RESUMO
Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.
RESUMO
Microglia are essential for maintenance of normal brain function, with dysregulation contributing to numerous neurological diseases. Protocols have been developed to derive microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, primary microglia display major differences in morphology and gene expression when grown in culture, including down-regulation of signature microglial genes. Thus, in vitro differentiated microglia may not accurately represent resting primary microglia. To address this issue, we transplanted microglial precursors derived in vitro from hiPSCs into neonatal mouse brains and found that the cells acquired characteristic microglial morphology and gene expression signatures that closely resembled primary human microglia. Single-cell RNA-sequencing analysis of transplanted microglia showed similar cellular heterogeneity as primary human cells. Thus, hiPSCs-derived microglia transplanted into the neonatal mouse brain assume a phenotype and gene expression signature resembling that of resting microglia residing in the human brain, making chimeras a superior tool to study microglia in human disease.
Assuntos
Encéfalo/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Microglia/transplante , Animais , Encéfalo/metabolismo , Encéfalo/cirurgia , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Microglia/metabolismo , FenótipoRESUMO
Acetaminophen (APAP) is a widely used antipyretic analgesic which can lead to acute liver failure after overdoses. Chronic alcoholic fatty liver disease (AFLD) appears to enhance the risk and severity of APAP-induced liver injury, and the level of angiotensin II (Ang II) increased sharply at the same time. However, the underlying mechanisms remain unclear. Caveolin-1 (CAV1) has been proven to have a protective effect on AFLD. This study aimed to examine whether CAV1 can protect the APAP-induced hepatotoxicity of AFLD by affecting Ang II or its related targets. In vivo, the AFLD model was established according to the chronic-plus-binge ethanol model. Liver injury and hepatic lipid accumulation level were determined. The levels of Angiotensin converting enzyme 2 (ACE2), Ang II, CAV1, and other relevant proteins were evaluated by western blotting. In vitro, L02 cells were treated with alcohol and oleic acid mixture and APAP. CAV1 and ACE2 expression was downregulated in APAP-treated AFLD mice compared to APAP-treated mice. The overexpression of CAV1 in mice and L02 cells alleviated APAP-induced hepatotoxicity in AFLD and downregulated Ang II, p-EGFR/EGFR and P-ERK/ERK expression. Immunofluorescence experiments revealed interactions between CAV1, Ang II, and EGFR. The application of losartan (an Ang II receptor antagonist) and PD98059 (an ERK1/2 inhibitor) alleviated APAP-induced hepatotoxicity in AFLD. In conclusion, our findings verified that CAV1 alleviates APAP-aggravated hepatotoxicity in AFLD by downregulating the Ang II /EGFR/ERK axis, which could be a novel therapeutic target for its prevention or treatment.
Assuntos
Caveolina 1 , Doença Hepática Induzida por Substâncias e Drogas , Fígado Gorduroso Alcoólico , Acetaminofen/efeitos adversos , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Caveolina 1/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Receptores ErbB/metabolismo , Fígado Gorduroso Alcoólico/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Environmental aging of ubiquitous microplastics (MP) occurs through the action of biotic and abiotic factors, and aged MP exhibit different physicochemical properties and environmental behavior from virgin MP. This study aimed to investigate the aged micro-sized polystyrene (mPS) and polyvinyl chloride (mPVC), and the heavy metals copper (Cu) and cadmium (Cd), and examine the effects of their combined toxicities on microalga Chlorella vulgaris. Results showed that the presence of MP inhibited cell growth as compared with the control, the inhibition rate (I) decreased as concentrations of MP rose and aged MP exhibited stronger inhibition of cells than did virgin MP. The largest I was achieved in each culture with the MP concentration of 0.01 g/L, in which aged mPS with the maximal of 36.84% (Iaged mPS) followed by aged mPVC (Iaged mPVC = 30.03%), virgin mPS (Ivirgin mPS = 29.10%) and virgin mPVC (Ivirgin mPVC = 16.72%). Addition of the heavy metals Cu2+ and Cd2+ significantly inhibited cell growth, and toxicity increased with concentrations in a range of 0.5-2.0 mg/L; the maximum I values were 19.50% (ICu) and 85.14% (ICd), respectively. The combined toxicity of aged MP + Cu or aged MP + Cd was less than that of individual heavy metals. In particular, as compared with the maximal ICd of 85.14% achieved by single Cd2+, the toxicity of Cd2+ was greatly reduced when combined with aged mPS and mPVC, with the I value decreased to 27.55% (Iaged mPS) and 32.51% (Iaged mPVC), respectively. Both single and combined treatments caused cell damage to the microalga, accompanied by increased superoxide dismutase (SOD) and intracellular malonaldehyde (MDA) content.
Assuntos
Chlorella vulgaris/metabolismo , Metais Pesados/toxicidade , Microplásticos/toxicidade , Poluentes Químicos da Água/toxicidade , Disponibilidade Biológica , Cádmio/toxicidade , Chlorella vulgaris/efeitos dos fármacos , Cobre/toxicidade , Malondialdeído/farmacologia , Metais Pesados/metabolismo , Microalgas/metabolismo , Microplásticos/metabolismo , Plásticos , Poliestirenos/toxicidade , Cloreto de Polivinila , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/metabolismoRESUMO
BACKGROUND: Lupus nephritis (LN) is an inflammation of the kidneys and is a major cause of mortality in systemic lupus erythaematosus (SLE) patients. In addition, Th17/Treg balance is one of the most important factors that can promote the development of LN. It has been reported that vasoactive intestinal peptide (VIP) is associated with the downregulation of both inflammatory and autoimmune diseases through regulating T lymphocyte balance. Therefore, the aim of this study was to determine the role of VIP in modulating Th17/Treg balance in LN. METHODS: LN was induced in BALB/c female mice by injection pristane. After 3 months, mice were randomly divided into four groups: control, VIP + control, LN and VIP + LN. Autoantibody levels were tested by ELISA. The distribution of Th17/Treg cells in vivo and in vitro was detected by FC. Renal tissues were examined by PASM and DIF for pathology and Foxp3+CD3+. The mRNA and protein expression levels of pro- and anti-inflammatory cytokines were detected by qRT-PCR and western blotting. RESULTS: VIP can improve renal injury by regulating Th17/Treg imbalance in LN mice. Proteinuria, renal function defects and autoantibodies were significantly decreased, and Th17/Treg cell balance was restored in VIP compared with LN mice. In addition, VIP improved renal lesions by promoting the expression of Foxp3+CD3+ in renal tissue. Furthermore, VIP downregulated the mRNA and protein expression of IL-17, IL-6 and upregulated Foxp3, IL-10 expression. CONCLUSIONS: VIP reduced LN proteinuria and renal function defects and restored the Th17/Treg cell balance. Furthermore, VIP also downregulated autoantibody and inflammatory cytokine expression and upregulated Foxp3 and IL-10 expression.
Assuntos
Nefrite Lúpica/sangue , Nefrite Lúpica/tratamento farmacológico , Linfócitos T Reguladores/metabolismo , Terpenos/toxicidade , Células Th17/metabolismo , Peptídeo Intestinal Vasoativo/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Nefrite Lúpica/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
BACKGROUND: The transcriptional networks of Cyr61 and its function in cell injury are poorly understood. The present study depicted the lncRNA and mRNA profiles and the involvement in angiotensin II-induced injury after Cyr61 knockdown mediated by CRISPR/Cas9 in HEK293T cells. METHODS: HEK293T cells were cultured, and Cyr61 knockdown was achieved by transfection of the CRISPR/Cas9 KO plasmid. lncRNA and mRNA microarrays were used to identify differentially expressed genes (DEGs). Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine biofunctions and signaling pathways. RT-PCR was used to validate the microarray results. Cells were divided into four groups: control, Cyr61 knockdown, angiotensin II (Ang II) without Cyr61 knockdown, and Ang II with Cyr61 knockdown. CCK8, western blotting, and flow cytometry analysis were carried out to dissect cellular function. RESULTS: A total of 23184 lncRNAs and 28264 mRNAs were normalized. 26 lncRNAs and 212 mRNAs were upregulated, and 74 lncRNAs and 233 mRNAs were downregulated after Cyr61 knockdown. Analysis of cellular components, molecular functions, biological processes, and regulatory pathways associated with the differentially expressed mRNAs revealed downstream mechanisms of the Cyr61 gene. The differentially expressed genes were affected for small cell lung cancer, axon guidance, Fc gamma R-mediated phagocytosis, MAPK signaling pathway, focal adhesion, insulin resistance, and metabolic pathways. In addition, Cyr61 expression was increased in accordance with induction of cell cycle arrest and apoptosis and inhibition of cell proliferation induced by Ang II. Knockdown of Cyr61 in HEK293T cells promoted cell cycle procession, decreased apoptosis, and promoted cell proliferation. CONCLUSIONS: The Cyr61 gene is involved in Ang II-induced injury in HEK293T cells. Functional mechanisms of the differentially expressed lncRNAs and mRNAs as well as identification of metabolic pathways will provide new therapeutic targets for Cyr61-realated diseases.
Assuntos
Angiotensina II/farmacologia , Sistemas CRISPR-Cas/efeitos dos fármacos , Proteína Rica em Cisteína 61/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína Rica em Cisteína 61/genética , Ontologia Genética , Redes Reguladoras de Genes/genética , Células HEK293 , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismoRESUMO
The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (W(sh)/W(sh)), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse-human chimeric platform allows for a new approach to study NC development and diseases.
Assuntos
Quimera/metabolismo , Embrião de Mamíferos/citologia , Crista Neural/citologia , Pigmentação da Pele , Negro ou Afro-Americano , Animais , Animais Recém-Nascidos , Sobrevivência Celular , Células Cultivadas , DNA/metabolismo , Fibroblastos/citologia , Gastrulação , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos Endogâmicos C57BL , Microinjeções , Reação em Cadeia da Polimerase , Ratos , Especificidade da Espécie , Doadores de TecidosRESUMO
Alterations in DNA methylation have been associated with genome-wide hypomethylation and regional de novo methylation in numerous cancers. De novo methylation is mediated by the de novo methyltransferases Dnmt3a and 3b, but only Dnmt3b has been implicated in promoting cancer by silencing of tumor-suppressor genes. In this study, we have analyzed the role of Dnmt3a in lung cancer by using a conditional mouse tumor model. We show that Dnmt3a deficiency significantly promotes tumor growth and progression but not initiation. Changes in gene expression show that Dnmt3a deficiency affects key steps in cancer progression, such as angiogenesis, cell adhesion, and cell motion, consistent with accelerated and more malignant growth. Our results suggest that Dnmt3a may act like a tumor-suppressor gene in lung tumor progression and may be a critical determinant of lung cancer malignancy.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Neoplasias Pulmonares/patologia , Animais , Sequência de Bases , Divisão Celular , Metilação de DNA , DNA Metiltransferase 3A , Primers do DNA , Modelos Animais de Doenças , Progressão da Doença , Inativação Gênica , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Mir-290 through mir-295 (mir-290-295) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290-295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290-295-deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290-295(-/-) mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290-295(-/-) mice are unable to recover and are sterile, due to premature ovarian failure.
Assuntos
Perda do Embrião/genética , Perda do Embrião/patologia , Células Germinativas/metabolismo , Células Germinativas/patologia , MicroRNAs/metabolismo , Penetrância , Envelhecimento/patologia , Animais , Animais Recém-Nascidos , Apoptose , Contagem de Células , Ciclo Celular , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/crescimento & desenvolvimento , Gônadas/patologia , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , Masculino , Camundongos , Camundongos Mutantes , MicroRNAs/genéticaRESUMO
BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, following Alzheimer's. It is characterized by the aggregation of α-synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for α-synuclein has been linked to early-onset PD, and exposure to A53T-mutant human α-synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. METHODS: Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the cell-autonomous effects of the A53T mutation on human microglia. RESULTS: We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, transplanted A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. CONCLUSIONS: Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.
RESUMO
Acetaminophen (APAP) is one of the most widely used drugs in the world. The literature shows that excessive or long-term use of APAP can lead to increased cardiovascular dysfunction. An acute increase in angiotensin â ¡ (Ang â ¡) caused by APAP use in fatty liver disease may increase the risk and severity of vascular injury. However, the underlying mechanism remains unclear. Caveolin-1 (CAV1) is a broad-spectrum kinase inhibitor that significantly determines endothelial function. This study aimed to observe the effects of APAP on the vasculature in non-alcoholic fatty liver disease (NAFLD) and to determine whether CAV1 could alleviate vascular oxidative stress and inflammation by targeting Ang â ¡ or its downstream pathways. In this study, 7-week-old C57BL/6 male mice (18-20 g) were administered APAP by gavage after eight weeks of a high-fat diet. Any resulting vascular oxidative stress and inflammation were assessed. Levels of Ang â ¡, CAV1, and other related proteins were measured using ELISA and western blotting. In APAP-treated NAFLD mice, CAV1 expression was downregulated and Ang â ¡ expression was upregulated compared to normal APAP-treated mice. In vitro, HUVECs were incubated with Ang â ¡ (300 nM) for 48 h. Overexpression of CAV1 in HUVECs attenuated Ang â ¡-induced oxidative stress and inflammation and downregulated the expression of Protein kinase C (PKC) and p-P38/P38. After intervention with CAV1-siRNA, immunofluorescence results showed that the fluorescence intensity of PKC on mitochondria was further increased, and flow cytometry results showed that the mitochondrial membrane potential increased. PKC inhibitors alleviated Ang â ¡-induced endothelial injury. In conclusion, our findings confirmed that CAV1 exerts a protective effect against vascular injury by inhibiting oxidative stress and inflammation through the PKC/MAPK pathway. Therefore, restoration of CAV1 may have clinical benefits in reducing APAP-induced vascular damage in NAFLD patients.
Assuntos
Caveolina 1 , Doença Hepática Induzida por Substâncias e Drogas , Hepatopatia Gordurosa não Alcoólica , Lesões do Sistema Vascular , Animais , Masculino , Camundongos , Acetaminofen/efeitos adversos , Caveolina 1/genética , Caveolina 1/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Inflamação/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Estresse Oxidativo , Proteína Quinase C/metabolismo , Lesões do Sistema Vascular/metabolismoRESUMO
The overuse of acetaminophen (APAP) may cause more severe hepatotoxicity in patients with non-alcoholic fatty liver disease (NAFLD). Caveolin-1 (CAV1), is an essential regulator of metabolic function, which can alleviate liver damage by scavenging reactive oxygen species (ROS). Evidence suggests that the NOD-like receptor family pyrin domain-containing 3 (NLRP3) -mediated pyroptosis is involved in the development of NAFLD. Moreover, thioredoxin-interactive protein (TXNIP) activation is a key event linking ROS to NLRP3 inflammasome. However, whether CAV1 alleviates APAP-aggravated hepatotoxicity in NAFLD via the ROS/TXNIP/NLRP3 pathway remains unclear. An in vivo fatty liver model was established by feeding mice a high-fat diet for 56 days. Additionally, using in vitro approach, AML-12 cells were incubated with free fatty acids for 48 h and APAP was added during the last 24 h. We found that the overuse of APAP in NAFLD not only induced oxidative stress, but also increased TXNIP expression, NLRP3-mediated pyroptosis, and lipid deposition. In addition to inhibiting ROS generation and lipid deposition, overexpression of CAV1 reduced the elevated levels of TXNIP expression and NLRP3-mediated pyroptosis. However, the effect of CAV1 on TXNIP expression, NLRP3-mediated pyroptosis, and lipid deposition was reversed by CAV1 small interfering RNA (siRNA) intervention. Finally, N-acetyl cysteine (NAC) treatment reduced CAV1 siRNA-mediated changes in TXNIP expression and NLRP3-mediated pyroptosis levels. These results demonstrate that the inhibitory effect of CAV1 on NLRP3-mediated pyroptosis may be mediated through the ROS/TXNIP axis. Moreover, the current study provides novel mechanistic insights into the protective effects of CAV1 on APAP-aggravated hepatotoxicity in NAFLD.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Acetaminofen , Espécies Reativas de Oxigênio/metabolismo , Caveolina 1/metabolismo , Inflamassomos/metabolismo , Piroptose , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , RNA Interferente Pequeno , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Lipídeos , Proteínas de Transporte/genéticaRESUMO
Acetaminophen (APAP) is a common antipyretic and analgesic drug that can cause long-term liver damage after an overdose. Non-alcoholic fatty liver disease (NAFLD) increases susceptibility to APAP. In NAFLD, excessive accumulation of lipids leads to an abnormal increase in hypoxia-inducible factor-1α (HIF-1α). Caveolin-1 (CAV1) may protect against NAFLD by inhibiting HIF-1α. This research aimed to determine whether CAV1 could attenuate APAP-exacerbated liver injury in NAFLD by inhibiting oxidative stress involving HIF-1α. In this study, 7-week-old C57BL/6 mice were fed a high-fat diet (HFD) for eight weeks, followed by the instillation of APAP. Levels of oxidative stress and liver lipid deposition were determined, and p-ERK1/2 and HIF-1α protein expression were measured by the Western blot (WB) method. In the APAP-treated group, the level of CAV1 was decreased, while the levels of HIF-1α and reactive oxygen species (ROS) were significantly increased. AML12 cells were treated with a mixture of palmitic acid (PA) and oleic acid (OA) (1:2 mix) for 48 h, and APAP was added for the last 24 h. Overexpression of CAV1 in AML12 cells significantly inhibited the expression of ROS and HIF-1α. And the results of immunofluorescence after treatment with CAV1-SiRNA showed that the HIF-1α levels were significantly increased in mitochondria. In conclusion, our experimental results suggest that CAV1 has a protective function in the fatty liver based on preventing oxidative stress, which involves HIF-1α. Thus, upregulation of CAV1 may attenuate APAP-exacerbated liver injury in NAFLD.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo , Acetaminofen/efeitos adversos , Acetaminofen/metabolismo , Caveolina 1/metabolismo , Sistema de Sinalização das MAP Quinases , Espécies Reativas de Oxigênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismoRESUMO
Parkinson's disease (PD) is characterized by the aggregation of α-synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for α-synuclein has been linked to early-onset PD, and exposure to A53T-mutant human α-synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the effects of the A53T mutation on human microglia. We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, A53T mutant microglia showed a strong decrease in catalase expression in non-inflammatory conditions, and increased oxidative stress. Our results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.
RESUMO
BACKGROUND: Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. METHODS: To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. RESULTS: The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons. CONCLUSIONS: This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.
Assuntos
Síndrome do Cromossomo X Frágil , Células-Tronco Neurais , Humanos , Camundongos , Animais , Síndrome do Cromossomo X Frágil/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fenótipo , Encéfalo/metabolismo , Camundongos KnockoutRESUMO
Background: Retinal disorders cause substantial visual burden globally. Accurate estimates of the vision loss due to retinal diseases are pivotal to inform optimal eye health care planning and allocation of medical resources. The purpose of this study is to describe the proportion of visual impairment and blindness caused by major retinal diseases in China. Methods: A nationwide register-based study of vitreoretinal disease covering all 31 provinces (51 treating centres) of mainland China. A total of 28 320 adults diagnosed with retinal diseases were included. Participants underwent standardised ocular examinations, which included best-corrected visual acuity (BCVA), dilated-fundus assessments, and optical coherence tomography. Visual impairment and blindness are defined using BCVA according to the World Health Organization (WHO) (visual impairment: <20/63-≥20/400; blindness: <20/400) and the United States (visual impairment: <20/40-≥20/200; blindness: <20/200) definitions. The risk factors of vision loss were explored by logistic regression analyses. Results: Based on the WHO definitions, the proportions for unilateral visual impairment and blindness were 46% and 18%, respectively, whereas those for bilateral visual impairment and blindness were 31% and 3.3%, respectively. Diabetic retinopathy (DR) accounts for the largest proportion of patients with visual impairment (unilateral visual impairment: 32%, bilateral visual impairment: 60%) and blindness (unilateral blindness: 35%; bilateral blindness: 64%). Other retinal diseases that contributed significantly to vision loss included age-related macular degeneration, myopic maculopathy, retinal vein occlusion, and rhegmatogenous retinal detachment and other macular diseases. Women (bilateral vision loss: P = 0.011), aged patients (unilateral vision loss: 45-64 years: P < 0.001, ≥65 years: P < 0.001; bilateral vision loss: 45-64 years: P = 0.003, ≥65 years: P < 0.001 (reference: 18-44 years)) and those from Midwest China (unilateral and bilateral vision loss: both P < 0.001) were more likely to suffer from vision loss. Conclusions: Retinal disorders cause substantial visual burden among patients with retinal diseases in China. DR, the predominant retinal disease, is accountable for the most prevalent visual disabilities. Better control of diabetes and scaled-up screenings are warranted to prevent DR. Specific attention should be paid to women, aged patients, and less developed regions.
Assuntos
Retinopatia Diabética , Degeneração Macular , Doenças Retinianas , Baixa Visão , Pessoas com Deficiência Visual , Adulto , Humanos , Feminino , Idoso , Acuidade Visual , Cegueira/epidemiologia , Cegueira/etiologia , Baixa Visão/etiologia , Baixa Visão/complicações , Transtornos da Visão/etiologia , Transtornos da Visão/complicações , Doenças Retinianas/epidemiologia , Doenças Retinianas/complicações , Degeneração Macular/complicações , Degeneração Macular/epidemiologia , PrevalênciaRESUMO
Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process is made more efficient through the suppression of lineage specific gene expression.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Proteínas Repressoras/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Blastocisto/citologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Técnicas de Cocultura , Transferência Embrionária , Fibroblastos/citologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Fator 4 Semelhante a Kruppel , Antígenos CD15/metabolismo , Camundongos , Proteína Homeobox Nanog , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Teratoma/patologia , Quimeras de TransplanteRESUMO
To understand the mechanisms regulating the in vitro maturation of hPSC-derived hepatocytes, we developed a 3D differentiation system and compared gene regulatory elements in human primary hepatocytes with those in hPSC-hepatocytes that were differentiated in 2D or 3D conditions by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Regulome comparisons showed a reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and active enhancers without a reduced transcription of THRB. The addition of thyroid hormone T3 increased the binding of THRB to the CYP3A4 proximal enhancer, restored the super-enhancer status and gene expression of NFIC, and reduced the expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status, and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the hPSC-hepatocytes resulted in the engraftment of human hepatocytes into the mouse liver without disrupting normal liver histology. This work implicates the environmental factor-nuclear receptor axis in regulating the maturation of hPSC-hepatocytes.
Assuntos
Cromatina , Hepatócitos , Animais , Diferenciação Celular , Cromatina/metabolismo , Hepatócitos/metabolismo , Humanos , Camundongos , Polimorfismo de Nucleotídeo Único , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.
Assuntos
Encéfalo/patologia , Fibroblastos/citologia , Neurônios/citologia , Neurônios/fisiologia , Doença de Parkinson/terapia , Transplante de Células-Tronco/métodos , Animais , Comportamento Animal , Encéfalo/embriologia , Diferenciação Celular , Técnicas de Cultura , Eletrofisiologia , Fator 4 Semelhante a Kruppel , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , RatosRESUMO
To date, the degradation of microplastics (MPs; <5 mm) in different environments, particularly their adsorption characteristics for coexisted metal pollutants remains to be elucidated. Thus, this study investigated the effects of aging MPs, including polyamide (mPA), polyethylene terephthalate (mPET), polystyrene (mPS), and polyvinyl chloride (mPVC) for 3 months under UVA irradiation in four environmental media (air, seawater, sand, and soil) and adsorption of heavy metals (Cu, Cd) onto seawater-aged mPS and mPVC. The results showed that surface morphological changes, including cracks, oxidized particles, and wrinkles, appeared on aged MPs. The heavy metal adsorption capacity decreased in the order aged mPVC > aged mPS > unaged mPS > unaged mPVC, and the Cu2+ and Cd2+ ions competed for active adsorption sites on the MPs surfaces. Overall, the aging environment affected the physical and chemical properties of MPs and the aging of MPs enhanced their adsorption of coexisting metals tested.