RESUMO
We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism.SerpinE2 â¤HtrA1 protease â¤Syndecan-4 â NC cell migration.
Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Crista Neural , Serpina E2 , Animais , Movimento Celular/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Serpina E2/metabolismo , Transdução de Sinais , Xenopus laevis/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
The hippocampus is a primary region affected in Alzheimer's disease (AD). Because AD postmortem brain tissue is not available prior to symptomatic stage, we lack understanding of early cellular pathogenic mechanisms. To address this issue, we examined the cellular origin and progression of AD pathogenesis by comparing patient-based model systems including iPSC-derived brain cells transplanted into the mouse brain hippocampus. Proteomic analysis of the graft enabled the identification of pathways and network dysfunction in AD patient brain cells, associated with increased levels of Aß-42 and ß-sheet structures. Interestingly, the host cells surrounding the AD graft also presented alterations in cellular biological pathways. Furthermore, proteomic analysis across human iPSC-based models and human post-mortem hippocampal tissue projected coherent longitudinal cellular changes indicative of early to end stage AD cellular pathogenesis. Our data showcase patient-based models to study the cell autonomous origin and progression of AD pathogenesis.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Animais , Camundongos , Humanos , Proteômica , Autopsia , HipocampoRESUMO
SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Atrofia de Múltiplos Sistemas , Doença de Parkinson , Sinucleinopatias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Oligodendroglia/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Here, we examine the cellular changes triggered by tumor necrosis factor alpha (TNF-α) and different alpha-synuclein (αSYN) species in astrocytes derived from induced pluripotent stem cells. Human astrocytes treated with TNF-α display a strong reactive pro-inflammatory phenotype with upregulation of pro-inflammatory gene networks, activation of the nuclear factor κB (NF-κB) pathway, and release of pro-inflammatory cytokines, whereas those treated with high-molecular-weight αSYN fibrils acquire a reactive antigen (cross)-presenting phenotype with upregulation of major histocompatibility complex (MHC) genes and increased human leukocyte antigen (HLA) molecules at the cell surface. Surprisingly, the cell surface location of MHC proteins is abrogated by larger F110 fibrillar polymorphs, despite the upregulation of MHC genes. Interestingly, TNF-α and αSYN fibrils compete to drive the astrocyte immune reactive response. The astrocyte immune responses are accompanied by an impaired mitochondrial respiration, which is exacerbated in Parkinson's disease (PD) astrocytes. Our data provide evidence for astrocytic involvement in PD pathogenesis and reveal their complex immune reactive responses to exogenous stressors.
Assuntos
Astrócitos/imunologia , Mitocôndrias/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , alfa-Sinucleína/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apresentação de Antígeno , Astrócitos/metabolismo , Membrana Celular/metabolismo , Respiração Celular , Citocinas/metabolismo , Cadeias HLA-DRB1/química , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/metabolismo , Peso Molecular , Doença de Parkinson/patologia , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Lipid peroxidation-initiated ferroptosis is an iron-dependent mechanism of programmed cell death taking place in neurological diseases. Here we show that a condensed benzo[b]thiazine derivative small molecule with an arylthiazine backbone (ADA-409-052) inhibits tert-Butyl hydroperoxide (TBHP)-induced lipid peroxidation (LP) and protects against ferroptotic cell death triggered by glutathione (GSH) depletion or glutathione peroxidase 4 (GPx4) inhibition in neuronal cell lines. In addition, ADA-409-052 suppresses pro-inflammatory activation of BV2 microglia and protects N2a neuronal cells from cell death induced by pro-inflammatory RAW 264.7 macrophages. Moreover, ADA-409-052 efficiently reduces infarct volume, edema and expression of pro-inflammatory genes in a mouse model of thromboembolic stroke. Targeting ferroptosis may be a promising therapeutic strategy in neurological diseases involving severe neuronal death and neuroinflammation.
Assuntos
Morte Celular/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Apoptose/efeitos dos fármacos , Morte Celular/fisiologia , Ferroptose/fisiologia , Glutationa/metabolismo , Ferro/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neuroproteção/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/farmacologiaRESUMO
The hippocampus is important for memory formation and is severely affected in the brain with Alzheimer disease (AD). Our understanding of early pathogenic processes occurring in hippocampi in AD is limited due to tissue unavailability. Here, we report a chemical approach to rapidly generate free-floating hippocampal spheroids (HSs), from human induced pluripotent stem cells. When used to model AD, both APP and atypical PS1 variant HSs displayed increased Aß42/Aß40 peptide ratios and decreased synaptic protein levels, which are common features of AD. However, the two variants differed in tau hyperphosphorylation, protein aggregation, and protein network alterations. NeuroD1-mediated gene therapy in HSs-derived progenitors resulted in modulation of expression of numerous genes, including those involved in synaptic transmission. Thus, HSs can be harnessed to unravel the mechanisms underlying early pathogenic changes in the hippocampi of AD patients, and provide a robust platform for the development of therapeutic strategies targeting early stage AD.
Assuntos
Doença de Alzheimer/patologia , Hipocampo/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Esferoides Celulares/patologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Estudos de Casos e Controles , Terapia Genética , Humanos , Neurônios/patologia , Fenótipo , Presenilina-1/genética , Presenilina-1/metabolismo , Agregados Proteicos , Proteoma/metabolismo , Proteômica , Transcrição GênicaRESUMO
Here, we describe the generation of an induced pluripotent stem cell (iPSC) line, from a male patient diagnosed with Parkinson's disease (PD). The patient carries a heterozygous variation p.A53T in the SNCA gene. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated iPSC line (CSC-32) preserved the mutation, displayed expression of common pluripotency markers, differentiated into derivatives of the three germ layers, and exhibited a normal karyotype. The clone CSC-32B is presented thereafter; it can be used to study the mechanisms underlying PD pathogenesis.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/genética , Diferenciação Celular , Células Cultivadas , Heterozigoto , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Pessoa de Meia-IdadeRESUMO
Inflammation is a prominent feature of the neuropathology of amyotrophic lateral sclerosis (ALS). Emerging evidence suggests that inflammatory cascades contributing to the disease progression are not restricted to the central nervous system (CNS) but also occur peripherally. Indeed, alterations in T cell responses and their secreted cytokines have been detected in ALS patients and in animal models of ALS. One key cytokine responsible for the shift in T cell responses is interleukin-33 (IL-33), which stimulates innate type 2 immune cells to produce a large amount of Th2 cytokines that are possibly beneficial in the recovery processes of CNS injuries. Since the levels of IL-33 have been shown to be decreased in patients affected with ALS, we sought to determine whether a long-term recombinant IL-33 treatment of a transgenic mouse model of ALS expressing G93A-superoxide dismutase 1 (SOD1-G93A) alters the disease progression and ameliorates the ALS-like disease pathology. SOD1-G93A mice were treated with intraperitoneal injections of IL-33 and effects on disease onset and inflammatory status were determined. Spinal cord (SC) neurons, astrocytes and T-cells were exposed to IL-33 to evaluate the cell specific responses to IL-33. Treatment of SOD1-G93A mice with IL-33 delayed the disease onset in female mice, decreased the proportion of CD4+ and CD8 + T cell populations in the spleen and lymph nodes, and alleviated astrocytic activation in the ventral horn of the lumbar SC. Male SOD1-G93A mice were unresponsive to the treatment. In vitro studies showed that IL-33 is most likely not acting directly on neurons and astrocytes, but rather conveying its effects through peripheral T-cells. Our results suggest that strategies directed to the peripheral immune system may have therapeutic potential in ALS. The effect of gender dimorphisms to the treatment efficacy needs to be taken into consideration when designing new therapeutic strategies for CNS diseases.
RESUMO
Mutations in the glucocerebrosidase (GBA) gene have been associated with the development of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line was generated from a 60-year old patient diagnosed with PD and carrying a new mutation variant p.R301C in GBA. Using non-integrating Sendai virus-based technology, we utilized OCT3/4, SOX2, c-MYC and KLF4 transcription factors to reprogram skin fibroblasts into iPSCs. The generated iPSC line retained the mutation, displayed expression of common pluripotency markers, differentiated into the three germ layers, and exhibited normal karyotype. The iPSC line can be further used for studying PD pathogenesis.
Assuntos
Técnicas de Cultura de Células/métodos , Glucosilceramidase/genética , Células-Tronco Pluripotentes Induzidas/patologia , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Animais , Linhagem Celular , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
The leucine-rich repeat kinase 2 (LRRK2) p.G2019S mutation is the most common genetic cause of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line CSC-41 was generated from a 75-year old patient diagnosed with PD caused by a p.G2019S mutation in LRRK2. Skin fibroblasts were reprogrammed using a non-integrating Sendai virus-based technology to deliver OCT3/4, SOX2, c-MYC and KLF4 transcription factors. The generated iPSC line exhibits expression of common pluripotency markers, differentiates into the three germ layers and has a normal karyotype. The iPSC line can be used to explore the association between LRRK2 mutation and PD.
Assuntos
Técnicas de Cultura de Células/métodos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Idoso , Animais , Linhagem Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Fator 4 Semelhante a Kruppel , CamundongosRESUMO
Mutations in the PARK2 gene, which encodes PARKIN, are the most frequent cause of autosomal recessive Parkinson's disease (PD). We report the generation of an induced pluripotent stem cell (iPSC) line from a 78-year-old patient carrying a compound heterozygous mutation (c.823C>T and EX6del) in the PARK2 gene. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line CSC-44 exhibits expression of common pluripotency markers, in vitro differentiation into the three germ layers and normal karyotype. This iPSC line can be used to explore the association between PARK2 mutations and PD.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Feminino , Heterozigoto , Humanos , Fator 4 Semelhante a Kruppel , Mutação/genéticaRESUMO
Skin fibroblasts were collected from a 44-year-old patient with sporadic case of Parkinson's disease (PD). The non-integrating Sendai virus vector encoding OCT3/4, SOX2, c-MYC and KLF4 was used to reprogram fibroblasts into induced pluripotent stem cells (iPSCs). Generated iPSCs had normal karyotypes, expressed common stem cell markers, and were capable of differentiating into all three germ layers. Generated line could be used for PD modeling to understand the mechanisms that influence the disorder.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/metabolismo , Adulto , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Cariótipo , Fator 4 Semelhante a KruppelRESUMO
An induced pluripotent stem cell (iPSC) line was generated from a 36-year-old patient with sporadic Parkinson's disease (PD). Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated cell line (CSC-43) exhibits expression of common pluripotency markers, in vitro differentiation into three germ layers and normal karyotype. This iPSC line can be used to study the mechanisms underlying the development of PD.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/metabolismo , Adulto , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Parkinson's disease (PD) is a neurodegenerative disease with unknown etiology. Here we show the generation of an induced pluripotent stem cell (iPSC) line, named CSC-40, from dermal fibroblasts obtained from a 59-year-old male patient with a homozygous p.Q456X mutation in the PTEN-induced putative kinase 1 (PINK/PARK6) gene and a confirmed diagnosis of PD, which could be used to model familial PD. A non-integrating Sendai virus-based delivery of the reprogramming factors OCT3/4, SOX2, c-MYC and KLF4 was employed. The CSC-40 cell line showed normal karyotyping and fingerprinting following transduction as well as sustained expression of several pluripotency markers and the ability to differentiate into all three germ layers.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/genética , Proteínas Quinases/genética , Linhagem Celular , Células Cultivadas , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Doença de Parkinson/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Oligodendrocytes are part of the glial cells located in the central nervous system, capable of providing trophic support to neurons and ensheathing their axons. These cells can become dysfunctional under pathologic condition. Rodent and human pluripotent stem cells are inexhaustible sources for producing oligodendrocytes that can be used for disease modeling and cell replacement therapy studies. They also offer many opportunities to model the contribution of oligodendrocytes in non-genetic disorders such as multiple system atrophy. In this method article, we provide robust and reproducible differentiation protocols to obtain oligodendrocyte progenitor cells and purify them using fluorescence activated cell sorting.
Assuntos
Técnicas de Cultura de Células/métodos , Oligodendroglia/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Humanos , Proteína Básica da Mielina/metabolismo , Oligodendroglia/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Developing new therapies for stroke is urgently needed, as this disease is the leading cause of death and disability worldwide, and the existing treatment is only available for a small subset of patients. The interruption of blood flow to the brain during ischemic stroke launches multiple immune responses, characterized by infiltration of peripheral immune cells, the activation of brain microglial cells, and the accumulation of immune mediators. Copper is an essential trace element that is required for many critical processes in the brain. Copper homeostasis is disturbed in chronic neurodegenerative diseases and altered in stroke patients, and targeted copper delivery has been shown to be protective against chronic neurodegeneration. This study was undertaken to assess whether the copper bis(thiosemicarbazone) complex, CuII(atsm), is beneficial in acute brain injury, in preclinical mouse models of ischemic stroke. We demonstrate that the copper complex CuII(atsm) protects neurons from excitotoxicity and N2a cells from OGD in vitro, and is protective in permanent and transient ischemia models in mice as measured by functional outcome and lesion size. Copper delivery in the ischemic brains modulates the inflammatory response, specifically affecting the myeloid cells. It reduces CD45 and Iba1 immunoreactivity, and alters the morphology of Iba1 positive cells in the ischemic brain. CuII(atsm) also protects endogenous microglia against ischemic insult and reduces the proportion of invading monocytes. These results demonstrate that the copper complex CuII(atsm) is an inflammation-modulating compound with high therapeutic potential in stroke and is a strong candidate for the development of therapies for acute brain injury.
Assuntos
Isquemia Encefálica/metabolismo , Encefalite/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Compostos Organometálicos/administração & dosagem , Acidente Vascular Cerebral/metabolismo , Tiossemicarbazonas/administração & dosagem , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Isquemia Encefálica/prevenção & controle , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação , Modelos Animais de Doenças , Encefalite/prevenção & controle , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Acidente Vascular Cerebral/prevenção & controleRESUMO
ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs type 4) is a metalloprotease capable to degrade chondroitin sulfate proteoglycans leading to cartilage destruction during arthritis or to neuroplasticity during spinal cord injury (SCI). Although ADAMTS-4 is an inflammatory-regulated enzyme, its role during inflammation has never been investigated. The aim of this study was to investigate the role of ADAMTS-4 in neuroinflammation. First, we evidenced an increase of ADAMTS-4 expression in the ischemic brain hemisphere of mouse and human patients suffering from ischemic stroke. Then, we described that ADAMTS-4 has predominantly an anti-inflammatory effect in the CNS. Treatment of primary microglia or astrocyte cultures with low doses of a human recombinant ADAMTS-4 prior to LPS exposure decreased NO production and the synthesis/release of pro-inflammatory cytokines including NOS2, CCL2, TNF-α, IL-1ß and MMP-9. Accordingly, when cell cultures were transfected with silencing siRNA targeting ADAMTS-4 prior to LPS exposure, the production of NO and the synthesis/release of pro-inflammatory cytokines were increased. Finally, the feasibility of ADAMTS-4 to modulate neuroinflammation was investigated in vivo after permanent middle cerebral artery occlusion in mice. Although ADAMTS-4 treatment did not influence the lesion volume, it decreased astrogliosis and macrophage infiltration, and increased the number of microglia expressing arginase-1, a marker of alternatively activated cells with inflammation inhibiting functions. Additionally, ADAMTS-4 increased the production of IL-10 and IL-6 in the peri-ischemic area. By having anti-inflammatory and neuroregenerative roles, ADAMTS-4 may represent an interesting target to treat acute CNS injuries, such as ischemic stroke, SCI or traumatic brain injury. GLIA 2016;64:1492-1507.