RESUMO
Microglia continuously remodel synapses, which are embedded in the extracellular matrix (ECM). However, the mechanisms, which govern this process remain elusive. To investigate the influence of the neural ECM in synaptic remodeling by microglia, we disrupted ECM integrity by injection of chondroitinase ABC (ChABC) into the retrosplenial cortex of healthy adult mice. Using in vivo two-photon microscopy we found that ChABC treatment increased microglial branching complexity and ECM phagocytic capacity and decreased spine elimination rate under basal conditions. Moreover, ECM attenuation largely prevented synaptic remodeling following synaptic stress induced by photodamage of single synaptic elements. These changes were associated with less stable and smaller microglial contacts at the synaptic damage sites, diminished deposition of calreticulin and complement proteins C1q and C3 at synapses and impaired expression of microglial CR3 receptor. Thus, our findings provide novel insights into the function of the neural ECM in deposition of complement proteins and synaptic remodeling by microglia.
Assuntos
Condroitina ABC Liase , Complemento C1q , Matriz Extracelular , Camundongos Endogâmicos C57BL , Microglia , Sinapses , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Complemento C1q/metabolismo , Condroitina ABC Liase/farmacologia , Camundongos , Plasticidade Neuronal/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Complemento C3/metabolismo , Calreticulina/metabolismo , Masculino , Fagocitose/fisiologia , Fagocitose/efeitos dos fármacos , Camundongos Transgênicos , Antígeno de Macrófago 1/metabolismoRESUMO
Cervical level spinal cord injury (SCI) can severely impact upper limb muscle function, which is typically assessed in the clinic using electromyography (EMG). Here, we established novel preclinical methodology for EMG assessments of muscle function after SCI in awake freely moving animals. Adult female rats were implanted with EMG recording electrodes in bicep muscles and received bilateral cervical (C7) contusion injuries. Forelimb muscle activity was assessed by recording maximum voluntary contractions during a grip strength task and cortical motor evoked potentials in the biceps. We demonstrate that longitudinal recordings of muscle activity in the same animal are feasible over a chronic post-injury time course and provide a sensitive method for revealing post-injury changes in muscle activity. This methodology was utilized to investigate recovery of muscle function after a novel combination therapy. Cervical contused animals received intraspinal injections of a neuroplasticity-promoting agent (lentiviral-chondroitinase ABC) plus 11 weeks of cortical epidural electrical stimulation (3 h daily, 5 days/week) and behavioral rehabilitation (15 min daily, 5 days/week). Longitudinal monitoring of voluntary and evoked muscle activity revealed significantly increased muscle activity and upper limb dexterity with the combination treatment, compared to a single treatment or no treatment. Retrograde mapping of motor neurons innervating the biceps showed a predominant distribution across spinal segments C5-C8, indicating that treatment effects were likely due to neuroplastic changes in a mixture of intact and injured motor neurons. Thus, longitudinal assessments of muscle function after SCI correlate with skilled reach and grasp performance and reveal functional benefits of a novel combination therapy.
Assuntos
Condroitina ABC Liase , Traumatismos da Medula Espinal , Animais , Condroitina ABC Liase/farmacologia , Feminino , Membro Anterior/inervação , Membro Anterior/fisiologia , Músculo Esquelético , Ratos , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Extremidade SuperiorRESUMO
INTRODUCTION: Chondroitinase ABC (chABC) is an enzyme could improve regeneration and thereby improving functional recovery of spinal cord injury (SCI) in rodent models. Degradation of the active enzyme and diffusion away from the lesion are the causes of using hydrogels as a scaffold to deliver the chABC into the lesion site. In this meta-analysis, we investigated the effects of chABC embedded in a scaffold or hydrogel on the functional recovery after SCI. METHOD: Databases were searched based on keywords related to chABC and spinal cord injury (SCI). Primary and secondary screening was performed to narrow down study objectives and inclusion criteria, and finally the data were included in the meta-analysis. The standard mean difference of the score of the functional recovery that measured by Basso, Beattie, Bresnahan (BBB) test after SCI was used to analyze the results of the reported studies. Subgroup analysis was performed based on SCI model, severity of SCI, transplantation type, and the follow-up time. Quality control of articles was also specified. RESULTS: The results showed that embedding chABC within the scaffold increased significantly the efficiency of functional recovery after SCI in animal models (SMD = 1.95; 95% CI 0.71-3.2; p = 0.002) in 9 studies. SCI model, severity of SCI, injury location, transplantation type, and the follow-up time did not affect the overall results and in all cases scaffold effect could not be ignored. However, due to the small number of studies, this result is not conclusive and more studies are needed. CONCLUSION: The results could pave the way for the use of chABC embedded in the scaffold for the treatment of SCI and show that this method of administration is superior to chABC injection alone.
Assuntos
Condroitina ABC Liase , Traumatismos da Medula Espinal , Ratos , Animais , Condroitina ABC Liase/farmacologia , Ratos Sprague-Dawley , Recuperação de Função FisiológicaRESUMO
Purpose: Migration and integration remain critical challenges for stem cell replacement therapy. Glial barriers play an important role in preventing cell migration and integration. The purpose of this study was to investigate the effect and mechanisms of chondroitinase ABC on the migration of murine retinal progenitor cells (mRPCs) transplanted into the subretinal space of B6 mice. Methods: mRPCs were harvested from the neural retinas of P1 enhanced green fluorescent protein (GFP) B6 mice. Two µl containing 2 × 105 expanded RPCs alone or combined with chondroitinase ABC in suspension were injected into the subretinal space of the recipient B6 mice. Immunohistochemistry was performed on the recipient B6 retinas to evaluate the glial barrier formation and migration of the mRPCs. Western blotting was also used to check the expression of the glial barriers. Results: Glial fibrillary acidic protein (GFAP) and vimentin could be seen around the transplanted mRPCs in the B6 mice. Formation of glial barriers prevented the migration of donor cells into the retinal layers. Chondroitinase ABC promoted the migration and survival rates of the engrafted retinal progenitor cells in the retinal layers of recipient B6 mice. Injection induced upregulation of GFAP, chondroitin, and CD44 expression. Chondroitinase ABC disrupted the glial barriers. The CD44 around the mRPCs was much lower in the chondroitinase group. However, the CD44 in the retinal layers was considerably higher in the chondroitinase group. With the employment of chondroitinase ABC, more cells migrated into the outer nuclear layer or inner nuclear layer. The chondroitin and CD44 expression decreased 3 weeks after transplantation in the chondroitinase ABC group. Conclusions: Chondroitinase ABC degraded glial barriers and enhanced the migration of transplanted mouse retinal progenitor cells. Chondroitinase ABC may also have induced activation of the CD44 signaling pathway to exert the effect.
Assuntos
Movimento Celular/efeitos dos fármacos , Neuroglia/metabolismo , Retina/citologia , Células-Tronco/citologia , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Condroitina ABC Liase/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco , Vimentina/metabolismoRESUMO
The perineuronal net (PNN) is a specialized type of extracellular matrix found in the central nervous system. The PNN forms on fast spiking neurons during postnatal development but the ontogeny of PNN development has yet to be elucidated. By studying the development and prevalence of the PNN in the juvenile and adult rat brain, we may be able to understand the PNN's role in development and learning and memory. We show that the PNN is fully developed in the deep cerebellar nuclei (DCN) of rats by P18. By using enzymatic digestion of the PNN with chondroitinase ABC (ChABC), we are able to study how digestion of the PNN affects cerebellar-dependent eyeblink conditioning in vivo and perform electrophysiological recordings from DCN neurons in vitro. In vivo degradation of the PNN resulted in significant differences in eyeblink conditioning amplitude and area. Female animals in the vehicle group demonstrated higher levels of conditioning as well as significantly higher post-probe conditioned responses compared to males in that group, differences not present in the ChABC group. In vitro, we found that DCN neurons with a disrupted PNN following exposure to ChABC had altered membrane properties, fewer rebound spikes, and decreased intrinsic excitability. Together, this study further elucidates the role of the PNN in cerebellar learning in the DCN and is the first to demonstrate PNN degradation may erase sex differences in delay conditioning.
Assuntos
Cerebelo/fisiologia , Condicionamento Clássico/fisiologia , Neurônios/fisiologia , Animais , Piscadela/fisiologia , Núcleos Cerebelares/efeitos dos fármacos , Núcleos Cerebelares/fisiologia , Cerebelo/anatomia & histologia , Cerebelo/efeitos dos fármacos , Condroitina ABC Liase/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Eletrofisiologia , Feminino , Masculino , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Long-Evans , Fatores SexuaisRESUMO
Perineuronal nets (PNNs) are specialized extracellular matrix structures that surround subsets of neurons throughout the central nervous system (CNS). They are made up of chondroitin sulfate proteoglycans (CSPGs), hyaluronan, tenascin-R, and many other link proteins that together make up their rigid and lattice-like structure. Modulation of PNNs can alter synaptic plasticity and thereby affect learning, memory, and cognition. In the present study, we degraded PNNs in the medial prefrontal (mPFC) and posterior parietal (PPC) cortices of Long-Evans rats using the enzyme chondroitinase ABC (ChABC), which cleaves apart CSPGs. We then measured the consequences of PNN degradation on spatial working memory (WM) with a trial-unique, non-matching-to location (TUNL) automated touchscreen task. All rats were trained with a standard 6 sec delay and 20 sec inter-trial interval (ITI) and then tested under four different conditions: a 6 sec delay, a variable 2 or 6 sec delay, a 2 sec delay with a 1 sec ITI (interference condition), and a 20 sec delay. Rats that received mPFC ChABC treatment initially performed TUNL with higher accuracy, more selection trials completed, and fewer correction trials completed compared to controls in the 20 sec delay condition but did not perform differently from controls in any other condition. Rats that received PPC ChABC treatment did not perform significantly differently from controls in any condition. Posthumous immunohistochemistry confirmed an increase in CSPG degradation products (C4S stain) in the mPFC and PPC following ChABC infusions while WFA staining intensity and parvalbumin positive neuron number were decreased following mPFC, but not PPC, ChABC infusions. These findings suggest that PNNs in the mPFC play a subtle role in spatial WM, but PNNs in the PPC do not. Furthermore, it appears that PNNs in the mPFC are involved in adapting to a challenging novel delay, but that they do not play an essential role in spatial WM function.
Assuntos
Condroitina ABC Liase/farmacologia , Proteoglicanas de Sulfatos de Condroitina/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Memória de Curto Prazo/efeitos dos fármacos , Lobo Parietal/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Masculino , Ratos , Ratos Long-Evans , Fatores de TempoRESUMO
Genes and environmental conditions are thought to interact in the development of postnatal brain in schizophrenia (SZ). Genome wide association studies have identified that PPARGC1A being one of the top candidate genes for SZ. We previously reported GABAergic neuron-specific PGC-1α knockout mice (Dlx5/6-Cre:PGC-1αfl/fl) presented some characteristic features of SZ. However, there is a fundamental gap of the molecular mechanism by which PGC-1α gene involved in the developmental trajectory to SZ. To explore whether PGC-1α regulates environmental factors interacting with genetic susceptibility to trigger symptom onset and disease progression, PGC-1α deficient mice were utilized to model genetic effect and an additional oxidative stress was induced by GBR injection. We confirm that PGC-1α gene deletion prolongs critical period (CP) timing, as revealed by delaying maturation of PV interneurons (PVIs), including their perineuronal nets (PNNs). Further, we confirm that gene × environment (G × E) influences CP plasticity synergistically and the interaction varies as a function of age, with the most sensitive period being at preweaning stage, and the least sensitive one at early adult age in PGC-1α deficient mice. Along this line, we find that the synergic action of G × E is available in ChABC-infusion PGC-1α KO mice, even though during the adulthood, and the neuroplasticity seems to remain open to fluctuate. Altogether, these results refine the observations made in the PGC-1α deficient mice, a potential mouse model of SZ, and illustrate how PGC-1α regulates CP plasticity via G × E interaction in the developmental trajectory to SZ.
Assuntos
Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Parvalbuminas/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Esquizofrenia/metabolismo , Animais , Condroitina ABC Liase/farmacologia , Interação Gene-Ambiente , Giro do Cíngulo/citologia , Giro do Cíngulo/diagnóstico por imagem , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Estresse Oxidativo/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/deficiência , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Puberdade/metabolismo , Esquizofrenia/diagnóstico por imagem , Esquizofrenia/genética , Esquizofrenia/fisiopatologia , DesmameRESUMO
Selective chemonucleolytic effects of condoliase, a glycosaminoglycan degrading enzyme, was investigated histopathologically in cynomolgus monkeys. Condoliase was administered once into the lumber intervertebral disc (IVD), and as a comparative control, chymopapain, a proteolytic enzyme, was administered in a similar manner. Histopathological changes of the IVD and the adjacent vertebral body (VB) were examined at 1 to 26 weeks after administration. Major changes induced by condoliase in the IVD were degenerative and necrotic changes in the nucleus pulposus, annulus fibrosus, cartilaginous endplate (CEP), and epiphyseal growth plate (EGP); focal disappearance of the EGP; and neovascularization and ossification of the CEP. Decreased/necrosis of bone marrow cells with new bone formation was observed in the VB. Cellular regeneration in the IVD was observed as a recovery changes on and after week 4. The changes in the IVD and VB subsided at week 26. Chymopapain induced qualitatively similar but more widely extended changes. The degrees of the changes in the IVD and VB were more severe than those of condoliase, and the changes were exacerbated even at week 26. These results indicated that histopathological changes caused by condoliase were less severe and more selective than those by chymopapain.
Assuntos
Condroitina ABC Liase/farmacologia , Quimiólise do Disco Intervertebral , Disco Intervertebral/efeitos dos fármacos , Animais , Quimopapaína , Macaca fascicularisRESUMO
BACKGROUND: Parkinson's disease (PD) is characterised by dopaminergic cell loss within the substantia nigra pars compacta (SNc) that leads to reduced striatal dopamine content and resulting motor deficits. Identifying new strategies to protect these cells from degeneration and retain striatal dopaminergic innervation is therefore of great importance. Chondroitin sulphate proteoglycans (CSPGs) are recognised contributors to the inhibitory extracellular milieu known to hinder tissue recovery following CNS damage. Digestion of these molecules by the bacterial lyase chondroitinase ABC (ChABC) has been shown to promote functional recovery in animal models of neurological injury. Although ChABC has been shown to promote sprouting of dopaminergic axons following transection of the nigrostriatal pathway, its ability to protect against nigrostriatal degeneration in a toxin-based module with better construct validity for PD has yet to be explored. Here we examined the neuroprotective efficacy of ChABC treatment in the full and partial 6-hydroxydopamine (6-OHDA) lesion mouse models of PD. RESULTS: In mice bearing a full 6-OHDA lesion, ChABC treatment failed to protect against the loss of either nigral cells or striatal terminals. In contrast, in mice bearing a partial 6-OHDA lesion, ChABC treatment significantly protected cells of the rostral SNc, which remained at more than double the numbers seen in vehicle-treated animals. In the partial lesion model, ChABC treatment also significantly preserved dopaminergic fibres of the rostral dorsal striatum which increased from 15.3 ± 3.5% of the intact hemisphere in saline-treated animals to 36.3 ± 6.5% in the ChABC-treated group. These protective effects of ChABC treatment were not accompanied by improvements in either the cylinder or amphetamine-induced rotations tests of motor function. CONCLUSIONS: ChABC treatment provided significant protection against a partial 6-OHDA lesion of the nigrostriatal tract although the degree of protection was not sufficient to improve motor outcomes. These results support further investigations into the benefits of ChABC treatment for providing neuroprotection in PD.
Assuntos
Condroitina ABC Liase/farmacologia , Corpo Estriado/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos/tratamento farmacológico , Substância Negra/efeitos dos fármacos , Animais , Antiparkinsonianos/farmacologia , Morte Celular/efeitos dos fármacos , Corpo Estriado/patologia , Neurônios Dopaminérgicos/patologia , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Oxidopamina , Transtornos Parkinsonianos/patologia , Substância Negra/patologiaRESUMO
Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1+ innervation in spinal cord grey matter, particularly in lamina III-IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury.
Assuntos
Condroitina ABC Liase/administração & dosagem , Terapia Genética/métodos , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Condroitina ABC Liase/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Regeneração Nervosa/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Mutantes , Recuperação de Função Fisiológica/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Transgenes/genéticaRESUMO
After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI.
Assuntos
Axônios/efeitos dos fármacos , Axônios/fisiologia , Condroitina ABC Liase/farmacologia , Expressão Gênica , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Traumatismos da Medula Espinal/genética , Adenoviridae/genética , Animais , Comportamento Animal , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Genes Reporter , Genes fos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Atividade Motora , Neurônios/metabolismo , Ratos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Transdução GenéticaRESUMO
OBJECTIVE: The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. DESIGN: Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (µCT), and measurement of disc biomechanical properties. RESULTS: Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. CONCLUSIONS: A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.
Assuntos
Modelos Animais de Doenças , Degeneração do Disco Intervertebral/patologia , Animais , Condroitina ABC Liase/farmacologia , Discotomia Percutânea , Doenças das Cabras/patologia , Cabras , Humanos , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/cirurgia , Degeneração do Disco Intervertebral/diagnóstico por imagem , Masculino , Radiografia , Microtomografia por Raio-XRESUMO
After a spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to: (1) the presence of inhibitory molecules, e.g., chondroitin sulfate proteoglycans (CSPG), in the glial scar at the lesion; and (2) the diminished growth capacity of adult neurons. We sought to determine whether expressing a constitutively active form of the GTPase Rheb (caRheb) in adult neurons after a complete SCI in rats improves intrinsic growth potential to result in axon regeneration out of a growth-supportive peripheral nerve grafted (PNG) into the SCI cavity. We also hypothesized that treating the glial scar with chondroitinase ABC (ChABC), which digests CSPG, would further allow caRheb-transduced neurons to extend axons across the distal graft interface. We found that targeting this pathway at a clinically relevant post-SCI time point improves both sprouting and regeneration of axons. CaRheb increased the number of axons, but not the number of neurons, that projected into the PNG, indicative of augmented sprouting. We also saw that caRheb enhanced sprouting far rostral to the injury. CaRheb not only increased growth rostral and into the graft, it also resulted in significantly more regrowth of axons across a ChABC-treated scar into caudal spinal cord. CaRheb(+) neurons had higher levels of growth-associated-43, suggestive of a newly identified mechanism for mTOR-mediated enhancement of regeneration. Thus, we demonstrate for the first time that simultaneously addressing intrinsic and scar-associated, extrinsic impediments to regeneration results in significant regrowth beyond an extremely challenging, complete SCI site. SIGNIFICANCE STATEMENT: After spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to the diminished growth capacity of adult neurons and the presence of inhibitory molecules in the scar at the lesion. We sought to simultaneously counter both of these obstacles to achieve more robust regeneration after complete SCI. We transduced neurons postinjury to express a constitutively active Rheb to enhance their intrinsic growth potential, transplanted a growth supporting peripheral nerve graft into the lesion cavity, and enzymatically modulated the inhibitory glial scar distal to the graft. We demonstrate, for the first time, that simultaneously addressing neuron-related, intrinsic deficits in axon regrowth and extrinsic, scar-associated impediments to regeneration results in significant regeneration after SCI.
Assuntos
Condroitina ABC Liase/uso terapêutico , Cicatriz/terapia , Proteínas Monoméricas de Ligação ao GTP/genética , Regeneração Nervosa/genética , Neuropeptídeos/genética , Traumatismos da Medula Espinal/terapia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Condroitina ABC Liase/farmacologia , Cicatriz/tratamento farmacológico , Cicatriz/genética , Modelos Animais de Doenças , Feminino , Regeneração Nervosa/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/genética , Resultado do TratamentoRESUMO
There are lines of evidence demonstrating that NEDD9 (Cas-L, HEF-1) plays a key role in the development, progression, and metastasis of breast cancer cells. We previously reported that NEDD9 plays a critical role for promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, stable cells overexpressing NEDD9 were generated using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Importantly, enzymes generating chondroitin sulfate glycosaminoglycans (CS) such as CHST11, CHST15, and CSGALNACT1 were upregulated in HCC38(NEDD9) compared to HCC38(Vector). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to CD44 core protein. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent colony formation of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggests that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression and metastasis and possibly stem cell phenotypes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/patologia , Sulfatos de Condroitina/biossíntese , Fosfoproteínas/metabolismo , Esferoides Celulares/patologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Anticorpos Monoclonais/imunologia , Antígenos/biossíntese , Antígenos/metabolismo , Movimento Celular , Proliferação de Células , Condroitina ABC Liase/metabolismo , Condroitina ABC Liase/farmacologia , Regulação para Baixo , Feminino , Imunofluorescência , Humanos , Receptores de Hialuronatos/biossíntese , Glicoproteínas de Membrana/biossíntese , N-Acetilgalactosaminiltransferases/biossíntese , Metástase Neoplásica/patologia , Fosfoproteínas/biossíntese , Proteoglicanas/biossíntese , Proteoglicanas/metabolismo , Sulfotransferases/biossíntese , Sindecana-1/biossíntese , Sindecana-2/biossíntese , Células Tumorais Cultivadas , Regulação para Cima , Versicanas/biossíntese , Proteínas de Transporte Vesicular/biossínteseRESUMO
Ischemic stroke insults may lead to chronic functional limitations that adversely affect patient movements. Partial motor recovery is thought to be sustained by neuronal plasticity, particularly in areas close to the lesion site. It is still unknown if treatments acting exclusively on cortical plasticity of perilesional areas could result in behavioral amelioration. We tested whether enhancing plasticity in the ipsilesional cortex using local injections of chondroitinase ABC (ChABC) could promote recovery of skilled motor function in a focal cortical ischemia of forelimb motor cortex in rats. Using the skilled reaching test, we found that acute and delayed ChABC treatment induced recovery of impaired motor skills in treated rats. vGLUT1, vGLUT2, and vGAT staining indicated that functional recovery after acute ChABC treatment was associated with local plastic modification of the excitatory cortical circuitry positive for VGLUT2. ChABC effects on vGLUT2 staining were present only in rats undergoing behavioral training. Thus, the combination of treatments targeting the CSPG component of the extracellular matrix in perilesional areas and rehabilitation could be sufficient to enhance functional recovery from a focal stroke.
Assuntos
Isquemia Encefálica/terapia , Condroitina ABC Liase/uso terapêutico , Terapia por Exercício , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/terapia , Animais , Isquemia Encefálica/tratamento farmacológico , Condroitina ABC Liase/farmacologia , Terapia Combinada , Córtex Motor/efeitos dos fármacos , Córtex Motor/lesões , Córtex Motor/patologia , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Long-Evans , Acidente Vascular Cerebral/tratamento farmacológico , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Mammalian target of rapamycin (mTOR) functions as a master sensor of nutrients and energy, and controls protein translation and cell growth. Deletion of phosphatase and tensin homolog (PTEN) in adult CNS neurons promotes regeneration of injured axons in an mTOR-dependent manner. However, others have demonstrated mTOR-independent axon regeneration in different cell types, raising the question of how broadly mTOR regulates axonal regrowth across different systems. Here we define the role of mTOR in promoting collateral sprouting of spared axons, a key axonal remodeling mechanism by which functions are recovered after CNS injury. Using pharmacological inhibition, we demonstrate that mTOR is dispensable for the robust spontaneous sprouting of corticospinal tract axons seen after pyramidotomy in postnatal mice. In contrast, moderate spontaneous axonal sprouting and induced-sprouting seen under different conditions in young adult mice (i.e., PTEN deletion or degradation of chondroitin proteoglycans; CSPGs) are both reduced upon mTOR inhibition. In addition, to further determine the potency of mTOR in promoting sprouting responses, we coinactivate PTEN and CSPGs, and demonstrate that this combination leads to an additive increase in axonal sprouting compared with single treatments. Our findings reveal a developmental switch in mTOR dependency for inducing axonal sprouting, and indicate that PTEN deletion in adult neurons neither recapitulates the regrowth program of postnatal animals, nor is sufficient to completely overcome an inhibitory environment. Accordingly, exploiting mTOR levels by targeting PTEN combined with CSPG degradation represents a promising strategy to promote extensive axonal plasticity in adult mammals.
Assuntos
Axônios/fisiologia , Lesões Encefálicas/fisiopatologia , Regeneração Nervosa/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Lesões Encefálicas/patologia , Condroitina ABC Liase/farmacologia , Proteoglicanas de Sulfatos de Condroitina/antagonistas & inibidores , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Regeneração Nervosa/efeitos dos fármacos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/lesões , Tratos Piramidais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Extinction therapy has been suggested to suppress the conditioned motivational effect of drug cues to prevent relapse. However, extinction forms a new inhibiting memory rather than erasing the original memory trace and drug memories invariably return. Perineuronal nets (PNNs) are a specialized extracellular matrix around interneurons in the brain that have been suggested to be a permissive factor that allows synaptic plasticity in the adolescent brain. The degradation of PNNs caused by chondroitinase ABC (ChABC) may generate induced juvenile-like plasticity (iPlasticity) and promote experience-dependent plasticity in the adult brain. In the present study, we investigated the effect of removing PNNs in the amygdala of rat on the extinction of drug memories. We found that extinction combined with intra-amygdala injections of ChABC (0.01 U/side) prevented the subsequent priming-induced reinstatement of morphine-induced and cocaine-induced, but not food -induced, conditioned place preference (CPP). Intra-amygdala injections of ChABC alone had no effect on the retention, retrieval, or relearning of morphine-induced CPP and storage of acquired food-induced CPP. Moreover, we found that the procedure facilitated the extinction of heroin- and cocaine-seeking behavior and prevented the spontaneous recovery and drug-induced reinstatement of heroin- and cocaine-seeking behavior. We also found that the effect of PNNs degradation combined with extinction may be mediated by the potentiation of several plasticity-related proteins in the amygdala. Altogether, our findings demonstrate that a combination of extinction training with PNNs degradation in the amygdala erases drug memories and suggest that ChABC may be an attractive candidate for the prevention of relapse.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/fisiologia , Memória , Rede Nervosa/fisiologia , Transtornos Relacionados ao Uso de Substâncias/psicologia , Animais , Western Blotting , Condroitina ABC Liase/administração & dosagem , Condroitina ABC Liase/farmacologia , Transtornos Relacionados ao Uso de Cocaína/psicologia , Condicionamento Operante , Extinção Psicológica , Alimentos , Dependência de Heroína/psicologia , Masculino , Microinjeções , Dependência de Morfina/psicologia , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Reforço Psicológico , Prevenção SecundáriaRESUMO
Chondroitinase ABC (ChABC) has striking effects on promoting neuronal plasticity after spinal cord injury (SCI), but little is known about its involvement in other pathological mechanisms. Recent work showed that ChABC might also modulate the immune response by promoting M2 macrophage polarization. Here we investigate in detail the immunoregulatory effects of ChABC after SCI in rats. Initially, we examined the expression profile of 16 M1/M2 macrophage polarization markers at 3 h and 7 d postinjury. ChABC treatment had a clear effect on the immune signature after SCI. More specifically, ChABC increased the expression of the anti-inflammatory cytokine IL-10, accompanied by a reduction in the proinflammatory cytokine IL-12B in injured spinal tissue. These effects were associated with a distinct, IL-10-mediated anti-inflammatory response in ChABC-treated spinal cords. Mechanistically, we show that IL-10 expression is driven by tissue injury and macrophage infiltration, while the p38 MAPK is the central regulator of IL-10 expression in vivo. These findings provide novel insights into the effects of ChABC in the injured spinal cord and explain its immunoregulatory activity.
Assuntos
Condroitina ABC Liase/fisiologia , Regulação da Expressão Gênica , Imunomodulação/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-10/biossíntese , Traumatismos da Medula Espinal/imunologia , Animais , Condroitina ABC Liase/administração & dosagem , Condroitina ABC Liase/farmacologia , Imidazóis/farmacologia , Imunomodulação/fisiologia , Injeções Espinhais , Interleucina-12/biossíntese , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/fisiologia , Masculino , Proteoglicanas/metabolismo , Piridinas/farmacologia , Ratos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Recent studies have suggested that the perineuronal net (PNN), a specialised extracellular matrix structure, and parvalbumin (PV), an EF-hand calcium-binding protein, are involved in the regulation of plasticity of neural circuits. Here, we aimed to quantitatively estimate the relationship between the two plasticity regulators, PV and PNNs, in the hippocampus of young adult mice. Dual fluorescence staining for PV and Wisteria floribunda agglutinin (a broad PNN marker) showed that a substantial population of PV-expressing (PV(+) ) GABAergic neurons lacked PNNs. Optical disector analysis demonstrated that there were fewer PNN(+) neurons than PV(+) neurons. The ratio of PNN expression in PV(+) neurons was generally lower in the dendritic layers than in the principal cell layers, whereas the ratio of PV expression in PNN(+) neurons was effectively 100%. The mean PV fluorescence was significantly higher in PNN(+) /PV(+) neurons than in PNN(-) /PV(+) neurons. Cumulative frequencies for single-cell PV fluorescence indicated that intensely stained PV(+) neurons tend to be enwrapped by PNNs, whereas weakly stained PV(+) neurons are likely to lack PNNs. We digested the PNNs by a unilateral injection of chondroitinase ABC (chABC) into the dorsal CA1 region. Although the densities of PV(+) neurons remained unchanged, the PV fluorescence declined 7 days after chABC injection. Quantitative real-time polymerase chain reaction analysis demonstrated a reduction in PV mRNA expression following chABC injection. These findings indicate that the presence or absence of PNNs affects the relative PV expression in GABAergic neurons in the hippocampus.