RESUMEN
The mRNA transcript of the human STMN2 gene, encoding for stathmin-2 protein (also called SCG10), is profoundly impacted by TAR DNA-binding protein 43 (TDP-43) loss of function. The latter is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Using a combination of approaches, including transient antisense oligonucleotide-mediated suppression, sustained shRNA-induced depletion in aging mice, and germline deletion, we show that stathmin-2 has an important role in the establishment and maintenance of neurofilament-dependent axoplasmic organization that is critical for preserving the caliber and conduction velocity of myelinated large-diameter axons. Persistent stathmin-2 loss in adult mice results in pathologies found in ALS, including reduced interneurofilament spacing, axonal caliber collapse that drives tearing within outer myelin layers, diminished conduction velocity, progressive motor and sensory deficits, and muscle denervation. These findings reinforce restoration of stathmin-2 as an attractive therapeutic approach for ALS and other TDP-43-dependent neurodegenerative diseases.
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Esclerosis Amiotrófica Lateral , Animales , Ratones , Esclerosis Amiotrófica Lateral/metabolismo , Axones/fisiología , Desnervación , Proteínas de Unión al ADN/genética , Filamentos Intermedios/metabolismo , Filamentos Intermedios/patología , Neuronas Motoras/metabolismo , Estatmina/genética , Estatmina/metabolismoRESUMEN
One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.
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Reprogramación Celular , Células Madre Pluripotentes Inducidas , Inyecciones Espinales , Células-Madre Neurales , Trasplante de Células Madre , Adulto , Animales , Humanos , Ratas , Diferenciación Celular/fisiología , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Vectores Genéticos/genética , Supervivencia de Injerto/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Inyecciones Espinales/efectos adversos , Inyecciones Espinales/instrumentación , Inyecciones Espinales/métodos , Células-Madre Neurales/fisiología , Células-Madre Neurales/trasplante , Virus Sendai , Manejo de Especímenes/métodos , Trasplante de Células Madre/efectos adversos , Trasplante de Células Madre/instrumentación , Trasplante de Células Madre/métodos , Porcinos , Recolección de Tejidos y Órganos/métodos , Resultado del Tratamiento , Encéfalo , Médula EspinalRESUMEN
The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.
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Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Humanos , Ratas , Animales , Virus Sendai/genética , Leucocitos Mononucleares , Neuronas/metabolismo , Diferenciación CelularRESUMEN
Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.
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Neuralgia , Nociceptores , Animales , Técnicas de Transferencia de Gen , Ratones , Neuralgia/etiología , Neuralgia/terapia , Células del Asta Posterior , Médula Espinal , Asta Dorsal de la Médula Espinal , PorcinosRESUMEN
Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of DMPK. Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking. Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia. The sustained reversal of DM1 phenotypes provides further support that RNA-targeting Cas9 is a viable strategy for treating DM1 and other MRE-associated diseases.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edición Génica/métodos , Distrofia Miotónica/metabolismo , ARN/metabolismo , Adenoviridae/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/fisiología , Masculino , Ratones Transgénicos , Músculo Esquelético/metabolismo , Distrofia Miotónica/genética , FenotipoRESUMEN
Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.
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Esclerosis Amiotrófica Lateral/terapia , Dependovirus/metabolismo , Silenciador del Gen , Técnicas de Transferencia de Gen , Neuronas Motoras/patología , Degeneración Nerviosa/terapia , Piamadre/patología , Médula Espinal/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Atrofia , Progresión de la Enfermedad , Potenciales Evocados Motores , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/patología , Interneuronas/patología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Desarrollo de Músculos , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Piamadre/fisiopatología , Primates , Pliegue de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/administración & dosificación , Médula Espinal/diagnóstico por imagen , Médula Espinal/fisiopatología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , PorcinosRESUMEN
BACKGROUND: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.
Asunto(s)
Citometría de Flujo , Células Madre Multipotentes/citología , Células-Madre Neurales/citología , Línea Celular , HumanosRESUMEN
Interventions that preserve motor neurons or restore functional motor neuroplasticity may extend longevity in amyotrophic lateral sclerosis (ALS). Delivery of neurotrophins may potentially revive degenerating motor neurons, yet this approach is dependent on the proper subcellular localization of neurotrophin receptor (NTR) to plasmalemmal signaling microdomains, termed membrane/lipid rafts (MLRs). We previously showed that overexpression of synapsin-driven caveolin-1 (Cav-1) (SynCav1) increases MLR localization of NTR [e.g., receptor tyrosine kinase B (TrkB)], promotes hippocampal synaptic and neuroplasticity, and significantly improves learning and memory in aged mice. The present study crossed a SynCav1 transgene-positive (SynCav1+) mouse with the mutant human superoxide dismutase glycine to alanine point mutation at amino acid 93 (hSOD1G93A) mouse model of ALS. When compared with hSOD1G93A, hSOD1G93A/SynCav1+ mice exhibited greater body weight and longer survival as well as better motor function. Microscopic analyses of hSOD1G93A/SynCav1+ spinal cords revealed preserved spinal cord α-motor neurons and preserved mitochondrial morphology. Moreover, hSOD1G93A/SynCav1+ spinal cords contained more MLRs (cholera toxin subunit B positive) and MLR-associated TrkB and Cav-1 protein expression. These findings demonstrate that SynCav1 delays disease progression in a mouse model of ALS, potentially by preserving or restoring NTR expression and localization to MLRs.-Sawada, A., Wang, S., Jian, M., Leem, J., Wackerbarth, J., Egawa, J., Schilling, J. M., Platoshyn, O., Zemljic-Harpf, A., Roth, D. M., Patel, H. H., Patel, P. M., Marsala, M., Head, B. P. Neuron-targeted caveolin-1 improves neuromuscular function and extends survival in SOD1G93A mice.
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Caveolina 1/fisiología , Músculo Esquelético/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Superóxido Dismutasa-1/genética , Animales , Peso Corporal , Caveolina 1/metabolismo , Estimulación Eléctrica , Humanos , Longevidad , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/citología , Tasa de SupervivenciaRESUMEN
Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.
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Autofagia , Homeostasis , Lisosomas/metabolismo , Proteína FUS de Unión a ARN/biosíntesis , Proteína FUS de Unión a ARN/toxicidad , ARN/metabolismo , Animales , Perfilación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/genética , Proteínas Mutantes/toxicidad , Proteína FUS de Unión a ARN/genéticaRESUMEN
Current methods for bioprinting functional tissue lack appropriate biofabrication techniques to build complex 3D microarchitectures essential for guiding cell growth and promoting tissue maturation1. 3D printing of central nervous system (CNS) structures has not been accomplished, possibly owing to the complexity of CNS architecture. Here, we report the use of a microscale continuous projection printing method (µCPP) to create a complex CNS structure for regenerative medicine applications in the spinal cord. µCPP can print 3D biomimetic hydrogel scaffolds tailored to the dimensions of the rodent spinal cord in 1.6 s and is scalable to human spinal cord sizes and lesion geometries. We tested the ability of µCPP 3D-printed scaffolds loaded with neural progenitor cells (NPCs) to support axon regeneration and form new 'neural relays' across sites of complete spinal cord injury in vivo in rodents1,2. We find that injured host axons regenerate into 3D biomimetic scaffolds and synapse onto NPCs implanted into the device and that implanted NPCs in turn extend axons out of the scaffold and into the host spinal cord below the injury to restore synaptic transmission and significantly improve functional outcomes. Thus, 3D biomimetic scaffolds offer a means of enhancing CNS regeneration through precision medicine.
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Biomimética , Regeneración Nerviosa , Impresión Tridimensional , Traumatismos de la Médula Espinal/terapia , Andamios del Tejido/química , Animales , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/trasplante , Células-Madre Neurales/ultraestructura , RatasRESUMEN
The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.
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Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/trasplante , Médula Espinal/trasplante , Envejecimiento , Animales , Diferenciación Celular , Reprogramación Celular , Enfermedad Crónica , Fibroblastos/citología , Regulación de la Expresión Génica , Tolerancia Inmunológica , Inmunidad Humoral , Terapia de Inmunosupresión , Neostriado/patología , Células-Madre Neurales/citología , Neuronas/citología , Ratas , Piel/citología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Análisis de Supervivencia , Porcinos , Porcinos Enanos , Trasplante Homólogo , Trasplante IsogénicoRESUMEN
The loss of local spinal glycine-ergic tone has been postulated as one of the mechanisms contributing to the development of spinal injury-induced spasticity. In our present study using a model of spinal transection-induced muscle spasticity, we characterize the effect of spinally-targeted GlyT2 downregulation once initiated at chronic stages after induction of spasticity in rats. In animals with identified hyper-reflexia, the anti-spasticity effect was studied after intrathecal treatment with: i) glycine, ii) GlyT2 inhibitor (ALX 1393), and iii) GlyT2 antisense oligonucleotide (GlyT2-ASO). Administration of glycine and GlyT2 inhibitor led to significant suppression of spasticity lasting for a minimum of 45-60â¯min. Treatment with GlyT2-ASO led to progressive suppression of muscle spasticity seen at 2-3â¯weeks after treatment. Over the subsequent 4-12â¯weeks, however, the gradual appearance of profound spinal hyper-reflexia was seen. This was presented as spontaneous or slight-tactile stimulus-evoked muscle oscillations in the hind limbs (but not in upper limbs) with individual hyper-reflexive episodes lasting between 3 and 5â¯min. Chronic hyper-reflexia induced by GlyT2-ASO treatment was effectively blocked by intrathecal glycine. Immunofluorescence staining and Q-PCR analysis of the lumbar spinal cord region showed a significant (>90%) decrease in GlyT2 mRNA and GlyT2 protein. These data demonstrate that spinal GlyT2 downregulation provides only a time-limited therapeutic benefit and that subsequent loss of glycine vesicular synthesis resulting from chronic GlyT2 downregulation near completely eliminates the tonic glycine-ergic activity and is functionally expressed as profound spinal hyper-reflexia. These characteristics also suggest that chronic spinal GlyT2 silencing may be associated with pro-nociceptive activity.
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Regulación hacia Abajo/fisiología , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Espasticidad Muscular/metabolismo , Reflejo Anormal/fisiología , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Animales , Femenino , Espasticidad Muscular/fisiopatología , Ratas , Ratas Sprague-Dawley , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Vértebras Torácicas , Factores de TiempoRESUMEN
The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.
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Dependovirus/genética , Vectores Genéticos/metabolismo , Animales , Encéfalo/metabolismo , Femenino , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Médula Espinal/metabolismo , Grabación en VideoRESUMEN
Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Demencia Frontotemporal/tratamiento farmacológico , Factores de Intercambio de Guanina Nucleótido/genética , Oligonucleótidos Antisentido/farmacología , ARN/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteína C9orf72 , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , Ratones Transgénicos , Neuronas/metabolismo , Oligonucleótidos Antisentido/efectos adversos , Oligonucleótidos Antisentido/genéticaRESUMEN
The development of spinal hyper-reflexia as part of the spasticity syndrome represents one of the major complications associated with chronic spinal traumatic injury (SCI). The primary mechanism leading to progressive appearance of muscle spasticity is multimodal and may include loss of descending inhibitory tone, alteration of segmental interneuron-mediated inhibition and/or increased reflex activity to sensory input. Here, we characterized a chronic thoracic (Th 9) complete transection model of muscle spasticity in Sprague-Dawley (SD) rats. Isoflurane-anesthetized rats received a Th9 laminectomy and the spinal cord was transected using a scalpel blade. After the transection the presence of muscle spasticity quantified as stretch and cutaneous hyper-reflexia was identified and quantified as time-dependent changes in: i) ankle-rotation-evoked peripheral muscle resistance (PMR) and corresponding electromyography (EMG) activity, ii) Hoffmann reflex, and iii) EMG responses in gastrocnemius muscle after paw tactile stimulation for up to 8 months after injury. To validate the clinical relevance of this model, the treatment potency after systemic treatment with the clinically established anti-spastic agents baclofen (GABAB receptor agonist), tizanidine (α2-adrenergic agonist) and NGX424 (AMPA receptor antagonist) was also tested. During the first 3 months post spinal transection, a progressive increase in ankle rotation-evoked muscle resistance, Hoffmann reflex amplitude and increased EMG responses to peripherally applied tactile stimuli were consistently measured. These changes, indicative of the spasticity syndrome, then remained relatively stable for up to 8 months post injury. Systemic treatment with baclofen, tizanidine and NGX424 led to a significant but transient suppression of spinal hyper-reflexia. These data demonstrate that a chronic Th9 spinal transection model in adult SD rat represents a reliable experimental platform to be used in studying the pathophysiology of chronic spinal injury-induced spasticity. In addition a consistent anti-spastic effect measured after treatment with clinically effective anti-spastic agents indicate that this model can effectively be used in screening new anti-spasticity compounds or procedures aimed at modulating chronic spinal trauma-associated muscle spasticity.
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Espasticidad Muscular/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Modelos Animales de Enfermedad , Electromiografía , Femenino , Miembro Posterior/fisiopatología , Región Lumbosacra/patología , Región Lumbosacra/fisiopatología , Masculino , Espasticidad Muscular/etiología , Espasticidad Muscular/patología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Ratas Sprague-Dawley , Reflejo Anormal , Médula Espinal/patología , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/patología , Vértebras Torácicas/patología , Percepción del TactoRESUMEN
Vinculin (Vcl) links actin filaments to integrin- and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl co-immunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and ß1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.
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Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Miocitos Cardíacos/metabolismo , Vinculina/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Animales , Comunicación Celular , Membrana Celular/metabolismo , Células Cultivadas , Ratones , Ratones Noqueados , Necrosis , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Transporte de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
INTRODUCTION: Intraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury. METHODS: Three-month-old female Sprague-Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies. RESULTS: Intraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons. CONCLUSIONS: Peri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.
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Células-Madre Neurales/trasplante , Traumatismos de la Médula Espinal/terapia , Animales , Modelos Animales de Enfermedad , Femenino , Supervivencia de Injerto/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Imagen por Resonancia Magnética , Actividad Motora , Espasticidad Muscular/terapia , Células-Madre Neurales/citología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Tacrolimus/farmacología , Trasplante HeterólogoRESUMEN
Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43-dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.
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Esclerosis Amiotrófica Lateral/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Mutación , Empalme del ARN , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , UbiquitinaciónRESUMEN
BACKGROUND: Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA(B) receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. METHODS/PRINCIPAL FINDINGS: Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2-3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. CONCLUSIONS/SIGNIFICANCE: These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments.
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Agonistas del GABA/uso terapéutico , Terapia Genética , Glutamato Descarboxilasa/genética , Espasticidad Muscular/terapia , Columna Vertebral/metabolismo , Animales , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/efectos adversos , Anticonvulsivantes/uso terapéutico , Células Cultivadas , Terapia Combinada , Embrión de Mamíferos , Femenino , Agonistas del GABA/administración & dosificación , Agonistas del GABA/efectos adversos , Regulación de la Expresión Génica/fisiología , Terapia Genética/métodos , Glutamato Descarboxilasa/administración & dosificación , Glutamato Descarboxilasa/efectos adversos , Inyecciones Espinales , Masculino , Espasticidad Muscular/tratamiento farmacológico , Espasticidad Muscular/genética , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/efectos adversos , Fármacos Neuroprotectores/uso terapéutico , Ácidos Nipecóticos/administración & dosificación , Ácidos Nipecóticos/efectos adversos , Ácidos Nipecóticos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Columna Vertebral/patología , Porcinos , Porcinos Enanos , TiagabinaRESUMEN
The activity of voltage-gated ion channels is critical for the maintenance of cellular membrane potential and generation of action potentials. In turn, membrane potential regulates cellular ion homeostasis, triggering the opening and closing of ion channels in the plasma membrane and, thus, enabling ion transport across the membrane. Such transmembrane ion fluxes are important for excitation-contraction coupling in pulmonary artery smooth muscle cells (PASMC). Families of voltage-dependent cation channels known to be present in PASMC include voltage-gated K(+) (Kv) channels, voltage-dependent Ca(2+)-activated K(+) (Kca) channels, L- and T- type voltage-dependent Ca(2+) channels, voltage-gated Na(+) channels and voltage-gated proton channels. When cells are dialyzed with Ca(2+)-free K(+)- solutions, depolarization elicits four components of 4-aminopyridine (4-AP)-sensitive Kvcurrents based on the kinetics of current activation and inactivation. In cell-attached membrane patches, depolarization elicits a wide range of single-channel K(+) currents, with conductances ranging between 6 and 290 pS. Macroscopic 4-AP-sensitive Kv currents and iberiotoxin-sensitive Kca currents are also observed. Transcripts of (a) two Na(+) channel α-subunit genes (SCN5A and SCN6A), (b) six Ca(2+) channel α-subunit genes (α(1A), α(1B), α(1x), α(1D), α(1E) and α(1G)) and many regulatory subunits (α(2)δ(1), ß(1-4), and γ(6)), (c) 22 Kv channel α-subunit genes (Kv1.1 - Kv1.7, Kv1.10, Kv2.1, Kv3.1, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv5.1, Kv 6.1-Kv6.3, Kv9.1, Kv9.3, Kv10.1 and Kv11.1) and three Kv channel ß-subunit genes (Kv(ß1-3) and (d) four Kca channel α-subunit genes (Sloα1 and SK2-SK4) and four Kca channel (ß-subunit genes (Kca(ß1-4) have been detected in PASMC. Tetrodotoxin-sensitive and rapidly inactivating Na(+) currents have been recorded with properties similar to those in cardiac myocytes. In the presence of 20 mM external Ca(2+), membrane depolarization from a holding potential of -100 mV elicits a rapidly inactivating T-type Ca(2+) current, while depolarization from a holding potential of -70 mV elicits a slowly inactivating dihydropyridine-sensitive L-type Ca(2+) current. This review will focus on describing the electrophysiological properties and molecular identities of these voltage-dependent cation channels in PASMC and their contribution to the regulation of pulmonary vascular function and its potential role in the pathogenesis of pulmonary vascular disease.