RESUMEN
Genetic and biochemical evidence points to an association between mitochondrial dysfunction and Parkinson's disease (PD). PD-associated mutations in several genes have been identified and include those encoding PTEN-induced putative kinase 1 (PINK1) and parkin. To identify genes, pathways, and pharmacological targets that modulate the clearance of damaged or old mitochondria (mitophagy), here we developed a high-content imaging-based assay of parkin recruitment to mitochondria and screened both a druggable genome-wide siRNA library and a small neuroactive compound library. We used a multiparameter principal component analysis and an unbiased parameter-agnostic machine-learning approach to analyze the siRNA-based screening data. The hits identified in this analysis included specific genes of the ubiquitin proteasome system, and inhibition of ubiquitin-conjugating enzyme 2 N (UBE2N) with a specific antagonist, Bay 11-7082, indicated that UBE2N modulates parkin recruitment and downstream events in the mitophagy pathway. Screening of the compound library identified kenpaullone, an inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3, as a modulator of parkin recruitment. Validation studies revealed that kenpaullone augments the mitochondrial network and protects against the complex I inhibitor MPP+. Finally, we used a microfluidics platform to assess the timing of parkin recruitment to depolarized mitochondria and its modulation by kenpaullone in real time and with single-cell resolution. We demonstrate that the high-content imaging-based assay presented here is suitable for both genetic and pharmacological screening approaches, and we also provide evidence that pharmacological compounds modulate PINK1-dependent parkin recruitment.
Asunto(s)
Mitocondrias/metabolismo , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Benzazepinas/química , Benzazepinas/metabolismo , Benzazepinas/farmacología , Células HeLa , Humanos , Hidrazonas/química , Hidrazonas/metabolismo , Hidrazonas/farmacología , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitofagia/efectos de los fármacos , Análisis de Componente Principal , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Interferencia de ARN , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The medial prefrontal cortex (mPFC) is known to be critical for specific forms of long-term recognition memory, however the cellular mechanisms in the mPFC that underpin memory maintenance have not been well characterized. This study examined the importance of phosphorylation of cAMP responsive element binding protein (CREB) in the mPFC for different forms of long-term recognition memory in the rat. Adenoviral transduction of the mPFC with a dominant-negative inhibitor of CREB impaired object-in-place memory following a 6 or 24 h retention delay, but no impairment was observed following delays of 5 min or 3 h. Long-term object temporal order memory and spatial temporal order memory was also impaired. In contrast, there were no impairments in novel object recognition or object location memory. These results establish, for the first time, the importance of CREB phosphorylation within the mPFC for memory of associative and temporal information crucial to recognition.
Asunto(s)
Asociación , Proteína de Unión a CREB/fisiología , Memoria a Largo Plazo/fisiología , Corteza Prefrontal/metabolismo , Reconocimiento en Psicología/fisiología , Memoria Espacial/fisiología , Transcripción Genética/genética , Animales , Conducta Animal/fisiología , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Dependovirus , Masculino , Fosforilación/fisiología , RatasRESUMEN
BACKGROUND: The potential of microRNAs (miRNAs) as bedside biomarkers in selecting newborns with hypoxic-ischemic encephalopathy (HIE) for neuroprotection has yet to be explored. Commonly, blood-based biomarker tests use plasma or serum which don't allow evaluation of both intracellular and extracellular changes. METHODS: We describe a technique to extract and compare expression of miRNAs from a single small 6-mm-diameter dried blood spot (DBS) stored at room temperature with those from EDTA-blood, plasma, and urine. Three miRNAs (RNU6B, let7b, and miR-21) were quantified via extraction and quantitative RT-PCR performed from a DBS and compared with levels from EDTA-blood, plasma, and urine. Secondarily, candidate miRNAs let7b, miR-21, miR-29b, miR-124, and miR-155 in DBS were evaluated as potential biomarkers for HIE. RESULTS: Candidate miRNAs were extractable in all biosamples from newborns, with the highest expression in DBS. There was a good correlation between miRNAs' levels in DBS and EDTA-blood at -80 °C. No significant difference was observed in the miRNA levels between the favorable and unfavorable outcome groups for babies with HIE. CONCLUSION: DBS may be useful for studying the potential of miRNAs as biomarkers for brain injury.
Asunto(s)
Asfixia Neonatal/sangre , Asfixia Neonatal/genética , Pruebas con Sangre Seca , MicroARNs/metabolismo , Tamizaje Neonatal/métodos , Biomarcadores/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Hipoxia-Isquemia Encefálica/genética , Recién Nacido , Pulmón/metabolismo , MasculinoRESUMEN
Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.
Asunto(s)
Expresión Génica , Factores de Crecimiento Nervioso/genética , Enfermedad de Parkinson/genética , Sustancia Negra/metabolismo , Animales , Conducta Animal , Línea Celular , Modelos Animales de Enfermedad , Orden Génico , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Lentivirus/genética , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Oxidopamina/efectos adversos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/terapia , Ratas , Proteínas Recombinantes de Fusión , Sustancia Negra/patología , Transducción Genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
We show that a single gene locus gives rise to two fully processed and functional miRNAs, i.e. that due to imperfect base pairing, two distinct microRNAs (miRNAs) can be produced from the fully complementary DNA strands. The antisense strand encodes miR-214, which is transcribed by its own promoter, whereas a novel miRNA, miR-3120, is co-expressed with its host gene mRNA. We also found that miR-3120 regulates important aspects of cellular function that are similar to that of its host gene, dynamin-3. miR-3120 was found to be located in neuronal cell bodies and to target Hsc70 and auxilin, and its lentivirus-mediated expression inhibited the uncoating of clathrin-coated vesicles. Finally, mirror miRNAs are likely to represent a new group of miRNAs with complex roles in coordinating gene expression.
Asunto(s)
Auxilinas/biosíntesis , Vesículas Cubiertas por Clatrina/metabolismo , Proteínas HSP70 de Choque Térmico/biosíntesis , MicroARNs/biosíntesis , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Animales , Auxilinas/genética , Vesículas Cubiertas por Clatrina/genética , Dinamina III/biosíntesis , Dinamina III/genética , Regulación de la Expresión Génica/fisiología , Proteínas HSP70 de Choque Térmico/genética , MicroARNs/genética , Neuronas/citología , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST) normally have low levels of the neuronal calcium sensor-1 (NCS1) protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system.
Asunto(s)
Proteínas Sensoras del Calcio Neuronal/genética , Plasticidad Neuronal/genética , Neuropéptidos/genética , Traumatismos de la Médula Espinal/fisiopatología , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Cartilla de ADN , Activación Enzimática , Vectores Genéticos , Inmunohistoquímica , Lentivirus/genética , Masculino , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Proteínas Quinasas/metabolismo , Ratas , Ratas WistarRESUMEN
We previously reported that kenpaullone, which inhibits GSK-3a/b and CDKs inhibited CCCP mediated mitochondrial depolarisation and augments the mitochondrial network. To investigate the actions of this class of drug further, we compared the ability of kenpaullone, alsterpaullone, 1-azakenapaullone, AZD5438, AT7519 (CDK and GSK-3a/b inhibitors) and dexpramipexole and olesoxime (mitochondrial permeability transition pore inhibitors) to prevent CCCP mediated mitochondrial depolarisation and found that AZD5438 and AT7519, were the most effective. Furthermore, treatment with AZD5438 alone increased the complexity of the mitochondrial network. We also found that AZD5438 prevented the rotenone induced decrease in PGC-1alpha and TOM20 levels and that it mediated powerful anti-apoptotic effects and promoted glycolytic respiration. Importantly, experiments in human iPSC derived cortical and midbrain neurons showed AZD5438 mediated significant protective effects, preventing the neuronal cell death, and collapse in the neurite and mitochondrial network associated with rotenone treatment. These results suggest drugs that target GSK-3a/b and CDKs should be developed and assessed further as they may have significant therapeutic potential.
Asunto(s)
Neuronas , Rotenona , Humanos , Carbonil Cianuro m-Clorofenil Hidrazona , Imidazoles , Inhibidores de Proteínas Quinasas , Quinasas Ciclina-DependientesRESUMEN
Evidence suggests that the acquisition of recognition memory depends upon CREB-dependent long-lasting changes in synaptic plasticity in the perirhinal cortex.The CREB-responsive microRNA miR-132 has been shown to regulate synaptic transmission and we set out to investigate a role for this microRNA in recognition memory and its underlying plasticity mechanisms. To this end we mediated the specific overexpression of miR-132 selectively in the rat perirhinal cortex and demonstrated impairment in short-term recognition memory. This functional deficit was associated with a reduction in both long-term depression and long-term potentiation. These results confirm that microRNAs are key coordinators of the intracellular pathways that mediate experience-dependent changes in the brain. In addition, these results demonstrate a role for miR-132 in the neuronal mechanisms underlying the formation of short-term recognition memory.
Asunto(s)
Corteza Cerebral/fisiología , Regulación de la Expresión Génica , Potenciación a Largo Plazo/genética , Memoria a Corto Plazo/fisiología , MicroARNs/metabolismo , Reconocimiento en Psicología/fisiología , Animales , Corteza Cerebral/metabolismo , Potenciales Postsinápticos Excitadores , Células HeLa , Humanos , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Masculino , MicroARNs/genética , Ratas , Ratas WistarRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the selective death of motor neurons in the brain and spinal cord. Some familial cases of ALS are caused by dominant mutations in the gene encoding superoxide dismutase (SOD1). The emergence of interfering RNA (RNAi) for specific gene silencing could be therapeutically beneficial for the treatment of such dominantly inherited diseases. We generated a lentiviral vector to mediate expression of RNAi molecules specifically targeting the human SOD1 gene (SOD1). Injection of this vector into various muscle groups of mice engineered to overexpress a mutated form of human SOD1 (SOD1(G93A)) resulted in an efficient and specific reduction of SOD1 expression and improved survival of vulnerable motor neurons in the brainstem and spinal cord. Furthermore, SOD1 silencing mediated an improved motor performance in these animals, resulting in a considerable delay in the onset of ALS symptoms by more than 100% and an extension in survival by nearly 80% of their normal life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi and provide the highest therapeutic efficacy observed in this field to date.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Degeneración Nerviosa , Interferencia de ARN , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/terapia , Animales , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos , Humanos , Lentivirus/genética , Ratones , Ratones Transgénicos , Mutación , ARN Interferente Pequeño , Tasa de Supervivencia , TransfecciónRESUMEN
Spinal cord injury (SCI) can cause irreversible paralysis, with no regenerative treatment clinically available. Dogs with natural SCI present an established model and can facilitate translation of experimental findings in rodents to people. We conducted a prospective, single arm clinical safety study in companion dogs with chronic SCI to characterize the feasibility of intraspinal transplantation of hydrogel-encapsulated autologous mucosal olfactory ensheathing cell (mOEC) populations expressing chondroitinase ABC (chABC). mOECs and chABC are both promising therapies for SCI, and mOECs expressing chABC drive greater voluntary motor recovery than mOECs alone after SCI in rats. Canine mOECs encapsulated in collagen hydrogel can be matched in stiffness to canine SCI. Four dogs with complete and chronic loss of function caudal to a thoraco-lumbar lesion were recruited. After baseline measures, olfactory mucosal biopsy was performed and autologous mOECs cultured and transduced to express chABC, then hydrogel-encapsulated and percutaneously injected into the spinal cord. Dogs were monitored for 6 months with repeat clinical examinations, spinal MRI, kinematic gait and von Frey assessment. No adverse effects or significant changes on neurological examination were detected. MRI revealed large and variable lesions, with no spinal cord compression or ischemia visible after hydrogel transplantation. Owners reported increased pelvic-limb reflexes with one dog able to take 2-3 unsupported steps, but gait-scoring and kinematic analysis showed no significant improvements. This novel combination approach to regeneration after SCI is therefore feasible and safe in paraplegic dogs in a clinical setting. A randomised-controlled trial in this translational model is proposed to test efficacy.
Asunto(s)
Mascotas , Traumatismos de la Médula Espinal , Animales , Trasplante de Células , Condroitina ABC Liasa/farmacología , Condroitinasas y Condroitín Liasas/uso terapéutico , Perros , Estudios de Factibilidad , Humanos , Hidrogeles/uso terapéutico , Regeneración Nerviosa , Estudios Prospectivos , Ratas , Recuperación de la Función , Traumatismos de la Médula Espinal/patologíaRESUMEN
Spinal cord injury (SCI) can cause chronic paralysis and incontinence and remains a major worldwide healthcare burden, with no regenerative treatment clinically available. Intraspinal transplantation of olfactory ensheathing cells (OECs) and injection of chondroitinase ABC (chABC) are both promising therapies but limited and unpredictable responses are seen, particularly in canine clinical trials. Sustained delivery of chABC presents a challenge due to its thermal instability; we hypothesised that transplantation of canine olfactory mucosal OECs genetically modified ex vivo by lentiviral transduction to express chABC (cOEC-chABC) would provide novel delivery of chABC and synergistic therapy. Rats were randomly divided into cOEC-chABC, cOEC, or vehicle transplanted groups and received transplant immediately after dorsal column crush corticospinal tract (CST) injury. Rehabilitation for forepaw reaching and blinded behavioural testing was conducted for 8 weeks. We show that cOEC-chABC transplanted animals recover greater forepaw reaching accuracy on Whishaw testing and more normal gait than cOEC transplanted or vehicle control rats. Increased CST axon sprouting cranial to the injury and serotonergic fibres caudal to the injury suggest a mechanism for recovery. We therefore demonstrate that cOECs can deliver sufficient chABC to drive modest functional improvement, and that this genetically engineered cellular and molecular approach is a feasible combination therapy for SCI.
Asunto(s)
Condroitinasas y Condroitín Liasas/administración & dosificación , Mucosa Olfatoria/fisiología , Mucosa Olfatoria/trasplante , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/enzimología , Traumatismos de la Médula Espinal/rehabilitación , Animales , Células Cultivadas , Condroitinasas y Condroitín Liasas/biosíntesis , Perros , Masculino , Mucosa Olfatoria/citología , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/patologíaRESUMEN
The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor beta2 (RARbeta2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARbeta2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARbeta2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARbeta2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARbeta2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration.
Asunto(s)
Regeneración Nerviosa/fisiología , Neuronas Aferentes/metabolismo , Receptores de Ácido Retinoico/metabolismo , Médula Espinal/metabolismo , Animales , Células Cultivadas , Ganglios Espinales/lesiones , Ganglios Espinales/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratas , Ratas Wistar , Recuperación de la Función/fisiologíaRESUMEN
Safe hydrogel delivery requires stiffness-matching with host tissues to avoid iatrogenic damage and reduce inflammatory reactions. Hydrogel-encapsulated cell delivery is a promising combinatorial approach to spinal cord injury therapy, but a lack of in vivo clinical spinal cord injury stiffness measurements is a barrier to their use in clinics. We demonstrate that ultrasound elastography - a non-invasive, clinically established tool - can be used to measure spinal cord stiffness intraoperatively in canines with spontaneous spinal cord injury. In line with recent experimental reports, our data show that injured spinal cord has lower stiffness than uninjured cord. We show that the stiffness of hydrogels encapsulating a clinically relevant transplant population (olfactory ensheathing cells) can also be measured by ultrasound elastography, enabling synthesis of hydrogels with comparable stiffness to canine spinal cord injury. We therefore demonstrate proof-of-principle of a novel approach to stiffness-matching hydrogel-olfactory ensheathing cell implants to 'real-life' spinal cord injury values; an approach applicable to multiple biomaterial implants for regenerative therapies.
RESUMEN
SAFB1 is a DNA and RNA binding protein that is highly expressed in the cerebellum and hippocampus and is involved in the processing of coding and non-coding RNAs, splicing and dendritic function. We analyzed SAFB1 expression in the post-mortem brain tissue of spinocerebellar ataxia (SCA), Huntington's disease (HD), Multiple sclerosis (MS), Parkinson's disease patients and controls. In SCA cases, the expression of SAFB1 in the nucleus was increased and there was abnormal and extensive expression in the cytoplasm where it co-localized with the markers of Purkinje cell injury. Significantly, no SAFB1 expression was found in the cerebellar neurons of the dentate nucleus in control or MS patients; however, in SCA patients, SAFB1 expression was increased significantly in both the nucleus and cytoplasm of dentate neurons. In HD, we found that SAFB1 expression was increased in the nucleus and cytoplasm of striatal neurons; however, there was no SAFB1 staining in the striatal neurons of controls. In PD substantia nigra, we did not see any changes in neuronal SAFB1 expression. iCLIP analysis found that SAFB1 crosslink sites within ATXN1 RNA were adjacent to the start and within the glutamine repeat sequence. Further investigation found increased binding of SAFB1 to pathogenic ATXN1-85Q mRNA. These novel data strongly suggest SAFB1 contributes to the etiology of SCA and Huntington's chorea and that it may be a pathological marker of polyglutamine repeat expansion diseases.
Asunto(s)
Encéfalo/metabolismo , Enfermedad de Huntington/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Neuronas/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Receptores de Estrógenos/metabolismo , Ataxias Espinocerebelosas/metabolismo , Anciano , Anciano de 80 o más Años , Encéfalo/patología , Cerebelo/metabolismo , Cerebelo/patología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Femenino , Humanos , Enfermedad de Huntington/patología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Células de Purkinje/metabolismo , Células de Purkinje/patología , Ataxias Espinocerebelosas/patologíaRESUMEN
Olfactory ensheathing cells are thought to support regeneration and remyelination of damaged axons when transplanted into spinal cord injuries. Following transplantation, improved locomotion has been detected in many laboratory models and in dogs with naturally-occurring spinal cord injury; safety trials in humans have also been completed. For widespread clinical implementation, it will be necessary to derive large numbers of these cells from an accessible and, preferably, autologous, source making olfactory mucosa a good candidate. Here, we compared the yield of olfactory ensheathing cells from the olfactory mucosa using 3 different techniques: rhinotomy, frontal sinus keyhole approach and rhinoscopy. From canine clinical cases with spinal cord injury, 27 biopsies were obtained by rhinotomy, 7 by a keyhole approach and 1 with rhinoscopy. Biopsy via rhinoscopy was also tested in 13 cadavers and 7 living normal dogs. After 21 days of cell culture, the proportions and populations of p75-positive (presumed to be olfactory ensheathing) cells obtained by the keyhole approach and rhinoscopy were similar (~4.5 x 106 p75-positive cells; ~70% of the total cell population), but fewer were obtained by frontal sinus rhinotomy. Cerebrospinal fluid rhinorrhea was observed in one dog and emphysema in 3 dogs following rhinotomy. Blepharitis occurred in one dog after the keyhole approach. All three biopsy methods appear to be safe for harvesting a suitable number of olfactory ensheathing cells from the olfactory mucosa for transplantation within the spinal cord but each technique has specific advantages and drawbacks.
Asunto(s)
Trasplante de Células/métodos , Regeneración Nerviosa , Mucosa Olfatoria/citología , Mucosa Olfatoria/trasplante , Recuperación de la Función , Traumatismos de la Médula Espinal/terapia , Animales , Células Cultivadas , Perros , LocomociónRESUMEN
Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients.
Asunto(s)
Axones , Condroitina ABC Liasa/biosíntesis , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Enfermedades de los Perros/metabolismo , Mucosa Olfatoria/trasplante , Traumatismos de la Médula Espinal/veterinaria , Animales , Enfermedades de los Perros/patología , Perros , Mucosa Olfatoria/citología , Mucosa Olfatoria/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patologíaRESUMEN
Episodic memory formation depends on information about a stimulus being integrated within a precise spatial and temporal context, a process dependent on the hippocampus and prefrontal cortex. Investigations of putative functional interactions between these regions are complicated by multiple direct and indirect hippocampal-prefrontal connections. Here application of a pharmacogenetic deactivation technique enabled us to investigate the mnemonic contributions of two direct hippocampal-medial prefrontal cortex (mPFC) pathways, one arising in the dorsal CA1 (dCA1) and the other in the intermediate CA1 (iCA1). While deactivation of either pathway impaired episodic memory, the resulting pattern of mnemonic deficits was different: deactivation of the dCA1âmPFC pathway selectively disrupted temporal order judgments while iCA1âmPFC pathway deactivation disrupted spatial memory. These findings reveal a previously unsuspected division of function among CA1 neurons that project directly to the mPFC. Such subnetworks may enable the distinctiveness of contextual information to be maintained in an episodic memory circuit.
Asunto(s)
Hipocampo/fisiología , Memoria Episódica , Vías Nerviosas/fisiología , Neuronas/fisiología , Corteza Prefrontal/fisiología , Animales , Masculino , Red Nerviosa/fisiología , Ratas , Memoria Espacial/fisiologíaRESUMEN
Following peripheral axon injury, dysregulation of non-coding microRNAs (miRs) occurs in dorsal root ganglia (DRG) sensory neurons. Here we show that DRG neuron cell bodies release extracellular vesicles, including exosomes containing miRs, upon activity. We demonstrate that miR-21-5p is released in the exosomal fraction of cultured DRG following capsaicin activation of TRPV1 receptors. Pure sensory neuron-derived exosomes released by capsaicin are readily phagocytosed by macrophages in which an increase in miR-21-5p expression promotes a pro-inflammatory phenotype. After nerve injury in mice, miR-21-5p is upregulated in DRG neurons and both intrathecal delivery of a miR-21-5p antagomir and conditional deletion of miR-21 in sensory neurons reduce neuropathic hypersensitivity as well as the extent of inflammatory macrophage recruitment in the DRG. We suggest that upregulation and release of miR-21 contribute to sensory neuron-macrophage communication after damage to the peripheral nerve.
Asunto(s)
Exosomas/metabolismo , Ganglios Espinales/metabolismo , Macrófagos/inmunología , MicroARNs/metabolismo , Neuralgia/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Axones/metabolismo , Exosomas/genética , Ganglios Espinales/citología , Ganglios Espinales/lesiones , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Neuralgia/genética , Neuralgia/inmunología , Fagocitosis , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
The management of disorders of the nervous system remains a medical challenge. The key goals are to understand disease mechanisms, to validate therapeutic targets, and to develop new therapeutic strategies. Viral vector-mediated gene transfer can meet these goals and vectors based on lentiviruses have particularly useful features. Lentiviral vectors can deliver 8 kb of sequence, they mediate gene transfer into any neuronal cell type, expression and therapy are sustained, and normal cellular functions in vitro and in vivo are not compromised. After delivery into the nervous system they induce no significant immune responses, there are no unwanted side effects of the vectors per se to date, and manufacturing and safety testing for clinical applications are well advanced. There are now numerous examples of effective long-term treatment of animal models of neurological disorders, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, motor neuron diseases, lysosomal storage diseases, and spinal injury, using a range of therapeutic genes expressed in lentiviral vectors. Significant issues remain in some areas of neural gene therapy including defining the optimum therapeutic gene(s), increasing the specificity of delivery, regulating expression of potentially toxic genes, and designing clinically relevant strategies. We discuss the applications of lentiviral vectors in therapy and research and highlight the essential features that will ensure their translation to the clinic in the near future.