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1.
Mol Cell ; 77(4): 875-886.e7, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31836389

RESUMO

Dysregulation of cellular protein synthesis is linked to a variety of diseases. Mutations in EIF2S3, encoding the γ subunit of the heterotrimeric eukaryotic translation initiation factor eIF2, cause MEHMO syndrome, an X-linked intellectual disability disorder. Here, using patient-derived induced pluripotent stem cells, we show that a mutation at the C terminus of eIF2γ impairs CDC123 promotion of eIF2 complex formation and decreases the level of eIF2-GTP-Met-tRNAiMet ternary complexes. This reduction in eIF2 activity results in dysregulation of global and gene-specific protein synthesis and enhances cell death upon stress induction. Addition of the drug ISRIB, an activator of the eIF2 guanine nucleotide exchange factor, rescues the cell growth, translation, and neuronal differentiation defects associated with the EIF2S3 mutation, offering the possibility of therapeutic intervention for MEHMO syndrome.


Assuntos
Acetamidas/farmacologia , Cicloexilaminas/farmacologia , Epilepsia/genética , Fator de Iniciação 2 em Eucariotos/genética , Genitália/anormalidades , Hipogonadismo/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Microcefalia/genética , Mutação , Obesidade/genética , Biossíntese de Proteínas/efeitos dos fármacos , Apoptose , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/citologia
2.
BMC Biol ; 21(1): 55, 2023 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-36941669

RESUMO

BACKGROUND: The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. RESULTS: We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. CONCLUSIONS: TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.


Assuntos
Pancreatite , Adulto , Humanos , Animais , Camundongos , Doença Aguda , Pancreatite/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Pâncreas/metabolismo , Organogênese/genética
3.
Gut ; 70(4): 743-760, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32873698

RESUMO

OBJECTIVE: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC). DESIGN: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy. RESULTS: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance. CONCLUSION: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.


Assuntos
Adenocarcinoma/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Carcinoma Ductal Pancreático/genética , Recombinação Homóloga , Neoplasias Pancreáticas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Adenocarcinoma/tratamento farmacológico , Animais , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular , Variações do Número de Cópias de DNA , Dano ao DNA , Reparo do DNA , Resistência a Múltiplos Medicamentos/genética , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal , Genótipo , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Prognóstico
4.
Cells Tissues Organs ; 209(4-6): 155-164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33486479

RESUMO

Usually, pandemic COVID-19 disease, caused by SARS-CoV2, presents with mild respiratory symptoms such as fever, cough, but frequently also with anosmia and neurological symptoms. Virus-cell fusion is mediated by angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) with their organ expression pattern determining viral tropism. Clinical presentation suggests rapid viral dissemination to the central nervous system leading frequently to severe symptoms including viral meningitis. Here, we provide a comprehensive expression landscape of ACE2 and TMPRSS2 proteins across human postmortem nasal and olfactory tissue. Sagittal sections through the human nose complemented with immunolabelling of respective cell types represent different anatomically defined regions including olfactory epithelium, respiratory epithelium of the nasal conchae and the paranasal sinuses along with the hardly accessible human olfactory bulb. ACE2 can be detected in the olfactory epithelium as well as in the respiratory epithelium of the nasal septum, the nasal conchae, and the paranasal sinuses. ACE2 is located in the sustentacular cells and in the glandular cells in the olfactory epithelium as well as in the basal cells, glandular cells, and epithelial cells of the respiratory epithelium. Intriguingly, ACE2 is not expressed in mature or immature olfactory receptor neurons and basal cells in the olfactory epithelium. Similarly, ACE2 is not localized in the olfactory receptor neurons albeit the olfactory bulb is positive. Vice versa, TMPRSS2 can also be detected in the sustentacular cells and the glandular cells of the olfactory epithelium. Our findings provide the basic anatomical evidence for the expression of ACE2 and TMPRSS2 in the human nose, olfactory epithelium, and olfactory bulb. Thus, they are substantial for future studies that aim to elucidate the symptom of SARS-CoV2 induced anosmia via the olfactory pathway.


Assuntos
Enzima de Conversão de Angiotensina 2/análise , COVID-19/patologia , Mucosa Nasal/patologia , Bulbo Olfatório/patologia , SARS-CoV-2/isolamento & purificação , Serina Endopeptidases/análise , COVID-19/diagnóstico , Humanos , Mucosa Nasal/virologia , Nariz/patologia , Nariz/virologia , Bulbo Olfatório/virologia , Mucosa Olfatória/patologia , Mucosa Olfatória/virologia
5.
Int J Mol Sci ; 21(4)2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085662

RESUMO

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Edição de Genes , Perfilação da Expressão Gênica , Adulto , Animais , Ciclo Celular/genética , Regulação da Expressão Gênica , Genoma , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mamíferos/genética , Camundongos , Células Fotorreceptoras de Vertebrados/metabolismo
6.
Am J Hum Genet ; 98(3): 500-513, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26942284

RESUMO

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.


Assuntos
Mitofagia/genética , Transtornos Parkinsonianos/genética , Proteínas Quinases/genética , Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Animais , Células COS , Estudos de Casos e Controles , Consanguinidade , Feminino , Inativação Gênica , Heterogeneidade Genética , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Parkinsonianos/diagnóstico , Linhagem , Fenótipo , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Reprodutibilidade dos Testes , Turquia , Ubiquitina-Proteína Ligases/metabolismo
7.
Gut ; 66(3): 473-486, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27633923

RESUMO

OBJECTIVE: The generation of acinar and ductal cells from human pluripotent stem cells (PSCs) is a poorly studied process, although various diseases arise from this compartment. DESIGN: We designed a straightforward approach to direct human PSCs towards pancreatic organoids resembling acinar and ductal progeny. RESULTS: Extensive phenotyping of the organoids not only shows the appropriate marker profile but also ultrastructural, global gene expression and functional hallmarks of the human pancreas in the dish. Upon orthotopic transplantation into immunodeficient mice, these organoids form normal pancreatic ducts and acinar tissue resembling fetal human pancreas without evidence of tumour formation or transformation. Finally, we implemented this unique phenotyping tool as a model to study the pancreatic facets of cystic fibrosis (CF). For the first time, we provide evidence that in vitro, but also in our xenograft transplantation assay, pancreatic commitment occurs generally unhindered in CF. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activation in mutated pancreatic organoids not only mirrors the CF phenotype in functional assays but also at a global expression level. We also conducted a scalable proof-of-concept screen in CF pancreatic organoids using a set of CFTR correctors and activators, and established an mRNA-mediated gene therapy approach in CF organoids. CONCLUSIONS: Taken together, our platform provides novel opportunities to model pancreatic disease and development, screen for disease-rescuing agents and to test therapeutic procedures.


Assuntos
Fibrose Cística/terapia , Modelos Animais de Doenças , Organoides/crescimento & desenvolvimento , Organoides/transplante , Pâncreas/citologia , RNA Mensageiro/uso terapêutico , Células Acinares/citologia , Animais , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Perfilação da Expressão Gênica , Terapia Genética , Humanos , Camundongos , Organoides/citologia , Organoides/metabolismo , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Ductos Pancreáticos/citologia , Fenótipo , Células-Tronco Pluripotentes
8.
Stem Cells ; 34(6): 1563-75, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26946488

RESUMO

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/metabolismo
10.
Neural Plast ; 2016: 3760702, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27247802

RESUMO

Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function, given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus, to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons, using a protocol for motor neuron differentiation, we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes, cell survival, cell fate, and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival, altered neuronal differentiation, and, in particular, synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation.


Assuntos
Homeostase/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Zinco/metabolismo , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Transdução de Sinais/fisiologia
11.
Neurobiol Dis ; 82: 420-429, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26253605

RESUMO

Autosomal-dominant mutations within the gene FUS (fused in sarcoma) are responsible for 5% of familial cases of amyotrophic lateral sclerosis (ALS). The FUS protein is physiologically mainly located in the nucleus, while cytoplasmic FUS aggregates are pathological hallmarks of FUS-ALS. Data from non-neuronal cell models and/or models using heterologous expression of FUS mutants suggest cytoplasmic FUS translocation as a pivotal initial event which leads to neurodegeneration depending on a second hit. Here we present the first human model of FUS-ALS using patient-derived neurons carrying endogenous FUS mutations leading to a benign (R521C) or a more severe clinical phenotype (frameshift mutation R495QfsX527). We thereby showed that the severity of the underlying FUS mutation determines the amount of cytoplasmic FUS accumulation and cellular vulnerability to exogenous stress. Cytoplasmic FUS inclusions formed spontaneously depending on both, severity of FUS mutation and neuronal aging. These aggregates showed typical characteristics of FUS-ALS including methylated FUS. Finally, neurodegeneration was not specific to layer V cortical neurons perfectly in line with the current model of disease spreading in ALS. Our study highlights the value and usefulness of patient-derived cell models in FUS-ALS.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios/patologia , Proteína FUS de Ligação a RNA/genética , Adulto , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Progressão da Doença , Feminino , Humanos , Corpos de Inclusão/patologia , Corpos de Inclusão/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Pessoa de Meia-Idade , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Mutação , Neurônios/fisiologia , Fenótipo , Proteína FUS de Ligação a RNA/metabolismo , Índice de Gravidade de Doença , Medula Espinal/patologia , Medula Espinal/fisiopatologia
12.
Gastroenterology ; 142(4): 907-17, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22245845

RESUMO

BACKGROUND & AIMS: Ectopic expression of certain transcription factors can reprogram somatic cells to a pluripotent state. Hematopoietic and muscle stem cells can be more efficiently reprogrammed than differentiated blood or muscle cells, yet similar findings have not been shown in other primary organ systems. Moreover, molecular characteristics of the cellular hierarchy of tissues that influence reprogramming capacities need to be delineated. We analyzed the effect of differentiation stage of freshly isolated, mouse liver cells on the reprogramming efficiency. METHODS: Liver progenitor cell (LPC)-enriched cell fractions were isolated from adult (6-8 wk) and fetal (embryonic day 14.5) livers of mice and reprogrammed to become induced pluripotent stem (iPS) cells. Different transcription factors were expressed in liver cells, and markers of pluripotency were examined, along with the ability of iPS cells to differentiate, in vitro and in vivo, into different germ layers. RESULTS: Fetal and adult LPCs had significantly greater reprogramming efficiency after transduction with 3 or 4 reprogramming factors. Transduction efficiency-corrected reprogramming rates of fetal LPCs were 275-fold higher, compared with unsorted fetal liver cells, when 3 reprogramming factors were transduced. The increased reprogramming efficiency of LPCs, compared with differentiated liver cells, occurred independently of proliferation rates, but was associated with endogenous expression of reprogramming factors (Klf4 and c-Myc) and BAF (Brg1/Brm associated factor)-complex members Baf155 and Brg1, which mediate epigenetic changes during reprogramming. Knockdown of BAF complex members negated the increased reprogramming efficiency of LPCs, compared with non-LPCs. CONCLUSIONS: LPCs have intrinsic, cell proliferation-independent characteristics resulting in an increased reprogramming capacity compared to differentiated liver cells.


Assuntos
Diferenciação Celular , DNA Helicases/metabolismo , Hepatócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , DNA Helicases/genética , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/embriologia , Camundongos , Proteínas Nucleares/genética , Fenótipo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Fatores de Tempo , Fatores de Transcrição/genética , Transdução Genética , Transfecção
13.
FASEB J ; 26(12): 4990-5001, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22935140

RESUMO

Inactivation of FoxO proteins by phosphorylation is the result of a number of stimuli, including the insulin/IGF pathway. We were interested in the consequence of blunting this pathway by employing transgenic mice with tetracycline-controllable conditional expression of a constitutively active allele of FOXO3 under the control of the forebrain-specific CaMKIIα promoter. Although transgene-expressing mice were viable, brain weight was reduced by 30% in adult animals. Brains showed an isocortex compression with normal cortical layering, and a size reduction in regions known to depend on adult neurogenesis, i.e., the olfactory bulbs and the dentate gyrus. On postnatal activation of the transgene, adult neurogenesis was also severely affected. Investigating the molecular basis of this phenotype, we observed enhanced apoptosis starting from embryonic day E10.5 and a subsequent loss of progenitors in the ventricular/subventricular zones, but not in the isocortex or the striatum of adult mice. The enhanced apoptosis was accompanied by increased expression of PIK3IP1, which we identified as a direct transcriptional target of FOXO3. Transfection of Pik3ip1 into differentiating neural progenitors resulted in a significant reduction of viable cells. We therefore conclude that neural progenitors are particularly vulnerable to FOXO3-induced apoptosis, which is mediated by PIK3IP1, a negative PI3 kinase regulator.


Assuntos
Fatores de Transcrição Forkhead/genética , Células-Tronco Neurais/metabolismo , Prosencéfalo/metabolismo , Animais , Apoptose/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Análise por Conglomerados , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
14.
J Neural Transm (Vienna) ; 120(5): 785-98, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23143281

RESUMO

The dynactin p150glued subunit, encoded by the gene DCTN1 is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport. This subunit is a candidate modifier for neurodegenerative diseases, in particular motoneuron and extrapyramidal diseases. Based on an extensive screening effort of all 32 exons in more than 2,500 ALS/MND patients, patients suffering from Parkinsonian Syndromes and controls, we investigated 24 sequence variants of p150 in cell-based studies. We used both non-neuronal cell lines and primary rodent spinal motoneurons and report on cell biological abnormalities in five of these sequence alterations and also briefly report on the clinical features. Our results suggest the presence of biological changes caused by some p150 mutants pointing to a potential pathogenetic significance as modifier of the phenotype of the human disease.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Associadas aos Microtúbulos/genética , Neurônios Motores/metabolismo , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , Proteínas Adaptadoras de Transdução de Sinal , Esclerose Lateral Amiotrófica/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Complexo Dinactina , Embrião de Mamíferos , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Mutação/genética , Gravidez , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Estudos Retrospectivos , Medula Espinal/citologia , Fatores de Tempo
15.
Cell Tissue Res ; 350(1): 13-26, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22777741

RESUMO

The dynactin p150(Glued) subunit, encoded by the gene DCTN1, is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport in motor neurons. The p150 subunit is a candidate gene for neurodegenerative diseases, in particular motor neuron and extrapyramidal diseases. Tubulin-binding cofactors are believed to be involved in tubulin biogenesis and degradation and therefore to contribute to microtubule functional diversity and regulation. A yeast-two-hybrid screen for putative interacting proteins of dynactin p150(Glued) has revealed tubulin-folding cofactor B (TBCB). We analyzed the interaction of these proteins and investigated the impact of this complex on the microtubule network in cell lines and primary hippocampal neurons in vitro. We especially concentrated on neuronal morphology and synaptogenesis. Overexpression of both proteins or depletion of TBCB alone does not alter the microtubule network and/or neuronal morphology. The demonstration of the interaction of the transport molecule dynactin and the tubulin-regulating factor TBCB is thought to have an impact on several cellular mechanisms. TBCB expression levels have been found to have only a subtle influence on the microtubule network and neuronal morphology. However, overexpression of TBCB leads to the decreased localization of p150 to the microtubule network that might result in a functional modulation of this protein complex.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Regulação para Baixo/genética , Complexo Dinactina , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sinapses/metabolismo
16.
Cells ; 11(12)2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35741085

RESUMO

Various somatic cell types are suitable for induced pluripotency reprogramming, such as dermal fibroblasts, mesenchymal stem cells or hair keratinocytes. Harvesting primary epithelial keratinocytes from plucked human hair follicles (HFs) represents an easy and non-invasive alternative to a fibroblast culture from invasive skin biopsies. Nevertheless, to facilitate and simplify the process, which can be divided into three main steps (collecting, culturing and reprogramming), the whole procedure of generating hair keratinocytes has to be revised and upgraded continuously. In this study, we address advancements and approaches which improve the generation and handling of primary HF-derived keratinocytes tremendously, e.g., for iPSCs reprogramming. We not only evaluated different serum- and animal-origin-free media, but also supplements and coating solutions for an enhanced protocol. Here, we demonstrate the importance of speed and accuracy in the collecting step, as well as the choice of the right transportation medium. Our results lead to a more defined approach that further increases the reliability of downstream experiments and inter-laboratory reproducibility. These improvements will make it possible to obtain keratinocytes from plucked human hair for the generation of donor-specific iPSCs easier and more efficient than ever before, whilst preserving a non-invasive capability.


Assuntos
Células-Tronco Pluripotentes Induzidas , Queratinócitos , Animais , Cabelo , Folículo Piloso , Queratinócitos/metabolismo , Reprodutibilidade dos Testes
17.
Commun Biol ; 5(1): 52, 2022 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-35027657

RESUMO

Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies.


Assuntos
Produtos Biológicos/farmacologia , Corioide/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Procedimentos Analíticos em Microchip , Anticorpos Biespecíficos/efeitos dos fármacos , Anticorpos Biespecíficos/metabolismo , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo
18.
Circulation ; 122(18): 1823-36, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-20956206

RESUMO

BACKGROUND: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. METHODS AND RESULTS: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects. CONCLUSIONS: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.


Assuntos
Diferenciação Celular/fisiologia , Sistema de Condução Cardíaco/citologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Canais de Potássio Cálcio-Ativados/fisiologia , Animais , Benzimidazóis/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Linhagem Celular , Proliferação de Células , Citoesqueleto/fisiologia , Sistema de Condução Cardíaco/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Transdução de Sinais/fisiologia
19.
J Microsc ; 244(3): 259-72, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21883208

RESUMO

Here we present a set of methods for documenting (exo-)morphology by applying autofluorescence imaging. For arthropods, but also for other taxa, autofluorescence imaging combined with composite imaging is a fast documentation method with high-resolution capacities. Compared to conventional micro- and macrophotography, the illumination is much more homogenous, and structures are often better contrasted. Applying different wavelengths to the same object can additionally be used to enhance distinct structures. Autofluorescence imaging can be applied to dried and embedded specimens, but also directly on specimens within their storage liquid. This has an enormous potential for the documentation of rare specimens and especially type specimens without the need of preparation. Also for various fossils, autofluorescence can be used to enhance the contrast between the fossil and the matrix significantly, making even smallest details visible. 'Life-colour' fluorescence especially is identified as a technique with great potential. It provides additional information for which otherwise more complex methods would have to be applied. The complete range of differences and variations between fluorescence macrophotography and different types of fluorescence microscopy techniques are here explored and evaluated in detail. Also future improvements are suggested. In summary, autofluorescence imaging is a powerful, easy and fast-to-apply tool for morphological studies.


Assuntos
Artrópodes/anatomia & histologia , Equinodermos/anatomia & histologia , Fluorescência , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Moluscos/anatomia & histologia , Fotografação/métodos , Animais , Artrópodes/química , Artrópodes/citologia , Equinodermos/química , Equinodermos/citologia , Moluscos/química , Moluscos/citologia
20.
Exp Cell Res ; 316(17): 2760-78, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20599952

RESUMO

Tissue-specific stem cells, such as bone-marrow-derived human mesenchymal stem cells (hMSCs), are thought to be lineage restricted and therefore, could only be differentiated into cell types of the tissue of origin. Several recent studies however have suggested that these types of stem cells might be able to break barriers of germ layer commitment and differentiate in vitro into cells with neuroectodermal properties. We reported earlier about efficient conversion of adult hMSCs into a neural stem cell (NSC)-like population (hmNSCs, for human marrow-derived NSC-like cells) with all major properties of NSCs including functional neuronal differentiation capacity. Here we compared the transcriptomes from hMSCs and hmNSCs using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses to shed light on regulatory protein networks involved in this neuroectodermal conversion process. We found differential regulation of extracellular matrix protein transcripts, up-regulation of distinct neuroectodermal and NSCs marker genes and local chromosomal transcriptional up-regulation at chromosome 4q13.3. In comparison to hMSCs and primary adult hippocampal NSCs, the transcriptome of hmNSCs displayed minor overlap with both other cell populations. Advanced bioinformatics of regulated genes upon neuroectodermal conversion identified transcription factor networks with HIF-1 and microRNA miR-124a as potential major regulators. Together, transgerminal neuroectodermal conversion of hMSCs into NSC-like cells is accompanied by extensive changes of their global gene expression profile, which might be controlled in part by transcription factor networks related to HIF-1 and miR-124a.


Assuntos
Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Fator 1 Induzível por Hipóxia/genética , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Placa Neural/citologia , Adolescente , Adulto , Células da Medula Óssea , Linhagem da Célula , Células Cultivadas , Humanos , Neurônios/citologia , Células-Tronco/citologia , Adulto Jovem
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