticity. In contrast to the corticospinal system, where sprouting of fibers and rearrangements of circuits in response to lesions have been well studied, structural adaptations within descending brainstem pathways and intraspinal networks are poorly investigated, despite the recognized physiological significance of these systems across species. In the present study, spontaneous neuroanatomical plasticity of severed bulbospinal systems and propriospinal neurons was investigated following unilateral C4 spinal hemisection in adult rats. Injection of retrograde tracer into the ipsilesional segments C3-C4 revealed a specific increase in the projection from the ipsilesional gigantocellular reticular nucleus in response to the injury. Substantial regenerative fiber sprouting of reticulospinal axons above the injury site was demonstrated by anterograde tracing. Regrowing reticulospinal fibers exhibited excitatory, vGLUT2-positive varicosities, indicating their synaptic integration into spinal networks. Reticulospinal fibers formed close appositions onto descending, double-midline crossing C3-C4 propriospinal neurons, which crossed the lesion site in the intact half of the spinal cord and recrossed to the denervated cervical hemicord below the injury. These propriospinal projections around the lesion were significantly enhanced after injury. Our results suggest that severed reticulospinal fibers, which are part of the phylogenetically oldest motor command system, spontaneously arborize and form contacts onto a plastic propriospinal relay, thereby bypassing the lesion. These rearrangements were accompanied by substantial locomotor recovery, implying a potential physiological relevance of the detour in restoration of motor function after spinal injury.
Asunto(s)
Bulbo Raquídeo/fisiología , Vías Nerviosas/fisiología , Plasticidad Neuronal/fisiología , Neuronas/patología , Formación Reticular/patología , Traumatismos de la Médula Espinal/patología , Animales , Axones , Recuento de Células , Modelos Animales de Enfermedad , Femenino , Lateralidad Funcional/fisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Actividad Motora/fisiología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Ratas , Ratas Endogámicas Lew , Recuperación de la Función , Formación Reticular/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The membrane protein Nogo-A is known as an inhibitor of axonal outgrowth and regeneration in the CNS. However, its physiological functions in the normal adult CNS remain incompletely understood. Here, we investigated the role of Nogo-A in cortical synaptic plasticity and motor learning in the uninjured adult rodent motor cortex. Nogo-A and its receptor NgR1 are present at cortical synapses. Acute treatment of slices with function-blocking antibodies (Abs) against Nogo-A or against NgR1 increased long-term potentiation (LTP) induced by stimulation of layer 2/3 horizontal fibers. Furthermore, anti-Nogo-A Ab treatment increased LTP saturation levels, whereas long-term depression remained unchanged, thus leading to an enlarged synaptic modification range. In vivo, intrathecal application of Nogo-A-blocking Abs resulted in a higher dendritic spine density at cortical pyramidal neurons due to an increase in spine formation as revealed by in vivo two-photon microscopy. To investigate whether these changes in synaptic plasticity correlate with motor learning, we trained rats to learn a skilled forelimb-reaching task while receiving anti-Nogo-A Abs. Learning of this cortically controlled precision movement was improved upon anti-Nogo-A Ab treatment. Our results identify Nogo-A as an influential molecular modulator of synaptic plasticity and as a regulator for learning of skilled movements in the motor cortex.
Asunto(s)
Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Corteza Motora/fisiología , Destreza Motora/fisiología , Proteínas de la Mielina/metabolismo , Animales , Masculino , Corteza Motora/metabolismo , Proteínas de la Mielina/genética , Proteínas Nogo , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismo , Sinapsis/fisiologíaRESUMEN
Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo-A/Nogo receptor (NgR1) signaling pathway. Nogo-A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody-mediated neutralization of Nogo-A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo-A KO vs. wild-type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4-positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth-inhibitory than WT but more than Nogo-A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo-A KO myelin as compared with WT neurons, supporting increased EphA4-mediated growth inhibition in Nogo-A KO mice. Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo-A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo-A.
Asunto(s)
Axones/fisiología , Corteza Cerebral/metabolismo , Ganglios Espinales/metabolismo , Proteínas de la Mielina/metabolismo , Regeneración de la Medula Espinal , Regulación hacia Arriba , Animales , Axones/metabolismo , Células Cultivadas , Corteza Cerebral/patología , Corteza Cerebral/fisiología , Efrina-A3/genética , Efrina-A3/metabolismo , Efrina-A4/genética , Efrina-A4/metabolismo , Ganglios Espinales/patología , Ganglios Espinales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina/genética , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Proteínas Nogo , Tractos Piramidales/metabolismo , Tractos Piramidales/patología , Tractos Piramidales/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Traumatismos de la Médula Espinal/metabolismoRESUMEN
Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.
RESUMEN
Cervical incomplete spinal cord injuries often lead to severe and persistent impairments of sensorimotor functions and are clinically the most frequent type of spinal cord injury. Understanding the motor impairments and the possible functional recovery of upper and lower extremities is of great importance. Animal models investigating motor dysfunction following cervical spinal cord injury are rare. We analysed the differential spontaneous recovery of fore- and hindlimb locomotion by detailed kinematic analysis in adult rats with unilateral C4/C5 hemisection, a lesion that leads to the Brown-Séquard syndrome in humans. The results showed disproportionately better performance of hindlimb compared with forelimb locomotion; hindlimb locomotion showed substantial recovery, whereas the ipsilesional forelimb remained in a very poor functional state. Such a differential motor recovery pattern is also known to occur in monkeys and in humans after similar spinal cord lesions. On the lesioned side, cortico-, rubro-, vestibulo- and reticulospinal tracts and the important modulatory serotonergic, dopaminergic and noradrenergic fibre systems were interrupted by the lesion. In an attempt to facilitate locomotion, different monoaminergic agonists were injected intrathecally. Injections of specific serotonergic and noradrenergic agonists in the chronic phase after the spinal cord lesion revealed remarkable, although mostly functionally negative, modulations of particular parameters of hindlimb locomotion. In contrast, forelimb locomotion was mostly unresponsive to these agonists. These results, therefore, show fundamental differences between fore- and hindlimb spinal motor circuitries and their functional dependence on remaining descending inputs and exogenous spinal excitation. Understanding these differences may help to develop future therapeutic strategies to improve upper and lower limb function in patients with incomplete cervical spinal cord injuries.
Asunto(s)
Síndrome de Brown-Séquard/fisiopatología , Lateralidad Funcional/fisiología , Trastornos del Movimiento/etiología , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/complicaciones , 8-Hidroxi-2-(di-n-propilamino)tetralin/uso terapéutico , Animales , Apomorfina/uso terapéutico , Clonidina/uso terapéutico , Modelos Animales de Enfermedad , Agonistas de Dopamina/uso terapéutico , Interacciones Farmacológicas , Femenino , Metoxamina/uso terapéutico , Actividad Motora/efectos de los fármacos , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Trastornos del Movimiento/tratamiento farmacológico , Quipazina/uso terapéutico , Ratas , Ratas Endogámicas Lew , Recuperación de la Función/efectos de los fármacos , Serotonina/metabolismo , Agonistas de Receptores de Serotonina/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Simpaticolíticos/uso terapéutico , Simpatomiméticos/uso terapéutico , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The membrane protein Nogo-A, which is predominantly expressed by oligodendrocytes in the adult CNS and by neurons mainly during development, is well known for limiting neurite outgrowth and regeneration in the injured mammalian CNS. In addition, it has recently been proposed that abnormal Nogo-A expression or Nogo receptor (NgR) mutations may confer genetic risks for neuropsychiatric disorders of presumed neurodevelopmental origin, such as schizophrenia. We therefore evaluated whether Nogo-A deletion may lead to schizophrenia-like abnormalities in a mouse model of genetic Nogo-A deficiency. Here, we show that systemic, lifelong knock-out of the Nogo-A gene can lead to specific behavioral abnormalities resembling schizophrenia-related endophenotypes: deficient sensorimotor gating, disrupted latent inhibition, perseverative behavior, and increased sensitivity to the locomotor stimulating effects of amphetamine. These behavioral phenotypes were accompanied by altered monoaminergic transmitter levels in specific striatal and limbic structures, as well as changes in dopamine D2 receptor expression in the same brain regions. Nogo-A deletion was further associated with elevated expression of growth-related markers. In contrast, acute antibody-mediated Nogo-A neutralization in adult wild-type mice failed to produce such phenotypes, suggesting that the phenotypes observed in the knock-out mice might be of developmental origin, and that Nogo-A normally subserves critical functions in neurodevelopment. This study provides the first experimental demonstration that Nogo-A bears neuropsychiatric relevance, and alterations in its expression may be one etiological factor in schizophrenia and related disorders.
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Proteínas de la Mielina/deficiencia , Proteínas de la Mielina/fisiología , Fenotipo , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Eliminación de Secuencia/genética , Estimulación Acústica/efectos adversos , Anfetamina/farmacología , Análisis de Varianza , Animales , Anticuerpos/farmacología , Encéfalo/metabolismo , Encéfalo/patología , Estimulantes del Sistema Nervioso Central/farmacología , Cromatografía Líquida de Alta Presión/métodos , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Inhibición Psicológica , Relaciones Interpersonales , Aprendizaje/fisiología , Locomoción/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina/inmunología , Neurotransmisores/metabolismo , Proteínas Nogo , Corteza Prefrontal/metabolismo , Receptores de Dopamina D2/metabolismo , Esquizofrenia/patología , Filtrado Sensorial/genética , Filtrado Sensorial/fisiologíaRESUMEN
The adult, mature central nervous system (CNS) has limited plasticity. Physical exercising can counteract this limitation by inducing plasticity and fostering processes such as learning, memory consolidation and formation. Little is known about the molecular factors that govern these mechanisms, and how they are connected with exercise. In this study, we used immunohistochemical and behavioral analyses to investigate how running wheel exercise affects expression of the neuronal plasticity-inhibiting protein Nogo-A in the rat cortex, and how it influences motor learning in vivo. Following one week of exercise, rats exhibited a decrease in Nogo-A levels, selectively in motor cortex layer 2/3, but not in layer 5. Nogo-A protein levels returned to baseline after two weeks of running wheel exercise. In a skilled motor task (forelimb-reaching), administration of Nogo-A function-blocking antibodies over the course of the first training week led to improved motor learning. By contrast, Nogo-A antibody application over two weeks of training resulted in impaired learning. Our findings imply a bimodal, time-dependent function of Nogo-A in exercise-induced neuronal plasticity: While an activity-induced suppression of the plasticity-inhibiting protein Nogo-A appears initially beneficial for enhanced motor learning, presumably by allowing greater plasticity in establishing novel synaptic connections, this process is not sustained throughout continued exercise. Instead, upregulation of Nogo-A over the course of the second week of running wheel exercise in rats implies that Nogo-A is required for consolidation of acquired motor skills during the delayed memory consolidation process, possibly by inhibiting ongoing neuronal morphological reorganization to stabilize established synaptic pathways. Our findings suggest that Nogo-A downregulation allows leaning to occur, i.e. opens a 'learning window', while its later upregulation stabilizes the learnt engrams. These findings underline the importance of appropriately timing of application of Nogo-A antibodies in future clinical trials that aim to foster memory performance while avoiding adverse effects.
Asunto(s)
Aprendizaje , Actividad Motora/fisiología , Proteínas Nogo/metabolismo , Condicionamiento Físico Animal , Animales , Ratas , Factores de TiempoRESUMEN
Smaller spinal cord injuries often allow some degree of spontaneous behavioral improvements because of structural rearrangements within different descending fiber tracts or intraspinal circuits. In this study, we investigate whether rehabilitative training of the forelimb (forced limb use) influences behavioral recovery and plastic events after injury to a defined spinal tract, the corticospinal tract (CST). Female adult Lewis rats received a unilateral CST injury at the brainstem level. Use of the contralateral impaired forelimb was either restricted, by a cast, or forced, by casting the unimpaired forelimb immediately after injury for either 1 or 3 weeks. Forced use of the impaired forelimb was followed by full behavioral recovery on the irregular horizontal ladder, whereas animals that could not use their affected side remained impaired. BDA (biotinylated dextran amine) labeling of the intact CST showed lesion-induced growth across the midline where CST collaterals increased their innervation density and extended fibers toward the ventral and the dorsal horn in response to forced limb use. Gene chip analysis of the denervated ventral horn revealed changes in particular for growth factors, adhesion and guidance molecules, as well as components of synapse formation suggesting an important role for these factors in activity-dependent intraspinal reorganization after unilateral CST injury.
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Lesiones Encefálicas/terapia , Terapia por Ejercicio/métodos , Regeneración Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Tractos Piramidales/lesiones , Recuperación de la Función/fisiología , Animales , Biotina/análogos & derivados , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Mapeo Encefálico , Desnervación , Dextranos , Femenino , Miembro Anterior/inervación , Miembro Anterior/fisiopatología , Lateralidad Funcional/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Movimiento/fisiología , Tractos Piramidales/fisiopatología , Ratas , Ratas Endogámicas Lew , Restricción Física , Médula Espinal/citología , Médula Espinal/fisiología , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Myelin is a multilamellar membrane structure primarily composed of lipids and myelin proteins essential for proper neuronal function. Since myelin is a target structure involved in many pathophysiological conditions such as metabolic, viral, and autoimmune diseases and genetic myelin disorders, a reliable myelin detection technique is required that is equally suitable for light- and electron-microscopic analysis. Here, we report that single myelinated fibers are specifically stained by the gold phosphate complex, Black gold, which stains myelin in the brain, spinal cord, and peripheral nerve fibers in a reliable manner. Electron-microscopic and morphometric analyses have revealed that gold particles are equally distributed in the inner, compact, and outer myelin layers. In contrast to Luxol fast blue, the gold dye stains proteinase-sensitive myelin structures, indicating its selective labeling of myelin-specific proteins. Aiming at defining the target of gold staining, we performed staining in several mouse myelin mutants. Gold complex distribution and myelin staining in MBP(-/-)/shiverer mouse mutants was comparable with that seen in wild-type mice but revealed a more clustered Black gold distribution. This gold staining method thus provides a sensitive and specific high-resolution marker for both central and peripheral myelin sheaths; it also allows the quantitative analysis of myelinated fibers at the light- and electron-microscopic level suitable for investigations of myelin and axonal disorders.
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Sistema Nervioso Central/metabolismo , Inmunohistoquímica/métodos , Vaina de Mielina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso Periférico/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Transcripción/metabolismo , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/ultraestructura , Oro/química , Masculino , Ratones , Ratones Mutantes , Microscopía Electrónica de Transmisión , Proteína Básica de Mielina , Vaina de Mielina/ultraestructura , Glicoproteína Asociada a Mielina , Proteínas del Tejido Nervioso/genética , Sistema Nervioso Periférico/citología , Sistema Nervioso Periférico/ultraestructura , Ratas , Receptores de Superficie Celular/genética , Factores de Transcripción/genéticaRESUMEN
Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.