RESUMO
The fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) is characterized by a profound loss of motor neurons (MNs). Until now only riluzole minimally extends life expectancy in ALS, presumably by inhibiting glutamatergic neurotransmission and calcium overload of MNs. Therefore, the aim of this study was to investigate the glutamate receptor properties and key aspects of intracellular calcium dynamics in induced pluripotent stem cell (iPSC)-derived MNs from ALS patients with C9orf72 (n = 4 cell lines), fused in sarcoma (FUS) (n = 9), superoxide dismutase 1 (SOD1) (n = 3) or transactive response DNA-binding protein 43 (TDP43) (n = 3) mutations as well as healthy (n = 7 cell lines) and isogenic controls (n = 3). Using calcium imaging, we most frequently observed spontaneous transients in mutant C9orf72 MNs. Basal intracellular calcium levels and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced signal amplitudes were elevated in mutant TDP43 MNs. Besides, a majority of mutant TDP43 MNs responded to 3.5-dihydroxyphenylglycine as metabotropic glutamate receptor agonist. Quantitative real-time PCR demonstrated significantly increased expression levels of AMPA and kainate receptors in mutant FUS cells compared to healthy and isogenic controls. Furthermore, the expression of kainate receptors and voltage gated calcium channels in mutant C9orf72 MNs as well as metabotropic glutamate receptors in mutant SOD1 cells was markedly elevated compared to controls. Our data of iPSC-derived MNs from familial ALS patients revealed several mutation-specific alterations in glutamate receptor properties and calcium dynamics that could play a role in ALS pathogenesis and may lead to future translational strategies with individual stratification of neuroprotective ALS treatments.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Mutação , Receptores de Glutamato/metabolismo , Esclerose Lateral Amiotrófica/diagnóstico , Biomarcadores , Proteína C9orf72/genética , Sinalização do Cálcio , Proteínas de Ligação a DNA/genética , Suscetibilidade a Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteína FUS de Ligação a RNA/genética , Superóxido Dismutase-1/genéticaRESUMO
We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A -dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Astrócitos/enzimologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neurônios Motores/enzimologia , Músculo Esquelético/enzimologia , Superóxido Dismutase-1/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/patologia , Morte Celular , Células Cultivadas , Modelos Animais de Doenças , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/deficiência , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/patologia , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/genéticaRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron disorder. It is supposed that ALS is at least in part an axonopathy. Neuropilin 1 is an important receptor of the axon repellent Semaphorin 3A and a co-receptor of vascular endothelial growth factor. It is probably involved in neuronal and axonal de-/regeneration and might be of high relevance for ALS pathogenesis and/or disease progression. To elucidate whether the expression of either Neuropilin1 or Semaphorin3A is altered in ALS we investigated these proteins in human brain, spinal cord and muscle tissue of ALS-patients and controls as well as transgenic SOD1G93A and control mice. Neuropilin1 and Semaphorin3A gene and protein expression were assessed by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. Groups were compared using either Student t-test or Mann-Whitney U test. We observed a consistent increase of Neuropilin1 expression in the spinal cord and decrease of Neuropilin1 and Semaphorin3A in muscle tissue of transgenic SOD1G93A mice at the mRNA and protein level. Previous studies have shown that damage of neurons physiologically causes Neuropilin1 and Semaphorin3A increase in the central nervous system and decrease in the peripheral nervous system. Our results indicate that this also occurs in ALS. Pharmacological modulation of expression and function of axon repellents could be a promising future therapeutic option in ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Músculo Esquelético/metabolismo , Neuropilina-1/metabolismo , Medula Espinal/metabolismo , Adulto , Idoso , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Semaforina-3A/metabolismoRESUMO
We have previously shown that total knockout of fibroblast growth factor-2 (FGF-2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1G93A ) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1G93A mice, with distinct regulation patterns of FGF-2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF-2-isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1G93A FGF-2 high molecular weight- and SOD1G93A FGF-2 low molecular weight-knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform-dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1G93A FGF-2 low molecular weight knockout mice compared to single mutant SOD1G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1G93A FGF-2 low molecular weight knockout mice implies that FGF-2 low molecular weight knockout (or the presence of the FGF-2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.
Assuntos
Esclerose Lateral Amiotrófica/genética , Fator 2 de Crescimento de Fibroblastos/genética , Longevidade/genética , Isoformas de Proteínas/genética , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Isoformas de Proteínas/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
Despite decades of research on amyotrophic lateral sclerosis (ALS), there is only one approved drug, which minimally extends patient survival. Here, we investigated pathophysiological mechanisms underlying ALS using motor neurons (MNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying mutations in FUS or SOD1. Patient-derived MNs were less active and excitable compared to healthy controls, due to reduced Na(+) /K(+) ratios in both ALS groups accompanied by elevated potassium channel (FUS) and attenuated sodium channel expression levels (FUS, SOD1). ALS iPSC-derived MNs showed elevated endoplasmic reticulum stress (ER) levels and increased caspase activation. Treatment with the FDA approved drug 4-Aminopyridine (4AP) restored ion-channel imbalances, increased neuronal activity levels and decreased ER stress and caspase activation. This study provides novel pathophysiological data, including a mechanistic explanation for the observed hypoexcitability in patient-derived MNs and a new therapeutic strategy to provide neuroprotection in MNs affected by ALS. Stem Cells 2016;34:1563-1575.
Assuntos
4-Aminopiridina/farmacologia , Esclerose Lateral Amiotrófica/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/genética , Caspases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Canais Iônicos/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , Neuroproteção/efeitos dos fármacos , Fenótipo , Proteína FUS de Ligação a RNA/genética , Superóxido Dismutase/genética , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1G93A transgenic ALS mouse model. We observed significant reduction of lifespan of animals treated by intraventricular hMSC injection compared with the vehicle treated control group, accompanied by changes in weight, general condition, and behavioural assessments. A potential explanation for these rather surprising deleterious effects lies in increased microgliosis detected in the hMSC treated animals. Repeated intraspinal injection at two time points resulted in a slight but not significant increase in survival and significant improvement of motor performance although no hMSC-induced changes of motor neuron numbers, astrogliosis, and microgliosis were detected. Quantitative real time polymerase chain reaction showed reduced expression of endothelial growth factor in animals having received hMSCs twice compared with the vehicle treated control group. hMSCs were detectable at the injection site at Day 20 after injection into the spinal cord but no longer at Day 70. Intraspinal injection of hMSCs may therefore be a more promising option for the treatment of ALS than intraventricular injection and repeated injections might be necessary to obtain substantial therapeutic benefit.