RESUMO
Sporadic amyotrophic lateral sclerosis (ALS) is a fatal disease with unknown etiology, characterized by a progressive loss of motor neurons leading to paralysis and death typically within 3-5 years of onset. Recently, there has been remarkable progress in understanding inherited forms of ALS in which well defined mutations are known to cause the disease. Rodent models in which the superoxide dismutase-1 (SOD1) mutation is overexpressed recapitulate hallmark signs of ALS in patients. Early anatomical changes in mouse models of fALS are seen in the neuromuscular junctions (NMJs) and lower motor neurons, and selective reduction of toxic mutant SOD1 in the spinal cord and muscle of these models has beneficial effects. Therefore, much of ALS research has focused on spinal motor neuron and NMJ aspects of the disease. Here we show that, in the SOD1(G93A) rat model of ALS, spinal motor neuron loss occurs presymptomatically and before degeneration of ventral root axons and denervation of NMJs. Although overt cell death of corticospinal motor neurons does not occur until disease endpoint, we wanted to establish whether the upper motor neuron might still play a critical role in disease progression. Surprisingly, the knockdown of mutant SOD1 in only the motor cortex of presymptomatic SOD1(G93A) rats through targeted delivery of AAV9-SOD1-shRNA resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of NMJs. This datum suggests an early dysfunction and thus an important role of the upper motor neuron in this animal model of ALS and perhaps patients with the disease.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Córtex Motor/enzimologia , Córtex Motor/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/fisiologia , Esclerose Lateral Amiotrófica/mortalidade , Animais , Morte Celular/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Herpesvirus Suídeo 1/genética , Humanos , Masculino , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Superóxido Dismutase-1 , TransfecçãoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.
Assuntos
Esclerose Lateral Amiotrófica/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Longevidade/genética , Fator A de Crescimento do Endotélio Vascular/genética , Esclerose Lateral Amiotrófica/mortalidade , Animais , Sobrevivência Celular/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , RatosRESUMO
RATIONALE: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES: To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS: SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Insuficiência Respiratória/prevenção & controle , Terapia Respiratória/métodos , Transplante de Células-Tronco , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hipóxia , Capacidade Inspiratória , Masculino , Neurônios Motores/metabolismo , Nervo Frênico/metabolismo , Nervo Frênico/fisiopatologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Superóxido DismutaseRESUMO
Amyotrophic lateral sclerosis (ALS) involves progressive motor neuron loss, leading to paralysis and death typically within 3-5 years of diagnosis. Dysfunctional astrocytes may contribute to disease and glial cell line-derived neurotrophic factor (GDNF) can be protective. Here we show that human neural progenitor cells transduced with GDNF (CNS10-NPC-GDNF) differentiated to astrocytes protected spinal motor neurons and were safe in animal models. CNS10-NPC-GDNF were transplanted unilaterally into the lumbar spinal cord of 18 ALS participants in a phase 1/2a study (NCT02943850). The primary endpoint of safety at 1 year was met, with no negative effect of the transplant on motor function in the treated leg compared with the untreated leg. Tissue analysis of 13 participants who died of disease progression showed graft survival and GDNF production. Benign neuromas near delivery sites were common incidental findings at post-mortem. This study shows that one administration of engineered neural progenitors can provide new support cells and GDNF delivery to the ALS patient spinal cord for up to 42 months post-transplantation.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Neurais , Esclerose Lateral Amiotrófica/terapia , Animais , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Medula Espinal , Superóxido DismutaseRESUMO
Microglia are the immune cells of the brain. Here we show a massive infiltration of highly ramified and elongated microglia within the core of amyloid plaques in transgenic mouse models of Alzheimer's disease (AD). Many of these cells originate from the bone marrow, and the beta-amyloid-40 and -42 isoforms are able to trigger this chemoattraction. These newly recruited cells also exhibit a specific immune reaction to both exogenous and endogenous beta-amyloid in the brain. Creation of a new AD transgenic mouse that expresses the thymidine kinase protein under the control of the CD11b promoter allowed us to show that blood-derived microglia and not their resident counterparts have the ability to eliminate amyloid deposits by a cell-specific phagocytic mechanism. These bone marrow-derived microglia are thus very efficient in restricting amyloid deposits. Therapeutic strategies aiming to improve their recruitment could potentially lead to a new powerful tool for the elimination of toxic senile plaques.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Células da Medula Óssea/fisiologia , Microglia/fisiologia , Placa Amiloide/patologia , Fatores Etários , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Transplante de Medula Óssea/métodos , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imageamento Tridimensional/métodos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Indóis , Injeções Intraventriculares/métodos , Interleucina-1/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína Cofatora de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos , Microglia/efeitos dos fármacos , Microscopia Confocal/métodos , Fragmentos de Peptídeos/farmacologia , Fagocitose/fisiologia , Placa Amiloide/metabolismo , Presenilina-1 , RNA Mensageiro/metabolismo , Fatores de Tempo , Receptor 2 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Irradiação Corporal Total/métodosRESUMO
Approximately 10% of the cases of amyotrophic lateral sclerosis (ALS) are inherited, with the majority of identified linkages in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies showed that human wild-type SOD1 (SOD1(WT)) overexpression accelerated disease in mice expressing human SOD1 mutants linked to ALS. However, there is a controversy whether the exacerbation mechanism occurs through coaggregation of human SOD1(WT) with SOD1 mutants, stabilization by SOD1(WT) of toxic soluble SOD1 species, or conversion of SOD1(WT) into toxic species through oxidative damage. To further address whether the exacerbation of disease requires misfolding, modifications, and/or interaction of SOD1(WT) with pathogenic forms of SOD1 species, we have studied the effect of human SOD1(WT) overexpression in mice expressing the murine mutant Sod1(G86R). Surprisingly, unlike a previous report with SOD1(G85R) mice, SOD1(WT) overexpression did not affect the life span of Sod1(G86R) mice. Our analysis of spinal cord extracts revealed a lack of heterodimerization or aggregation between human SOD1(WT) and mouse Sod1(G86R) proteins. Moreover, there was no evidence of conversion of SOD1(WT) into misfolded or abnormal SOD1 isoforms based on immunoreactivity with monoclonal antibodies specific to misfolded forms of SOD1 mutants and on analysis of SOD1 isoforms after two-dimensional gel electrophoresis. We conclude that a direct interaction between wild type and mutant forms of SOD1 is required for exacerbation of ALS disease by SOD1(WT) protein.
Assuntos
Modelos Animais de Doenças , Doença dos Neurônios Motores/enzimologia , Doença dos Neurônios Motores/genética , Medula Espinal/enzimologia , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Progressão da Doença , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Doença dos Neurônios Motores/patologia , Mutação , Medula Espinal/patologia , Superóxido Dismutase/fisiologia , Superóxido Dismutase-1RESUMO
The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TK(mt-30) mice expressing a mutated HSV-1 thymidine kinase (TK) gene regulated by the myeloid-specific CD11b promoter. Continuous delivery of ganciclovir at a sciatic nerve lesion site greatly decreased the number of granulocytes/inflammatory monocytes and macrophages in the distal stump of CD11b-TK(mt-30) mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TK(mt-30) mice. This was caused by an unsuitable growth environment rather than an altered regeneration capacity of neurons. In absence of CD11b+ cells, the clearance of inhibitory myelin debris was prevented, neurotrophin synthesis was abolished, and blood vessel formation/maintenance was severely compromised in the sciatic nerve distal stump. Spinal cord-injured axons also failed to regenerate through peripheral nerve grafts in the absence of CD11b+ cells. Therefore, myeloid cells support axonal regeneration and functional recovery by creating a growth-permissive milieu for injured axons.
Assuntos
Antígenos CD11/imunologia , Cones de Crescimento/imunologia , Células Mieloides/imunologia , Regeneração Nervosa/imunologia , Animais , Antivirais/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Ganciclovir/farmacologia , Sobrevivência de Enxerto/fisiologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Cones de Crescimento/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Neuropatia Ciática/imunologia , Neuropatia Ciática/fisiopatologia , Timidina Quinase/genética , Timidina Quinase/imunologia , Transplante de TecidosRESUMO
Microglial activation is a hallmark of all neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here, a detailed characterization of the microglial cell population within the spinal cord of a mouse model of familial ALS was performed. Using flow cytometry, we detected three distinct microglial populations within the spinal cord of mice overexpressing mutant superoxide dismutase (SOD1): mature microglial cells (CD11b(+), CD45(low)), myeloid precursor cells (CD11b(+), CD45(int)), and macrophages (CD11b(+), CD45(high)). Characterization of cell proliferation within the CNS of SOD1(G93A) mice revealed that the expansion in microglial cell population is mainly attributable to the proliferation of myeloid precursor cells. To assess the contribution of proliferating microglia in motor neuron degeneration, we generated CD11b-TK(mut-30); SOD1(G93A) doubly transgenic mice that allow the elimination of proliferating microglia on administration of ganciclovir. Surprisingly, a 50% reduction in reactive microglia specifically in the lumbar spinal cord of CD11b-TK(mut-30); SOD1(G93A) doubly transgenic mice had no effect on motor neuron degeneration. This suggests that proliferating microglia-expressing mutant SOD1 are not central contributors of the neurodegenerative process in ALS caused by mutant SOD1.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Microglia/patologia , Neurônios Motores/patologia , Mutação , Degeneração Neural/patologia , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/complicações , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Contagem de Células , Proliferação de Células , Senescência Celular , Células Dendríticas/metabolismo , Progressão da Doença , Camundongos , Camundongos Transgênicos , Microglia/imunologia , Degeneração Neural/etiologia , Neuroglia/patologia , Fenótipo , Medula Espinal/patologia , Células-Tronco/patologia , Superóxido Dismutase-1 , Linfócitos T/patologiaRESUMO
Macrophages can promote the growth of some tumors, such as those of the breast and lung, but it is unknown whether this is true for all tumors, including those of the nervous system. On the contrary, we have previously shown that macrophages can slow the progression of malignant gliomas through a tumor necrosis factor-dependent mechanism. Here, we provide evidence suggesting that this antitumor effect could be mediated by T lymphocytes, as their number was drastically reduced in tumor necrosis factor-deficient mice and inversely correlated with glioma volume. However, this correlation was only observed in allogeneic recipients, prompting a reevaluation of the role of macrophages in a nonimmunogenic context. Using syngeneic mice expressing the herpes simplex virus thymidine kinase under the control of the CD11b promoter, we show that macrophages can exert an antitumor effect without the help of T lymphocytes. Macrophage depletion achieved by ganciclovir treatment resulted in a 33% increase in glioma volume. The antitumor effect of macrophages was not likely due to a tumoricidal activity because phagocytosis or apoptosis of glioma cells, transduced ex vivo with a lentiviral vector expressing green fluorescent protein, was rarely observed. Their antitumor effect was also not due to a destructive action on the tumor vasculature because macrophage depletion resulted in a modest reduction in vascular density. Therefore, this study suggests that macrophages can attenuate glioma growth by an unconventional mechanism. This study also validates a new transgenic model to explore the role of macrophages in cancer.
Assuntos
Glioma/imunologia , Macrófagos/imunologia , Neoplasias do Sistema Nervoso/imunologia , Animais , Processos de Crescimento Celular/imunologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Glioma/irrigação sanguínea , Glioma/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Macrófagos/patologia , Masculino , Camundongos , Neovascularização Patológica/imunologia , Neoplasias do Sistema Nervoso/irrigação sanguínea , Neoplasias do Sistema Nervoso/patologia , Linfócitos T/imunologiaRESUMO
We generated mice with doxycycline control of a human neurofilament light (NF-L) transgene in the context of the absence (tTA;hNF-L;NF-L(-/-)) or presence (tTA;hNF-L;NF-L(+/-)) of endogenous mouse NF-L proteins. Doxycycline treatment caused the rapid disappearance of human NF-L (hNF-L) mRNA in tTA;hNF-L mice, but the hNF-L proteins remained with a half-life of 3 weeks in the brain. In the sciatic nerve, the disappearance of hNF-L proteins after doxycycline treatment occurred in synchrony along the sciatic nerve, suggesting a proteolysis of NF proteins along the entire axon. The presence of permanent NF network in tTA;hNF-L;NF-L(+/-) mice further stabilized and extended longevity of hNF-L proteins by several months. Surprisingly, after cessation of doxycycline treatment, there was no evidence of leading front of newly synthesized hNF-L proteins migrating into sciatic nerve axons devoid of NF structures. The hNF-L proteins detected at weekly intervals reappeared and accumulated in synchrony at similar rate along nerve segments, a phenomenon consistent with a fast hNF-L transport into axons. We estimated the hNF-L transport rate to be of approximately 10 mm/d in axons devoid of NF structures based on the use of an adenovirus encoding tet-responsive transcriptional activator to transactivate the hNF-L transgene in hypoglossal motor neurons. These results provide in vivo evidence that the stationary NF network in axons is a key determinant of half-life and transport rate of NF proteins.
Assuntos
Transporte Axonal/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Neurofilamentos/biossíntese , Proteínas de Neurofilamentos/genética , Animais , Meia-Vida , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/fisiologia , Transporte Proteico/genéticaRESUMO
Here we report in vivo evidence of a neuroprotective role of proliferating microglial cells in cerebral ischemia. Using transgenic mice expressing a mutant thymidine kinase form of herpes simplex virus driven by myeloid-specific CD11b promoter and ganciclovir treatment as a tool, we selectively ablated proliferating (Mac-2 positive) microglia after transient middle cerebral artery occlusion. The series of experiments using green fluorescent protein-chimeric mice demonstrated that within the first 72 h after ischemic injury, the Mac-2 marker [unlike Iba1 (ionized calcium-binding adapter molecule 1)] was preferentially expressed by the resident microglia. Selective ablation of proliferating resident microglia was associated with a marked alteration in the temporal dynamics of proinflammatory cytokine expression, a significant increase in the size of infarction associated with a 2.7-fold increase in the number of apoptotic cells, predominantly neurons, and a 1.8-fold decrease in the levels of IGF-1. A double-immunofluorescence analysis revealed a approximately 100% colocalization between IGF-1 positive cells and Mac-2, a marker of activated/proliferating resident microglia. Conversely, stimulation of microglial proliferation after cerebral ischemia by M-CSF (macrophage colony stimulating factor) resulted in a 1.9-fold increase in IGF-1 levels and a significant increase of Mac2+ cells. Our findings suggest that a postischemic proliferation of the resident microglial cells may serve as an important modulator of a brain inflammatory response. More importantly, our results revealed a marked neuroprotective potential of proliferating microglia serving as an endogenous pool of neurotrophic molecules such as IGF-1, which may open new therapeutic avenues in the treatment of stroke and other neurological disorders.
Assuntos
Isquemia Encefálica/patologia , Encéfalo/patologia , Proliferação de Células , Microglia/patologia , Animais , Antivirais/farmacologia , Apoptose , Encéfalo/metabolismo , Antígeno CD11b/genética , Proliferação de Células/efeitos dos fármacos , Infarto Cerebral/patologia , Citocinas/metabolismo , Citoproteção , Galectina 3/metabolismo , Ganciclovir/farmacologia , Mediadores da Inflamação/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos/virologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Mutação , Neurônios/patologia , Simplexvirus/genética , Timidina Quinase/genética , Distribuição TecidualRESUMO
Mutations in the gigaxonin gene are responsible for giant axonal neuropathy (GAN), a progressive neurodegenerative disorder associated with abnormal accumulations of Intermediate Filaments (IFs). Gigaxonin is the substrate-specific adaptor for a new Cul3-E3-ubiquitin ligase family that promotes the proteasome dependent degradation of its partners MAP1B, MAP8 and tubulin cofactor B. Here, we report the generation of a mouse model with targeted deletion of Gan exon 1 (Gan(Deltaexon1;Deltaexon1)). Analyses of the Gan(Deltaexon1;Deltaexon1) mice revealed increased levels of various IFs proteins in the nervous system and the presence of IFs inclusion bodies in the brain. Despite deficiency of full length gigaxonin, the Gan(Deltaexon1;Deltaexon1) mice do not develop overt neurological phenotypes and giant axons reminiscent of the human GAN disease. Nonetheless, at 6 months of age the Gan(Deltaexon1;Deltaexon1) mice exhibit a modest hind limb muscle atrophy, a 10% decrease of muscle innervation and a 27% axonal loss in the L5 ventral roots. This new mouse model should provide a useful tool to test potential therapeutic approaches for GAN disease.
Assuntos
Encéfalo/metabolismo , Proteínas do Citoesqueleto/deficiência , Atrofia Muscular/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Degeneração Walleriana/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Encéfalo/fisiopatologia , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Células-Tronco Embrionárias , Éxons/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Gânglios Espinais/fisiopatologia , Marcação de Genes/métodos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologia , Atrofia Muscular/genética , Atrofia Muscular/fisiopatologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/fisiopatologia , Fenótipo , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/patologia , Raízes Nervosas Espinhais/fisiopatologia , Degeneração Walleriana/genética , Degeneração Walleriana/fisiopatologiaRESUMO
Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a promising therapy for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is being used in a clinical trial for amyotrophic lateral sclerosis. However, chronic trophic factor delivery prohibits dose adjustment or cessation if side effects develop. To address this, we engineered a doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule. Human induced pluripotent stem cell (iPSC)-derived neural progenitors were stably transfected with the vector and transplanted into the adult mouse brain. Doxycycline can penetrate the graft, with addition and withdrawal providing inducible and reversible GDNF expression in vivo, over multiple cycles. Our findings provide proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Transplante de Células-Tronco , Terapia Baseada em Transplante de Células e Tecidos , Expressão Gênica , Genes Reporter , Terapia Genética , Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Plantas Geneticamente Modificadas , Transplante de Células-Tronco/métodos , Transdução Genética , TransgenesRESUMO
An increase in the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) has been observed in patients with amyotrophic lateral sclerosis (ALS) and in the mice models of the disease. TNF-alpha is a potent activator of macrophages and microglia and, under certain conditions, can induce or exacerbate neuronal cell death. Here, we assessed the contribution of TNF-alpha in motor neuron disease in mice overexpressing mutant superoxide dismutase 1 (SOD1) genes linked to familial ALS. This was accomplished by the generation of mice expressing SOD1(G37R) or SOD1(G93A) mutants in the context of TNF-alpha gene knock out. Surprisingly, the absence of TNF-alpha did not affect the lifespan or the extent of motor neuron loss in SOD1 transgenic mice. These results provide compelling evidence indicating that TNF-alpha does not directly contribute to motor neuron degeneration caused by SOD1 mutations.
Assuntos
Doença dos Neurônios Motores/genética , Mutação , Superóxido Dismutase/genética , Fator de Necrose Tumoral alfa/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Doença dos Neurônios Motores/enzimologia , Doença dos Neurônios Motores/patologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Ex vivo gene therapy involves the genetic modification of cells outside of the body to produce therapeutic factors and their subsequent transplantation back into patients. Various cell types can be genetically engineered. However, with the explosion in stem cell technologies, neural stem/progenitor cells and mesenchymal stem cells are most often used. The synergy between the effect of the new cell and the additional engineered properties can often provide significant benefits to neurodegenerative changes in the brain. In this review, we cover both preclinical animal studies and clinical human trials that have used ex vivo gene therapy to treat neurological disorders with a focus on Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, and stroke. We highlight some of the major advances in this field including new autologous sources of pluripotent stem cells, safer ways to introduce therapeutic transgenes, and various methods of gene regulation. We also address some of the remaining hurdles including tunable gene regulation, in vivo cell tracking, and rigorous experimental design. Overall, given the current outcomes from researchers and clinical trials, along with exciting new developments in ex vivo gene and cell therapy, we anticipate that successful treatments for neurological diseases will arise in the near future.
Assuntos
Terapia Genética , Doenças do Sistema Nervoso/terapia , Animais , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Transplante de Células-Tronco Mesenquimais , Doenças do Sistema Nervoso/genética , Células-Tronco Neurais , Transplante de Células-TroncoRESUMO
Age-associated health decline presents a significant challenge to healthcare, although there are few animal models that can be used to test potential treatments. Here, we show that there is a significant reduction in both spinal cord motor neurons and motor function over time in the aging rat. One explanation for this motor neuron loss could be reduced support from surrounding aging astrocytes. Indeed, we have previously shown using in vitro models that aging rat astrocytes are less supportive to rat motor neuron function and survival over time. Here, we test whether rejuvenating the astrocyte niche can improve the survival of motor neurons in an aging spinal cord. We transplanted fetal-derived human neural progenitor cells (hNPCs) into the aging rat spinal cord and found that the cells survive and differentiate into astrocytes with a much higher efficiency than when transplanted into younger animals, suggesting that the aging environment stimulates astrocyte maturation. Importantly, the engrafted astrocytes were able to protect against motor neuron loss associated with aging, although this did not result in an increase in motor function based on behavioral assays. We also transplanted hNPCs genetically modified to secrete glial cell line-derived neurotrophic factor (GDNF) into the aging rat spinal cord, as this combination of cell and protein delivery can protect motor neurons in animal models of ALS. During aging, GDNF-expressing hNPCs protected motor neurons, though to the same extent as hNPCs alone, and again had no effect on motor function. We conclude that hNPCs can survive well in the aging spinal cord, protect motor neurons and mature faster into astrocytes when compared to transplantation into the young spinal cord. While there was no functional improvement, there were no functional deficits either, further supporting a good safety profile of hNPC transplantation even into the older patient population.
Assuntos
Envelhecimento/fisiologia , Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Neurônios Motores/fisiologia , Transtornos dos Movimentos/cirurgia , Células-Tronco Neurais/fisiologia , Fatores Etários , Animais , Peso Corporal/fisiologia , Córtex Cerebral/citologia , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Feto/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Masculino , Transtornos dos Movimentos/patologia , Transtornos dos Movimentos/fisiopatologia , Força Muscular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/transplante , Junção Neuromuscular/fisiologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/transplanteRESUMO
Modeling amyotrophic lateral sclerosis (ALS) with human induced pluripotent stem cells (iPSCs) aims to reenact embryogenesis, maturation and aging of spinal motor neurons (spMNs) in vitro. As the maturity of spMNs grown in vitro compared to spMNs in vivo remains largely unaddressed, it is unclear to what extent this in vitro system captures critical aspects of spMN development and molecular signatures associated with ALS. Here, we compared transcriptomes among iPSC-derived spMNs, fetal spinal tissues and adult spinal tissues. This approach produced a maturation scale revealing that iPSC-derived spMNs were more similar to fetal spinal tissue than to adult spMNs. Additionally, we resolved gene networks and pathways associated with spMN maturation and aging. These networks enriched for pathogenic familial ALS genetic variants and were disrupted in sporadic ALS spMNs. Altogether, our findings suggest that developing strategies to further mature and age iPSC-derived spMNs will provide more effective iPSC models of ALS pathology.
Assuntos
Envelhecimento , Esclerose Lateral Amiotrófica/metabolismo , Neurônios Motores/citologia , Neurogênese/fisiologia , Esclerose Lateral Amiotrófica/fisiopatologia , Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurogênese/genéticaRESUMO
Innate immunity is a specific and organized immunological program engaged by peripheral organs and the CNS to maintain homeostasis after stress and injury. In neurodegenerative disorders, its putative deregulation, featured by inflammation and activation of glial cells resulting from inherited mutations or viral/bacterial infections, likely contributes to neuronal death. However, it remains unclear to what extent environmental factors and innate immunity cooperate to modulate the interactions between the neuronal and non-neuronal elements in the perturbed CNS. In the present study, we addressed the effects of acute and chronic administration of lipopolysaccharide (LPS), a Gram-negative bacterial wall component, in a genetic model of neurodegeneration. Transgenic mice expressing a mutant form of the superoxide dismutase 1 (SOD1(G37R)) linked to familial amyotrophic lateral sclerosis were challenged intraperitoneally with a single nontoxic or repeated injections of LPS (1 mg/kg). At different ages, SOD1(G37R) mice responded normally to acute endotoxemia. Remarkably, only a chronic challenge with LPS in presymptomatic 6-month-old SOD1(G37R) mice exacerbated disease progression by 3 weeks and motor axon degeneration. Closely associated with the severity of disease is the stronger and restricted upregulation of the receptor of innate immunity Toll-like receptor 2 and proinflammatory cytokines in degenerating regions of the ventral spinal cord and efferent fiber tracts of the brain from the LPS-treated SOD1(G37R) mice. This robust immune response was not accompanied by the establishment of acquired immunity. Our results provide solid evidence that environmental factors and innate immunity can cooperate to influence the course of disease of an inherited neuropathology.
Assuntos
Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/fisiopatologia , Imunidade Inata/imunologia , Imunização/métodos , Esclerose Lateral Amiotrófica/patologia , Animais , Axônios/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica/imunologia , Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Microglia/imunologia , Neurônios Motores/patologia , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Medula Espinal/metabolismo , Medula Espinal/patologia , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Taxa de Sobrevida , Receptor 2 Toll-Like , Receptores Toll-LikeRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease in which upper and lower motor neurons degenerate, leading to muscle atrophy, paralysis, and death within 3 to 5 years of onset. While a small percentage of ALS cases are genetically linked, the majority are sporadic with unknown origin. Currently, etiological links are associated with disease onset without mechanistic understanding. Of all the putative risk factors, however, head trauma has emerged as a consistent candidate for initiating the molecular cascades of ALS. Here, we test the hypothesis that traumatic brain injury (TBI) in the SOD1 (G93A) transgenic rat model of ALS leads to early disease onset and shortened lifespan. We demonstrate, however, that a one-time acute focal injury caused by controlled cortical impact does not affect disease onset or survival. Establishing the negligible involvement of a single acute focal brain injury in an ALS rat model increases the current understanding of the disease. Critically, untangling a single focal TBI from multiple mild injuries provides a rationale for scientists and physicians to increase focus on repeat injuries to hopefully pinpoint a contributing cause of ALS.
RESUMO
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.