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1.
Proc Natl Acad Sci U S A ; 119(12): e2111405119, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35294277

RESUMO

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/metabolismo
2.
J Neural Transm (Vienna) ; 124(4): 455-462, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28160153

RESUMO

Following the failure of a Phase II clinical study evaluating human retinal pigment epithelial (hRPE) cell implants as a potential treatment option for Parkinson's disease, speculation has centered on implant function and survival as possible contributors to the therapeutic outcomes. We recently reported that neonatal hRPE cells, similar to hRPE cells used in the Phase II clinical study, produced short-lived in vitro and limited in vivo trophic factors, which supports that assumption. We hypothesize that the switch from fetal to neonatal hRPE cells, between the Phase I and the Phase II clinical trial may be partly responsible for the later negative outcomes. To investigate this hypothesis, we used two neonatal hRPE cell lots, prepared in a similar manner to neonatal hRPE cells used in the Phase II clinical study, and compared them to previously evaluated fetal hRPE cells for behavioral changes following unilateral striatal implantation in 6-hydroxydopamine-lesioned rats. The results showed that only fetal, not neonatal, hRPE cell implants, were able to improve behavioral outcomes following striatal implantation in the lesioned rats. These data suggest that fetal hRPE cells may be preferential to neonatal hRPE cells in restoring behavioral deficits.


Assuntos
Transplante de Células , Transtornos Parkinsonianos/cirurgia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Anfetamina/farmacologia , Animais , Sobrevivência Celular , Senescência Celular , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/cirurgia , Células Epiteliais/transplante , Feminino , Humanos , Recém-Nascido , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Oxidopamina , Transtornos Parkinsonianos/fisiopatologia , Distribuição Aleatória , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Caminhada/fisiologia
3.
J Neural Transm (Vienna) ; 123(3): 167-77, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26546037

RESUMO

Human retinal pigment epithelial (hRPE) cell implants into the striatum have been investigated as a potential cell-based treatment for Parkinson's disease in a Phase II clinical trial that recently failed. We hypothesize that the trophic factor potential of the hRPE cells could potentially influence the function and/or survival of the implants and may be involved in an alternative mechanism of action. However, it is unclear if hRPE cells secreted trophic factors when handled in the manner used in the clinical Phase II trial. To address these questions, we investigated two neonatal hRPE cell lots, cultured in a similar manner to hRPE cells used in a Phase II clinical study, and longitudinally determined brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2), and pigment epithelium-derived factor concentrations in vitro and following striatal implantation into 6-hydroxydopamine-lesioned rats. The results demonstrate short-lived BDNF and FGF2 concentrations in vitro from hRPE cells grown alone or attached to gelatin microcarriers (GM)s as well as limited trophic factor concentration differences in vivo following striatal implantation of hRPE-GM in 6-hydroxydopamine lesioned rats compared to sham (GM-only). The data suggest that trophic factors from neonatal hRPE cell implants likely did not participate in an alternative mechanism of action, which adds supports to a hypothesis that additional factors may have been necessary for the survival and/or function of hRPE implants and potentially the success of the Phase II clinical trial.


Assuntos
Ensaios Clínicos Fase II como Assunto , Corpo Estriado/cirurgia , Fatores de Crescimento Neural/metabolismo , Transtornos Parkinsonianos/cirurgia , Epitélio Pigmentado da Retina/transplante , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Recém-Nascido , Masculino , Ratos , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
4.
Cell Rep ; 34(12): 108895, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33761362

RESUMO

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/metabolismo
5.
Stem Cell Reports ; 15(1): 256-273, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32589876

RESUMO

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 Gênica
6.
Acta Neuropathol Commun ; 7(1): 208, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31843010

RESUMO

Variations in the POLG1 gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma, have recently been associated with Parkinson's disease (PD), especially in patients diagnosed with progressive external ophthalmoplegia (PEO). However, the majority of the studies reporting this association mainly focused on the genetic identification of the variation in POLG1 in PD patient primary cells, and determination of mitochondrial DNA copy number, providing little information about the cellular alterations existing in patient brain cells, in particular dopaminergic neurons. Therefore, through the use of induced pluripotent stem cells (iPSCs), we assessed cellular alterations in novel p.Q811R POLG1 (POLG1Q811R) variant midbrain dopaminergic neuron-containing spheroids (MDNS) from a female patient who developed early-onset PD, and compared them to cultures derived from a healthy control of the same gender. Both POLG1 variant and control MDNS contained functional midbrain regionalized TH/FOXA2-positive dopaminergic neurons, capable of releasing dopamine. Western blot analysis identified the presence of high molecular weight oligomeric alpha-synuclein in POLG1Q811R MDNS compared to control cultures. In order to assess POLG1Q811R-related cellular alterations within the MDNS, we applied mass-spectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1Q811R and control samples. Pro- and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1Q811R MDNS was suggested by the increase in PFKM and LDHA levels and confirmed using functional analysis of glycolytic rate and oxygen consumption levels. Our results validate the use of iPSCs to assess cellular alterations in relation to PD pathogenesis, in a unique PD patient carrying a novel p.Q811R variation in POLG1, and identify several altered pathways that may be relevant to PD pathogenesis.


Assuntos
DNA Polimerase gama/genética , Variação Genética/genética , Oftalmoplegia Externa Progressiva Crônica/genética , Transtornos Parkinsonianos/genética , Células-Tronco Pluripotentes/fisiologia , Esferoides Celulares/fisiologia , Adulto , Feminino , Humanos , Mesencéfalo/patologia , Mesencéfalo/fisiologia , Oftalmoplegia Externa Progressiva Crônica/complicações , Oftalmoplegia Externa Progressiva Crônica/diagnóstico , Transtornos Parkinsonianos/complicações , Transtornos Parkinsonianos/diagnóstico , Células-Tronco Pluripotentes/patologia , Proteômica/métodos , Esferoides Celulares/patologia
7.
Stem Cell Res ; 34: 101373, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30640063

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-Idade
8.
Sci Rep ; 9(1): 9610, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270389

RESUMO

The glutamate transporter 1 (GLT1) is upregulated during astrocyte development and maturation in vivo and is vital for astrocyte function. Yet it is expressed at low levels by most cultured astrocytes. We previously showed that maturation of human and mouse stem cell-derived astrocytes - including functional glutamate uptake - could be enhanced by fibroblast growth factor (FGF)1 or FGF2. Here, we examined the specificity and mechanism of action of FGF2 and other FGF family members, as well as neurotrophic and differentiation factors, on mouse embryonic stem cell-derived astrocytes. We found that some FGFs - including FGF2, strongly increased GLT1 expression and enhanced astrocyte proliferation, while others (FGF16 and FGF18) mainly affected maturation. Interestingly, BMP4 increased astrocytic GFAP expression, and BMP4-treated astrocytes failed to promote the survival of motor neurons in vitro. Whole transcriptome analysis showed that FGF2 treatment regulated multiple genes linked to cell division, and that the mRNA encoding GLT1 was one of the most strongly upregulated of all astrocyte canonical markers. Since GLT1 is expressed at reduced levels in many neurodegenerative diseases, activation of this pathway is of potential therapeutic interest. Furthermore, treatment with FGFs provides a robust means for expansion of functionally mature stem cell-derived astrocytes for preclinical investigation.


Assuntos
Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Células-Tronco/citologia , Animais , Astrócitos/metabolismo , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Biologia Computacional/métodos , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ontologia Genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Família Multigênica
9.
Stem Cell Res ; 27: 78-81, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29334629

RESUMO

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 Kruppel
10.
Stem Cell Res ; 27: 61-64, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29331938

RESUMO

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/metabolismo
12.
Eur J Heart Fail ; 9(9): 892-6, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17613272

RESUMO

BACKGROUND: Brain natriuretic peptide (BNP) is increased in heart failure; however, the relative contribution of the right and left ventricles is largely unknown. AIM: To investigate if right ventricular function has an independent influence on plasma BNP concentration. METHODS: Right (RVEF), left ventricular ejection fraction (LVEF), and left ventricular end-diastolic volume index (LVEDVI) were determined in 105 consecutive patients by first-pass radionuclide ventriculography (FP-RNV) and multiple ECG-gated equilibrium radionuclide ventriculography (ERNV), respectively. BNP was analyzed by immunoassay. RESULTS: Mean LVEF was 0.51 (range 0.10-0.83) with 36% having a reduced LVEF (<0.50). Mean RVEF was 0.50 (range 0.26-0.78) with 43% having a reduced RVEF (<0.50). The mean LVEDVI was 92 ml/m2 with 22% above the upper normal limit (117 ml/m2). Mean BNP was 239 pg/ml range (0.63-2523). In univariate linear regression analysis LVEF, LVEDVI and RVEF all correlated significantly with log BNP (p<0.0001). In a multivariate analysis only RVEF and LVEF remained significant. The parameter estimates of the final adjusted model indicated that RVEF and LVEF influence on log BNP were of the same magnitude. CONCLUSION: BNP, which is a strong prognostic marker in heart failure, independently depends on both left and right ventricular systolic function. This might, at least in part, explain why BNP holds stronger prognostic value than LVEF alone.


Assuntos
Peptídeo Natriurético Encefálico/sangue , Disfunção Ventricular Esquerda/sangue , Disfunção Ventricular Direita/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
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