RESUMO
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is caused by the loss of the survival motor neuron-1 (SMN1) gene, which leads to motor neuron loss, muscle atrophy, respiratory distress, and death. Motor neurons exhibit the most profound loss, but the mechanisms underlying disease pathogenesis are not fully understood. Recent evidence suggests that motor neuron extrinsic influences, such as those arising from astrocytes, contribute to motor neuron malfunction and loss. Here we investigated both loss-of-function and toxic gain-of-function astrocyte mechanisms that could play a role in SMA pathology. We had previously found that glial derived neurotrophic factor (GDNF) is reduced in SMA astrocytes. However, reduced GDNF expression does not play a major role in SMA pathology as viral-mediated GDNF re-expression did not improve astrocyte function or motor neuron loss. In contrast, we found that SMA astrocytes increased microRNA (miR) production and secretion compared to control astrocytes, suggesting potential toxic gain-of-function properties. Specifically, we found that miR-146a was significantly upregulated in SMA induced pluripotent stem cell (iPSC)-derived astrocytes and SMNΔ7 mouse spinal cord. Moreover, increased miR-146a was sufficient to induce motor neuron loss in vitro, whereas miR-146a inhibition prevented SMA astrocyte-induced motor neuron loss. Together, these data indicate that altered astrocyte production of miR-146a may be a contributing factor in astrocyte-mediated SMA pathology.
Assuntos
MicroRNAs/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , MicroRNAs/genética , Neurônios Motores/metabolismo , Degeneração Neural/patologia , Medula Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Regulação para CimaRESUMO
Spinal muscular atrophy (SMA) is a genetic disorder characterized by loss of motor neurons in the spinal cord leading to muscle atrophy and death. Although motor neurons (MNs) are the most obviously affected cells in SMA, recent evidence suggest dysfunction in multiple cell types. Astrocytes are a crucial component of the motor circuit and are intimately involved with MN health and maintenance. We have previously shown that SMA astrocytes are altered both morphologically and functionally early in disease progression, though it is unclear what causes astrocytes to become reactive. Oxidative stress is a common feature among neurodegenerative diseases. Oxidative stress can both induce apoptosis in neurons and can cause astrocytes to become reactive, which are features observed in the SMA induced pluripotent stem cell (iPSC) cultures. Therefore, we asked if oxidative stress contributes to SMA astrocyte pathology. We examined mitochondrial bioenergetics, transcript and protein levels of oxidative and anti-oxidant factors, and reactive oxygen species (ROS) production and found little evidence of oxidative stress. We did observe a significant increase in endogenous catalase expression in SMA iPSCs. While catalase knockdown in SMA iPSCs increased ROS production above basal levels, levels of ROS remained lower than in controls, further arguing against robust oxidative stress in this system. Viral delivery of survival motor neuron (SMN) reversed astrocyte activation and restored catalase levels to normal, without changing mitochondrial respiration or expression of oxidative stress markers. Taken together, these data indicate that SMN deficiency induces astrocyte reactivity, but does not do so through an oxidative stress-mediated process.
Assuntos
Astrócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/metabolismo , Células-Tronco Neurais/metabolismo , Astrócitos/patologia , Catalase/antagonistas & inibidores , Catalase/genética , Catalase/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Células-Tronco Neurais/patologia , Fosforilação Oxidativa , Estresse Oxidativo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Glutationa Peroxidase GPX1RESUMO
Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA.
Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Atrofia Muscular Espinal/patologia , Animais , Diferenciação Celular , Dependovirus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Fenótipo , Medula Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Loss of Survival Motor Neuron-1 (SMN1) causes Spinal Muscular Atrophy, a devastating neurodegenerative disease. SMN2 is a nearly identical copy gene; however SMN2 cannot prevent disease development in the absence of SMN1 since the majority of SMN2-derived transcripts are alternatively spliced, encoding a truncated, unstable protein lacking exon 7. Nevertheless, SMN2 retains the ability to produce low levels of functional protein. Previously we have described a splice-switching Morpholino antisense oligonucleotide (ASO) sequence that targets a potent intronic repressor, Element1 (E1), located upstream of SMN2 exon 7. In this study, we have assessed a novel panel of Morpholino ASOs with the goal of optimizing E1 ASO activity. Screening for efficacy in the SMNΔ7 mouse model, a single ASO variant was more active in vivo compared with the original E1(MO)-ASO. Sequence variant eleven (E1(MOv11)) consistently showed greater efficacy by increasing the lifespan of severe Spinal Muscular Atrophy mice after a single intracerebroventricular injection in the central nervous system, exhibited a strong dose-response across an order of magnitude, and demonstrated excellent target engagement by partially reversing the pathogenic SMN2 splicing event. We conclude that Morpholino modified ASOs are effective in modifying SMN2 splicing and have the potential for future Spinal Muscular Atrophy clinical applications.
Assuntos
Íntrons , Morfolinos/genética , Atrofia Muscular Espinal/genética , Elementos de Resposta , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Marcação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/mortalidade , Mutação , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Transcrição GênicaRESUMO
Spinal muscular atrophy (SMA) is a genetic disorder caused by the deletion of the survival motor neuron 1 (SMN1) gene that leads to loss of motor neurons in the spinal cord. Although motor neurons are selectively lost during SMA pathology, selective replacement of SMN in motor neurons does not lead to full rescue in mouse models. Due to the ubiquitous expression of SMN, it is likely that other cell types besides motor neurons are affected by its disruption and therefore may contribute to disease pathology. Here we show that astrocytes in SMAΔ7 mouse spinal cord and from SMA-induced pluripotent stem cells exhibit morphological and cellular changes indicative of activation before overt motor neuron loss. Furthermore, our in vitro studies show mis-regulation of basal calcium and decreased response to adenosine triphosphate stimulation indicating abnormal astrocyte function. Together, for the first time, these data show early disruptions in astrocytes that may contribute to SMA disease pathology.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Atrofia Muscular Espinal/patologia , Medula Espinal/citologia , Trifosfato de Adenosina/farmacologia , Fatores Etários , Aldeído Desidrogenase/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Linhagem Celular Transformada , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/genética , Mutação/genética , Nestina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Células-Tronco Pluripotentes/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Proteínas S100/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
INTRODUCTION: The gender gap in professorship and leadership roles persists in academic medicine, whereas reasons for these disparities remain unclear. MATERIALS AND METHODS: Open-ended text responses to a 2013 faculty engagement survey were analyzed by using the grounded theory and consensual qualitative analysis techniques. The authors grouped 491 faculty's text responses into descriptive codes and three themes: (1) No Obstacles, (2) Barriers to Success, and (3) Concerns Regarding Processes. Demographics of codes were compared by using chi-square analysis. RESULTS: Male faculty identified barriers that included negative views of leadership or leaders. Female faculty, especially those in clinical roles, expressed barriers related to role overload, including that the demands of their current positions prevented advancement or addition of further roles, no matter how desirable further roles may be. Women also shared that considerable self-promotion was required to receive acknowledgement of their work and support by leadership. CONCLUSION: A proposed framework depicts male and female faculty's concerns on a continuum. No Obstacle and Process Concerns were relatively gender neutral, whereas large gender disparities occurred within the Barriers to Success theme. Women's barriers largely revolved around internal obstacles (I can't do any more), and men's barriers largely revolved around external factors (leaders are impeding my progress). Resources are needed to mitigate work overload specifically for female faculty, and to ensure that all faculty are both engaged in advanced career opportunities and encouraged to pursue leadership positions.
Assuntos
Mobilidade Ocupacional , Docentes de Medicina/organização & administração , Liderança , Médicas/psicologia , Sexismo , Centros Médicos Acadêmicos/organização & administração , Feminino , Humanos , Satisfação no Emprego , Masculino , Tutoria , Pesquisa Qualitativa , Inquéritos e QuestionáriosRESUMO
PURPOSE: Albinism is associated with disrupted foveal development, though intersubject variability is becoming appreciated. We sought to quantify this variability, and examine the relationship between foveal cone specialization and pit morphology in patients with a clinical diagnosis of albinism. METHODS: We recruited 32 subjects with a clinical diagnosis of albinism. DNA was obtained from 25 subjects, and known albinism genes were analyzed for mutations. Relative inner and outer segment (IS and OS) lengthening (fovea-to-perifovea ratio) was determined from manually segmented spectral domain-optical coherence tomography (SD-OCT) B-scans. Foveal pit morphology was quantified for eight subjects from macular SD-OCT volumes. Ten subjects underwent imaging with adaptive optics scanning light ophthalmoscopy (AOSLO), and cone density was measured. RESULTS: We found mutations in 22 of 25 subjects, including five novel mutations. All subjects lacked complete excavation of inner retinal layers at the fovea, though four subjects had foveal pits with normal diameter and/or volume. Peak cone density and OS lengthening were variable and overlapped with that observed in normal controls. A fifth hyper-reflective band was observed in the outer retina on SD-OCT in the majority of the subjects with albinism. CONCLUSIONS: Foveal cone specialization and pit morphology vary greatly in albinism. Normal cone packing was observed in the absence of a foveal pit, suggesting a pit is not required for packing to occur. The degree to which retinal anatomy correlates with genotype or visual function remains unclear, and future examination of larger patient groups will provide important insight on this issue.
Assuntos
Albinismo Oculocutâneo/diagnóstico , Fóvea Central/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Acuidade Visual , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Albinismo Oculocutâneo/genética , Albinismo Oculocutâneo/fisiopatologia , Contagem de Células , Criança , DNA/genética , Eletrorretinografia , Proteínas do Olho/genética , Feminino , Fóvea Central/fisiopatologia , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Oftalmoscopia , Células Fotorreceptoras Retinianas Cones/metabolismo , Tomografia de Coerência Óptica , Adulto JovemRESUMO
We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.