RESUMO
Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100ß, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.
Assuntos
Astrócitos/fisiologia , Engenharia Celular/métodos , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Animais , Astrócitos/transplante , Sobrevivência Celular/fisiologia , Células Cultivadas , Desoxirribonucleases , Dependovirus/genética , Fibroblastos/fisiologia , Genes Reporter , Vetores Genéticos , Substância Cinzenta/citologia , Substância Cinzenta/fisiologia , Substância Cinzenta/cirurgia , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Regiões Promotoras Genéticas , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/fisiologia , Medula Espinal/cirurgia , Dedos de ZincoRESUMO
The astrocyte glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake in the adult central nervous system (CNS), thereby regulating extracellular glutamate homeostasis and preventing excitotoxicity. Glutamate dysregulation plays a central role in outcome following traumatic spinal cord injury (SCI). To determine the role of GLT1 in secondary cell loss following SCI, mice heterozygous for the GLT1 astrocyte glutamate transporter (GLT1+/-) and wild-type mice received thoracic crush SCI. Compared with wild-type controls, GLT1+/- mice had an attenuated recovery in hindlimb motor function, increased lesion size, and decreased tissue sparing. GLT1+/- mice showed a decrease in intraspinal GLT1 protein and functional glutamate uptake compared with wild-type mice, accompanied by increased apoptosis and neuronal loss following crush injury. These results suggest that astrocyte GLT1 plays a role in limiting secondary cell death following SCI, and also show that compromise of key astrocyte functions has significant effects on outcome following traumatic CNS injury. These findings also suggest that increasing intraspinal GLT1 expression may represent a therapeutically relevant target for SCI treatment.
Assuntos
Astrócitos/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Gliose/metabolismo , Ácido Glutâmico/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Animais , Astrócitos/patologia , Modelos Animais de Doenças , Regulação para Baixo/genética , Transportador 2 de Aminoácido Excitatório/deficiência , Gliose/genética , Camundongos , Camundongos Knockout , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologiaRESUMO
IMPORTANCE: Newborn screening (NBS) identifies infants with specific congenital disorders for which earlier intervention cannot only prevent a lifetime of chronic disability but also, most importantly, save lives. In this article, we discuss complexities associated with NBS processes in the United States, with a focus on challenges in neuromuscular disorders. OBSERVATIONS: As new interventions for neuromuscular disorders become available, the clinical community must prepare to overcome the challenges of adding new diseases to screening panels and understand the rigorous evidence review at the federal level and the complex process of state-level implementation. In this regard, NBS programs for Pompe disease and spinal muscular atrophy can guide the path of Duchenne muscular dystrophy and other neuromuscular disorders as future candidates for NBS. CONCLUSIONS AND RELEVANCE: The availability of advanced screening methods, the emergence of effective treatment, and the support of professional organizations may facilitate the expansion of NBS, such that an increasing number of infants can be identified in the newborn period who will benefit from life-saving interventions.
RESUMO
BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of alpha motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron 1 gene (SMN1). In humans, a nearly identical copy gene, SMN2, is present. Because SMN2 has been shown to decrease disease severity in a dose-dependent manner, SMN2 copy number is predictive of disease severity. OBJECTIVE: To develop a treatment algorithm for SMA-positive infants identified through newborn screening based upon SMN2 copy number. METHODS: A working group comprised of 15 SMA experts participated in a modified Delphi process, moderated by a neutral third-party expert, to develop treatment guidelines. RESULTS: The overarching recommendation is that all infants with two or three copies of SMN2 should receive immediate treatment (nâ=â13). For those infants in which immediate treatment is not recommended, guidelines were developed that outline the timing and appropriate screens and tests to be used to determine the timing of treatment initiation. CONCLUSIONS: The identification SMA affected infants via newborn screening presents an unprecedented opportunity for achievement of maximal therapeutic benefit through the administration of treatment pre-symptomatically. The recommendations provided here are intended to help formulate treatment guidelines for infants who test positive during the newborn screening process.
Assuntos
Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos/uso terapêutico , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Assistência ao Convalescente , Algoritmos , Desenvolvimento Infantil , Variações do Número de Cópias de DNA , Técnica Delphi , Gerenciamento Clínico , Intervenção Médica Precoce , Eletromiografia , Dosagem de Genes , Humanos , Lactente , Recém-Nascido , Destreza Motora , Triagem Neonatal , Guias de Prática Clínica como Assunto , Índice de Gravidade de Doença , Atrofias Musculares Espinais da Infância/diagnóstico , Atrofias Musculares Espinais da Infância/genética , Atrofias Musculares Espinais da Infância/fisiopatologia , Proteína 2 de Sobrevivência do Neurônio Motor/genética , TempoRESUMO
ALS is a neurodegenerative disease with a prevalence rate of up to 7.4/100,000 and the overall risk of developing ALS over a lifetime is 1:400. Most patients die from respiratory failure following a course of progressive weakness. To date, only one traditional pharmaceutical agent-riluzole, has been shown to afford a benefit on survival but numerous pharmaceutical interventions have been studied in preclinical models of ALS without subsequent translation to patient efficacy. Despite the relative selectivity of motor neuron cell death, animal and tissue culture models of familial ALS suggest that non-neuronal cells significantly contribute to neuronal dysfunction and death. Early efforts to transplant stem cells had focused on motor neuron replacement. More practically for this aggressive neurodegenerative disease, recent studies, preclinical efforts, and early clinical trials have focused on the transplantation of neural stem cells, mesenchymal stem cells, or glial progenitors. Using transgenic mouse or rat models of ALS, a number of studies have shown neuroprotection through a variety of different mechanisms that have included neurotrophic factor secretion, glutamate transporter regulation, and modulation of neuroinflammation, among others. However, given that cell replacement could involve a number of biologically relevant factors, identifying the key pathway(s) that may contribute to neuroprotection remains a challenge. Nevertheless, given the abundant data supporting the interplay between non-neuronal cell types and motor neuron disease propagation, the replacement of disease-carrying host cells by normal cells may be sufficient to confer neuroprotection. Key preclinical issues that currently are being addressed include the most appropriate methods and routes for delivery of cells to disease-relevant regions of the neuraxis, cell survival and migration, and tracking the cells following transplantation. Central to the initial development of stem cell transplantation into patients with ALS is the demonstration that transplanted cells lack tumorigenicity and have the appropriate biodistribution to ensure the safety of ALS patients receiving these therapies. Here, we review preclinical and clinical studies focusing on the transplantation of neural and glial progenitor cells as a promising neuroprotective therapy for ALS. The rationale for stem cell transplantation for neuroprotection, proof-of-concept animal studies, and current challenges facing translation of these therapies to the clinic is presented. Lastly, we discuss advancements on the horizon including induced pluripotent stem cell technology and developments for cellular tracking and detection post-transplantation. With the safe completion of the first-in-human Phase I clinical trial for intraspinal stem cell transplantation for ALS in the United States, the time is ripe for stem cell therapies to be translated to the clinic and excitingly, evaluated for neuroprotection for ALS. This article is part of a Special Issue entitled SI: Neuroprotection.
Assuntos
Esclerose Lateral Amiotrófica/cirurgia , Neuroglia/fisiologia , Neurônios/fisiologia , Transplante de Células-Tronco/métodos , Animais , HumanosRESUMO
Although Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease, basic research studies have highlighted that astrocytes contribute to the disease process. Therefore, strategies which replace the diseased astrocyte population with healthy astrocytes may protect against motor neuron degeneration. Our studies have sought to evaluate astrocyte replacement using glial-restricted progenitors (GRPs), which are lineage-restricted precursors capable of differentiating into astrocytes after transplantation. The goal of our current study was to evaluate how transplantation to the diseased ALS spinal cord versus a healthy, wild-type spinal cord may affect human GRP engraftment and selected gene expression. Human GRPs were transplanted into the spinal cord of either an ALS mouse model or wild-type littermate mice. Mice were sacrificed for analysis at either the onset of disease course or at the endstage of disease. The transplanted GRPs were analyzed by immunohistochemistry and NanoString gene profiling which showed no gross differences in the engraftment or gene expression of the cells. Our data indicate that human glial progenitor engraftment and gene expression is independent of the neurodegenerative ALS spinal cord environment. These findings are of interest given that human GRPs are currently in clinical development for spinal cord transplantation into ALS patients.
Assuntos
Esclerose Lateral Amiotrófica/cirurgia , Regulação da Expressão Gênica/fisiologia , Neuroglia/fisiologia , Neuroglia/transplante , Transplante de Células-Tronco/métodos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Antígenos Nucleares/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cadáver , Movimento Celular , Proliferação de Células/genética , Modelos Animais de Doenças , Feto , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Medula Espinal/metabolismo , Medula Espinal/patologia , Células-Tronco , Superóxido Dismutase/genéticaRESUMO
The generation of human induced pluripotent stem cells (hiPSCs) represents an exciting advancement with promise for stem cell transplantation therapies as well as for neurological disease modeling. Based on the emerging roles for astrocytes in neurological disorders, we investigated whether hiPSC-derived astrocyte progenitors could be engrafted to the rodent spinal cord and how the characteristics of these cells changed between in vitro culture and after transplantation to the in vivo spinal cord environment. Our results show that human embryonic stem cell- and hiPSC-derived astrocyte progenitors survive long-term after spinal cord engraftment and differentiate to astrocytes in vivo with few cells from other lineages present. Gene profiling of the transplanted cells demonstrates the astrocyte progenitors continue to mature in vivo and upregulate a variety of astrocyte-specific genes. Given this mature astrocyte gene profile, this work highlights hiPSCs as a tool to investigate disease-related astrocyte biology using in vivo disease modeling with significant implications for human neurological diseases currently lacking animal models.
Assuntos
Astrócitos , Diferenciação Celular , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Medula Espinal , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Regulação da Expressão Gênica , Xenoenxertos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Neuroinflammation is one of the most striking hallmarks of amyotrophic lateral sclerosis (ALS). Nuclear factor-kappa B (NF-κB), a master regulator of inflammation, is upregulated in spinal cords of ALS patients and SOD1-G93A mice. In this study, we show that selective NF-κB inhibition in ALS astrocytes is not sufficient to rescue motor neuron (MN) death. However, the localization of NF-κB activity and subsequent deletion of NF-κB signaling in microglia rescued MNs from microglial-mediated death in vitro and extended survival in ALS mice by impairing proinflammatory microglial activation. Conversely, constitutive activation of NF-κB selectively in wild-type microglia induced gliosis and MN death in vitro and in vivo. Taken together, these data provide a mechanism by which microglia induce MN death in ALS and suggest a novel therapeutic target that can be modulated to slow the progression of ALS and possibly other neurodegenerative diseases by which microglial activation plays a role.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Morte Celular/fisiologia , Microglia/citologia , Neurônios Motores/citologia , NF-kappa B/metabolismo , Fatores Etários , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microglia/metabolismo , Neurônios Motores/metabolismo , NF-kappa B/antagonistas & inibidores , Cultura Primária de Células , Transdução de Sinais/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
The role of glia as a contributing factor to motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) is becoming increasingly appreciated. However, most studies implicating astrocytes have focused solely on models of ALS caused by superoxide dismutase 1 (SOD1) mutations. The goal of our study was to determine whether astrocytes contribute to wild-type MN death in the case of ALS caused by mutations in tar-DNA binding protein 43 (TDP-43). Since it is currently unknown how TDP-43 mutations cause disease, we derived astrocytes for study from both gain and loss of function mouse models of TDP-43. Astrocytes overexpressing mutant TDP-43(A315T) as well as astrocytes lacking TDP-43 were morphologically indistinguishable from wild-type astrocytes in vitro. Furthermore, astrocytes with these TDP-43 alterations did not cause the death of wild-type MNs in co-culture. To investigate the in vivo effects of TDP-43 alterations in astrocytes, glial-restricted precursors were transplanted to the wild-type rat spinal cord where they differentiated into astrocytes and interacted with host MNs. Astrocytes with TDP-43 alterations did not cause host wild-type MN damage although they were capable of engrafting and interacting with host MNs with the same efficiency as wild-type astrocytes. These data indicate that astrocytes do not adopt the same toxic phenotype as mutant SOD1 astrocytes when TDP-43 is mutated or expression levels are modified. Our study reinforces the heterogeneity in ALS disease mechanisms and highlights the potential for future screening subsets of ALS patients prior to treatment with cell type-directed therapies.
Assuntos
Esclerose Lateral Amiotrófica/genética , Astrócitos/metabolismo , Proteínas de Ligação a DNA/genética , Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Sobrevivência Celular , Técnicas de Cocultura , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-DawleyRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.