RESUMO
Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (hiPSCs) by the overexpression of ATOH7, BRN3B, and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers by whole transcriptome profiling. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças do Nervo Óptico , Humanos , Células Ganglionares da Retina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doenças do Nervo Óptico/metabolismo , Apoptose , Etambutol/farmacologia , Etambutol/metabolismo , Fatores de Transcrição SOXC/metabolismoRESUMO
Microglia, the immune cells of the central nervous system, play critical roles in brain physiology and pathology. We report a novel approach that produces, within 10 days, the differentiation of human induced pluripotent stem cells (hiPSCs) into microglia (iMG) by forced expression of both SPI1 and CEBPA. High-level expression of the main microglial markers and the purity of the iMG cells were confirmed by RT-qPCR, immunostaining, and flow cytometry analyses. Whole-transcriptome analysis demonstrated that these iMGs resemble human fetal/adult microglia but not human monocytes. Moreover, these iMGs exhibited appropriate physiological functions, including various inflammatory responses, ADP/ATP-evoked migration, and phagocytic ability. When co-cultured with hiPSC-derived neurons, the iMGs respond and migrate toward injured neurons. This study has established a protocol for the rapid conversion of hiPSCs into functional iMGs, which should facilitate functional studies of human microglia using different disease models and also help with drug discovery.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Microglia/citologia , Fatores de Transcrição/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Biomarcadores/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microglia/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) is the most common type of dementia and also one of the leading causes of death worldwide. However, the underlying mechanisms remain unclear, and currently there is no drug treatment that can prevent or cure AD. Here, we have applied the advantages of using induced pluripotent stem cell (iPSC)-derived neurons (iNs) from AD patients, which are able to offer human-specific drug responsiveness, in order to evaluate therapeutic candidates for AD. Using approach involving an inducible neurogenin-2 transgene, we have established a robust and reproducible protocol for differentiating human iPSCs into glutamatergic neurons. The AD-iN cultures that result have mature phenotypic and physiological properties, together with AD-like biochemical features that include extracellular ß-amyloid (Aß) accumulation and Tau protein phosphorylation. By screening using a gene set enrichment analysis (GSEA) approach, Graptopetalum paraguayense (GP) has been identified as a potential therapeutic agent for AD from among a range of Chinese herbal medicines. We found that administration of a GP extract caused a significantly reduction in the AD-associated phenotypes of the iNs, including decreased levels of extracellular Aß40 and Aß42, as well as reduced Tau protein phosphorylation at positions Ser214 and Ser396. Additionally, the effect of GP was more prominent in AD-iNs compared to non-diseased controls. These findings provide valuable information that suggests moving extracts of GP toward drug development, either for treating AD or as a health supplement to prevent AD. Furthermore, our human iN-based platform promises to be a useful strategy when it is used for AD drug discovery.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/genética , Crassulaceae/química , Fragmentos de Peptídeos/genética , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/efeitos dos fármacos , Descoberta de Drogas , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/patologiaRESUMO
Based on the protein kinase A (PKA)/GSK3ß interaction protein (GSKIP)/glycogen synthase kinase 3ß (GSK3ß) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3ß-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3ß with PKA but not Ca2+/calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer's disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA-Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein (APPWT/D678H) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3ß, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3ß function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis.
RESUMO
Congenital heart defects, dysmorphic facial features and intellectual developmental disorders (CHDFIDD) syndrome in humans was recently associated with mutation in CDK13 gene. In order to assess the loss of function of Cdk13 during mouse development, we employed gene trap knock-out (KO) allele in Cdk13 gene. Embryonic lethality of Cdk13-deficient animals was observed by the embryonic day (E) 16.5, while live embryos were observed on E15.5. At this stage, improper development of multiple organs has been documented, partly resembling defects observed in patients with mutated CDK13. In particular, overall developmental delay, incomplete secondary palate formation with variability in severity among Cdk13-deficient animals or complete midline deficiency, kidney failure accompanied by congenital heart defects were detected. Based on further analyses, the lethality at this stage is a result of heart failure most likely due to multiple heart defects followed by insufficient blood circulation resulting in multiple organs dysfunctions. Thus, Cdk13 KO mice might be a very useful model for further studies focused on delineating signaling circuits and molecular mechanisms underlying CHDFIDD caused by mutation in CDK13 gene.
RESUMO
Mutations in RAB18, a member of small G protein, cause Warburg micro syndrome (WARBM), whose clinical features include vision impairment, postnatal microcephaly, and lower limb spasticity. Previously, our Rab18-/- mice exhibited hind limb weakness and spasticity as well as signs of axonal degeneration in the spinal cord and lumbar spinal nerves. However, the cellular and molecular function of RAB18 and its roles in the pathogenesis of WARBM are still not fully understood. Using immunofluorescence staining and expression of Rab18 and organelle markers, we find that Rab18 associates with lysosomes and actively traffics along neurites in cultured neurons. Interestingly, Rab18-/- neurons exhibit impaired lysosomal transport. Using autophagosome marker LC3-II, we show that Rab18 dysfunction leads to aberrant autophagy activities in neurons. Electron microscopy further reveals accumulation of lipofuscin-like granules in the dorsal root ganglion of Rab18-/- mice. Surprisingly, Rab18 colocalizes, cofractionates, and coprecipitates with the lysosomal regulator Rab7, mutations of which cause Charcot-Marie-Tooth (CMT) neuropathy type 2B. Moreover, Rab7 is upregulated in Rab18-deficient neurons, suggesting a compensatory effect. Together, our results suggest that the functions of RAB18 and RAB7 in lysosomal and autophagic activities may constitute an overlapping mechanism underlying WARBM and CMT pathogenesis in the nervous system.
Assuntos
Anormalidades Múltiplas/metabolismo , Autofagia , Catarata/congênito , Doença de Charcot-Marie-Tooth/metabolismo , Córnea/anormalidades , Hipogonadismo/metabolismo , Deficiência Intelectual/metabolismo , Lisossomos/metabolismo , Microcefalia/metabolismo , Sistema Nervoso/metabolismo , Atrofia Óptica/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Catarata/metabolismo , Córnea/metabolismo , Epistasia Genética , Células HEK293 , Humanos , Laminopatias , Camundongos , Neurônios/metabolismo , Células PC12 , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
DNA damage response (DDR) pathways are critical for ensuring that replication stress and various types of DNA lesion do not perturb production of neural cells during development. Cdk12 maintains genomic stability by regulating expression of DDR genes. Mutant mice in which Cdk12 is conditionally deleted in neural progenitor cells (NPCs) die after birth and exhibit microcephaly with a thinner cortical plate and an aberrant corpus callosum. We show that NPCs of mutant mice accumulate at G2 and M phase, and have lower expression of DDR genes, more DNA double-strand breaks and increased apoptosis. In addition to there being fewer neurons, there is misalignment of layers IV-II neurons and the presence of abnormal axonal tracts of these neurons, suggesting that Cdk12 is also required for the migration of late-arising cortical neurons. Using in utero electroporation, we demonstrate that the migrating mutant cells remain within the intermediate zone and fail to adopt a bipolar morphology. Overexpression of Cdk5 brings about a partially restoration of the neurons reaching layers IV-II in the mutant mice. Thus, Cdk12 is crucial to the repair of DNA damage during the proliferation of NPCs and is also central to the proper migration of late-arising neurons.
Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Animais , Proliferação de Células/fisiologia , Córtex Cerebral/patologia , Quinase 5 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/genética , Dano ao DNA/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcefalia/metabolismo , Microcefalia/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurônios/patologia , Tamanho do Órgão , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
The pathogenesis of Parkinson's disease (PD) is not completely understood, Zinc (Zn(2+) ) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn(2+) and DA-induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn(2+) treatment and raised to an even higher level by Zn(2+) plus DA treatment. Zn(2+) plus DA treatment-induced PC12 cell death was enhanced when there was over-expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross-talk with Gadd45b during Zn(2+) and DA treatment. The synergistic effects of Zn(2+) and DA on PC12 cell death can be accounted for by an activation of the Gadd45b-induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn(2+) /DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD. Zinc and dopamine are implicated in the degeneration of dopaminergic neurons. We previously demonstrated that zinc and dopamine induced synergistic effects on PC12 cell death. Results from this study show that these synergistic effects can be accounted for by activation of the Gadd45b-induced cell death pathway and inhibition of the p38/JNK survival pathway. We provide in vitro and in vivo evidence to support a novel role for Gadd45b in the pathogenesis of Parkinson's disease.
Assuntos
Antígenos de Diferenciação/efeitos dos fármacos , Antígenos de Diferenciação/genética , Dopamina/toxicidade , Doença de Parkinson/genética , Doença de Parkinson/patologia , Zinco/toxicidade , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Morte Celular/efeitos dos fármacos , Sinergismo Farmacológico , Sequestradores de Radicais Livres/farmacologia , Técnicas de Silenciamento de Genes , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose/patologia , Proteínas Nucleares/genética , Células PC12 , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mutations in the gene of RAB18, a member of Ras superfamily of small G-proteins, cause Warburg Micro Syndrome (WARBM) which is characterized by defective neurodevelopmental and ophthalmological phenotypes. Despite loss of Rab18 had been reported to induce disruption of the endoplasmic reticulum structure and neuronal cytoskeleton organization, parts of the pathogenic mechanism caused by RAB18 mutation remain unclear. From the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis library, we identified a mouse line whose Rab18 was knocked out. This Rab18(-/-) mouse exhibited stomping gait, smaller testis and eyes, mimicking several features of WARBM. Rab18(-/-) mice were obviously less sensitive to pain and touch than WT mice. Histological examinations on Rab18(-/-) mice revealed progressive axonal degeneration in the optic nerves, dorsal column of the spinal cord and sensory roots of the spinal nerves while the motor roots were spared. All the behavioral and pathological changes that resulted from abnormalities in the sensory axons were prevented by introducing an extra copy of Rab18 transgene in Rab18(-/-) mice. Our results reveal that sensory axonal degeneration is the primary cause of stomping gait and progressive weakness of the hind limbs in Rab18(-/-) mice, and optic nerve degeneration should be the major pathology of progressive optic atrophy in children with WARBM. Our results indicate that the sensory nervous system is more vulnerable to Rab18 deficiency and WARBM is not only a neurodevelopmental but also neurodegenerative disease.
Assuntos
Anormalidades Múltiplas , Catarata/congênito , Córnea/anormalidades , Etilnitrosoureia/farmacologia , Hipogonadismo , Deficiência Intelectual , Microcefalia , Mutagênese/efeitos dos fármacos , Degeneração Neural/etiologia , Atrofia Óptica , Deleção de Sequência/genética , Proteínas rab de Ligação ao GTP/deficiência , Anormalidades Múltiplas/induzido quimicamente , Anormalidades Múltiplas/genética , Fatores Etários , Animais , Axônios/patologia , Axônios/ultraestrutura , Catarata/induzido quimicamente , Catarata/complicações , Catarata/genética , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Olho/patologia , Hipogonadismo/induzido quimicamente , Hipogonadismo/complicações , Hipogonadismo/genética , Deficiência Intelectual/induzido quimicamente , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcefalia/induzido quimicamente , Microcefalia/complicações , Microcefalia/genética , Microftalmia/etiologia , Microftalmia/genética , Degeneração Neural/patologia , Atrofia Óptica/induzido quimicamente , Atrofia Óptica/complicações , Atrofia Óptica/genética , Doenças do Nervo Óptico/etiologia , Doenças do Nervo Óptico/genética , Desempenho Psicomotor/efeitos dos fármacos , Testículo/patologia , Percepção do Tato/efeitos dos fármacos , Percepção do Tato/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
OBJECTIVE: To describe a novel mutation in TRK-fused gene (TFG) as a new cause of dominant axonal Charcot-Marie-Tooth disease (CMT) identified by exome sequencing and further characterized by in vitro functional studies. METHODS: Exome sequencing and linkage analysis were utilized to investigate a large Taiwanese family with a dominantly inherited adult-onset motor and sensory axonal neuropathy in which mutations in common CMT2-implicated genes had been previously excluded. Functional effects of the mutant gene products were investigated in vitro. RESULTS: Exome sequencing of 2 affected individuals in this family revealed a novel heterozygous mutation, c.806G>T (p.Gly269Val), in TFG that perfectly cosegregates with the CMT2 phenotype in all 27 family members. This mutation occurs at an evolutionarily conserved residue and is absent in the 1,140 ethnically matched control chromosomes. Genome-wide linkage study also supported its disease-causative role. Cell transfection studies showed that the TFG p.Gly269Val mutation increased the propensity of TFG proteins to form aggregates, resulting in sequestration of both mutant and wild-type TFG proteins and might thus deplete functional TFG molecules. The secreted Gaussia luciferase reporter assay demonstrated that inhibition of endogenous TFG compromised the protein secretion pathways, which could only be rescued by expressing wild-type TFG but not the p.Gly269Val altered proteins. TFG mutation was not found in 55 additional unrelated patients with CMT2, suggesting its rarity. CONCLUSION: This study identifies a new cause of dominant CMT2 and highlights the importance of TFG in the protein secretory pathways that are essential for proper functioning of the human peripheral nervous system.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteínas/genética , Adulto , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Exoma , Feminino , Ligação Genética , Humanos , Imageamento por Ressonância Magnética , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Fenótipo , Proteínas/metabolismo , TaiwanRESUMO
Cdk12 and Cdk13 are Cdc2-related proteins that share 92% identity in their kinase domains. Using in situ hybridization and Western blot analysis, we detected the expression of Cdk12 and Cdk13 mRNAs and their proteins in developing mouse embryos, especially during development of the nervous system. We explored the roles of Cdk12 and Cdk13 in neuronal differentiation using the P19 neuronal differentiation model. Upon knockdown of Cdk12 or Cdk13, no effect on differentiated cell numbers was detected, but a substantial decrease of numbers of neurons with long neurites was identified. Similarly, knockdown of Cdk12 or Cdk13 in primarily cultured cortical neurons shortens the averaged axonal length. A microarray analysis was used to examine changes in gene expression after knockdown or overexpression of Cdk12 and we identified Cdk5 as a molecule potentially involved in mediating the effect of Cdk12 and Cdk13. Depletion of Cdk12 or Cdk13 in P19 cells significantly reduces Cdk5 expression at both the mRNA and protein levels. Expression of Cdk5 protein in the developing mouse brain is also reduced in conditional Cdk12-knockout mice in proportion to the residual amount of Cdk12 protein present. This suggests that the reduced axonal outgrowth after knockdown of Cdk12 or Cdk13 might be due to lower Cdk5 expression. Furthermore, overexpression of Cdk5 protein in P19 cells was able to partially rescue the neurite outgrowth defect observed when Cdk12 or Cdk13 is depleted. Together, these findings suggest that Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression.
Assuntos
Axônios/fisiologia , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/citologia , Transdução de Sinais/fisiologia , Animais , Proteína Quinase CDC2 , Carcinoma/patologia , Contagem de Células , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral , Quinase 2 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: The axon guidance cue molecule Slit2 has been shown to suppress cancer cell invasion. However, the role of Slit2 in growth inhibition is still controversial. The authors identified a novel exon 15 (AKEQYFIP)-deleted slit2, located at the end of the second leucine-rich repeat (LRR2). Because LRR2 interacts with Robo1 receptor to inhibit invasion, they hypothesized that exon 15 plays an important role in modulating Slit2 function. METHODS: Slit2 expression was assessed via microarray analysis in 27 lung adenocarcinomas. Exon 15-deleted slit2 (slit2-ΔE15) and exon 15-containing slit2 (slit2-WT) were cloned and expressed in the CL1-5 lung cancer cell line. The effect of exon 15 on Slit2-mediated cell growth was evaluated by a xenografted model and in vitro cell growth assays. The effect of exon 15 on Slit2-mediated invasion was analyzed with a modified Boyden chamber in vitro. RESULTS: Tumor growth from CL1-5/Slit2-WT cells was comparable to that from CL1-5 cells bearing empty vector. However, tumor size from CL1-5/Slit2-ΔE15 cells was much smaller than that from Slit2-WT cells or vector control cells in the xenografted model. In vitro analyses demonstrated that Slit2-WT inhibits invasion of CL1-5 cells. In addition to inhibiting invasion, Slit2-ΔE15 greatly suppresses cell growth. CONCLUSIONS: The data demonstrated that exon 15 modulates Slit2 function in growth inhibition of lung cancer cells. Because slit2-ΔE15 splice variant is present in low invasive cancer cells and nontumor lung tissues, loss of this splice variant is an important event in tumor progression and invasion.
Assuntos
Adenocarcinoma/patologia , Divisão Celular/genética , Éxons , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Pulmonares/patologia , Proteínas do Tecido Nervoso/genética , Splicing de RNA , Adenocarcinoma/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Primers do DNA , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase , RNA Interferente PequenoRESUMO
Na(+)/Ca(2+) exchanger (NCX) is one of the major mechanisms for removing Ca(2+) from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca(2+). NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.
Assuntos
Creatina Quinase/metabolismo , Metabolismo Energético , Trocador de Sódio e Cálcio/metabolismo , Animais , Bovinos , Linhagem Celular , Creatina Quinase/química , Creatina Quinase Forma MM/química , Creatina Quinase Forma MM/metabolismo , Creatina Quinase Mitocondrial/química , Creatina Quinase Mitocondrial/metabolismo , Humanos , Espaço Intracelular/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Quinase C/metabolismo , Transporte Proteico , Sarcômeros/enzimologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Many Ig superfamily members are expressed in the developing nervous system, but the functions of these molecules during neurogenesis are not all clear. Here, we explore the expression and function of one of members of this superfamily, protogenin (PRTG), in the developing nervous system. Expression of PRTG protein is strong in the neural tube of mouse embryos between embryonic days 7.75 and 9.5 but disappears after embryonic day 10.5 when the neural progenitor marker nestin expresses prominently. Perturbation of PRTG activity in P19 embryonal carcinoma cells and in chick embryos, by either RNA interference or a dominant-negative PRTG mutant, increases neuronal differentiation. Using yeast two-hybrid screening and an in situ binding assay, we were able to identify ERdj3 (a stress-inducible endoplasmic reticulum DnaJ homolog) as a putative PRTG ligand. Addition of purified ERdj3 protein into the P19 differentiation assay reduced neurogenesis. This effect was blocked by addition of either a neutralizing antibody against PRTG or purified PRTG ectodomain protein, indicating that the effect of ERdj3 on neurogenesis is mediated through PRTG. Forced expression of ERdj3 in the chick neural tube also impairs neuronal differentiation. Together, these results suggest that expression of PRTG defines a stage between pluripotent epiblasts and committed neural progenitors, and its signaling plays a critical role in suppressing premature neuronal differentiation during early neural development.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/metabolismo , Tubo Neural/embriologia , Neurogênese/fisiologia , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/genética , Diferenciação Celular/genética , Linhagem Celular , Embrião de Galinha , Eletroporação/métodos , Embrião de Mamíferos , Humanos , Imunoprecipitação/métodos , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Tubo Neural/citologia , Neurogênese/genética , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção/métodosRESUMO
PURPOSE: To investigate OPA1 gene mutations in Chinese patients with autosomal dominant optic atrophy and sporadic optic atrophy. DESIGN: Molecular genetic studies and observational case series. PARTICIPANTS: Twenty-four patients from 10 unrelated Chinese pedigrees of autosomal-dominant optic atrophy, 35 isolated cases with bilateral optic atrophy of unknown cause, and 50 unrelated normal controls. METHODS: Genomic DNA was extracted from peripheral blood leukocytes. All 28 coding exons of the OPA1 gene and flanking intron splice sites were sequenced. Putative mutations were reexamined for segregation in the respective families by direct sequencing. Further characterization of selected splicing site mutations was performed by reverse transcription-polymerase chain reaction (PCR) of each patient's leukocyte mRNA. MAIN OUTCOME MEASURES: Direct sequencing of the OPA1 gene. RESULTS: Four OPA1 gene mutations were detected, including 2 splicing site mutations (c.1065+2T>C on intron 10 and c.1212+2insT on intron 12), 1 deletion (c.1776_1778delACT on exon 19), and 1 missense mutation (c.2846 T>C on exon 28). The c.1212+2insT, c.1776_1778delACT, and c.2846T>C mutations were newly identified OPA1 mutations. Reverse transcription (RT)-PCR and direct sequencing revealed that the splicing site mutations on c.1065+2T>C and c.1212+2insT caused skipping of exons 10 and 12, respectively. The c.1776_1778delACT mutation led to a deletion of the Leu amino acid on residue 593. OPA1 mutations were found in 4 of 10 familial cases (40 %) and in 1 of 35 sporadic cases of optic atrophy. CONCLUSIONS: OPA1 gene mutations are causative in Chinese autosomal-dominant optic atrophy and sporadic optic atrophy. Screening for OPA1 gene mutations in patients with childhood onset optic atrophy who have no affected relatives is useful in making the diagnosis.
Assuntos
Povo Asiático/genética , GTP Fosfo-Hidrolases/genética , Mutação , Atrofia Óptica Autossômica Dominante/genética , Adulto , Criança , China/epidemiologia , Análise Mutacional de DNA , Primers do DNA , Feminino , Genes Dominantes , Humanos , Masculino , Atrofia Óptica Autossômica Dominante/diagnóstico , Linhagem , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
In the present study, we characterized the Ca2+ responses and secretions induced by various secretagogues in mouse chromaffin cells. Activation of the acetylcholine receptor (AChR) by carbachol induced a transient intracellular Ca2+ concentration ([Ca2+](i)) increase followed by two phases of [Ca2+](i) decay and a burst of exocytic events. The contribution of the subtypes of AChRs to carbachol-induced responses was examined. Based on the results obtained by stimulating the cells with the nicotinic receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, high K(+) and the effects of thapsigargin, it appears that activation of nAChRs induces an extracellular Ca2+ influx, which in turn activate Ca(2+)-induced Ca2+ release via the ryanodine receptors. Muscarine, a muscarinic receptor (mAChRs) agonist, was found to induce [Ca2+](i) oscillation and sustained catecholamine release, possibly by activation of both the receptor- and store-operated Ca2+ entry pathways. The RT-PCR results showed that mouse chromaffin cells are equipped with messages for multiple subtypes of AChRs, ryanodine receptors and all known components of the receptor- and store-operated Ca2+ entry. Furthermore, results obtained by directly monitoring endoplasmic reticulum (ER) and mitochondrial Ca2+ concentration and by disabling mitochondrial Ca2+ uptake suggest that the ER acts as a Ca2+ source, while the mitochondria acts as a Ca2+ sink. Our results show that both nAChRs and mAChRs contribute to the initial carbachol-induced [Ca2+](i) increase which is further enhanced by the Ca2+ released from the ER mediated by Ca(2+)-induced Ca2+ release and mAChR activation. This information on the Ca2+ signaling pathways should lay a good foundation for future studies using mouse chromaffin cells as a model system.
Assuntos
Medula Suprarrenal/citologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Células Cromafins/metabolismo , Animais , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Catecolaminas/metabolismo , Colinérgicos/farmacologia , Células Cromafins/efeitos dos fármacos , Células Cromafins/ultraestrutura , Iodeto de Dimetilfenilpiperazina/farmacologia , Interações Medicamentosas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Muscarina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Cloreto de Potássio/farmacologia , RNA Mensageiro/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Tapsigargina/farmacologia , Fatores de TempoRESUMO
The molecular and cellular mechanisms behind glycogen metabolism and the energy metabolite translocation between mammal neurons and astrocytes have been well studied. A similar mechanism is proposed for rapid mobilization of local energy stores to support energy-dependent transepithelial ion transport in gills of the Mozambique tilapia (Oreochromis mossambicus). A novel gill glycogen phosphorylase isoform (tGPGG), which catalyzes the initial degradation of glycogen, was identified in branchial epithelial cells of O. mossambicus. Double in situ hybridization and immunocytochemistry demonstrated that tGPGG mRNA and glycogen were colocalized in glycogen-rich cells (GRCs), which surround ionocytes (labeled with a Na(+)-K(+)-ATPase antiserum) in gill epithelia. Concanavalin-A (a marker for the apical membrane) labeling indicated that GRCs and mitochondria-rich cells share the same apical opening. Quantitative real-time PCR analyses showed that tGPGG mRNA expression levels specifically responded to environmental salinity changes. Indeed, the glycogen content, glycogen phosphorylase (GP) protein level and total activity, and the density of tGPGG-expressing cells (i.e., GRCs) in fish acclimated to seawater (SW) were significantly higher than those in freshwater controls. Short-term acclimation to SW caused an evident depletion in the glycogen content of GRCs. Taken altogether, tGPGG expression in GRCs is stimulated by hyperosmotic challenge, and this may catalyze initial glycogen degradation to provide the adjacent ionocytes with energy to carry out iono- and osmoregulatory functions.
Assuntos
Metabolismo Energético/fisiologia , Células Epiteliais/metabolismo , Brânquias/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio/metabolismo , Tilápia/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Sequência de Aminoácidos , Animais , Astrócitos/enzimologia , Astrócitos/metabolismo , Western Blotting , Encéfalo/citologia , Encéfalo/enzimologia , Encéfalo/metabolismo , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Brânquias/citologia , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cloreto de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Due to the strong sequence homology it has been suggested that CDC2L5 and CDK12 belong to a high molecular weight subfamily of CDC2 family with PITAI/VRE motifs [F. Marques, J.L. Moreau, G. Peaucellier, J.C. Lozano, P. Schatt, A. Picard, I. Callebaut, E. Perret, A.M. Geneviere, A new subfamily of high molecular mass CDC2-related kinases with PITAI/VRE motifs, Biochem. Biophys. Res. Commun. 279 (2000) 832-837]. Recently, we reported that CDK12 interacts with L-type cyclins and is involved in alternative splicing regulation [H.-H. Chen, Y.-C. Wang, M.-J. Fann, Identification and characterization of the CDK12/Cyclin L1 complex involved in alternative splicing regulation, Mol. Cel. Biol. 26 (2006) 2736-2745]. Here, we provide evidence that CDC2L5 also interacts with L-type cyclins and thus rename it as cyclin-dependent kinase 13 (CDK13). The kinase domain of CDK13 is sufficient to bind the cyclin domains of L-type cyclins. Moreover, CDK13 and L-type cyclins modulate each other's subcellular localization. When CDK13 and an E1a minigene reporter construct were over-expressed in HEK293T cells, CDK13 alters the splicing pattern of E1a transcripts in a dose-dependent manner. Similar to effects of CDK12, effects of CDK13 on splicing pattern are counteracted by SF2/ASF and SC35. These findings strengthen CDK12 and CDK13 as a subfamily of cyclin-dependent kinases that regulate alternative splicing.
Assuntos
Processamento Alternativo/fisiologia , Proteína Quinase CDC2/metabolismo , Ciclinas/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Western Blotting , Proteína Quinase CDC2/química , Proteína Quinase CDC2/genética , Células Cultivadas , Ciclinas/química , Ciclinas/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Mutations in the optic atrophy type 1 (OPA1) gene give rise to human autosomal dominant optic atrophy. The purpose of this study is to investigate OPA1 protein expression in the human retina and optic nerve. A rabbit polyclonal antiserum was generated using a fusion protein covering amino acids 647 to 808 of the human OPA1 protein as the immunogenic antigen. Western blot and immunofluorescence staining were performed to examine OPA1 expression in the human retina and optic nerve. In human retina, we found that OPA1 expression was clearly present in retinal ganglion cells and photoreceptors. OPA1 immunoreactivity was also present in the nerve fiber layer, inner plexiform layer and outer plexiform layer. However, OPA1 protein was not detected in the choline acetyltransferase-positive, calretinin-positive, and calbindin-positive amacrine cells, nor in the calbindin-positive horizontal cells. In the human optic nerve, expression of OPA1 was present in the axonal tract that was labeled with neurofilament specific antibody. In conclusion, expression of OPA1 gene is present in the mitochondria-rich regions of the retina and optic nerve. This suggests that OPA1 protein might be involved in the functioning of the mitochondria that are present in both inner and outer retinal neurons.
Assuntos
Proteínas do Olho/análise , GTP Fosfo-Hidrolases/análise , Expressão Gênica/genética , Nervo Óptico/química , Retina/química , Adulto , Idoso , Axônios/química , Western Blotting/métodos , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Imunofluorescência/métodos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/imunologia , Humanos , Masculino , Disco Óptico/química , Células Fotorreceptoras de Vertebrados/química , Células Ganglionares da Retina/químicaRESUMO
CrkRS is a Cdc2-related protein kinase that contains an arginine- and serine-rich (SR) domain, a characteristic of the SR protein family of splicing factors, and is proposed to be involved in RNA processing. However, whether it acts together with a cyclin and at which steps it may function to regulate RNA processing are not clear. Here, we report that CrkRS interacts with cyclin L1 and cyclin L2, and thus rename it as the long form of cyclin-dependent kinase 12 (CDK12(L)). A shorter isoform of CDK12, CDK12(S), that differs from CDK12(L) only at the carboxyl end, was also identified. Both isoforms associate with cyclin L1 through interactions mediated by the kinase domain and the cyclin domain, suggesting a bona fide CDK/cyclin partnership. Furthermore, CDK12 isoforms alter the splicing pattern of an E1a minigene, and the effect is potentiated by the cyclin domain of cyclin L1. When expression of CDK12 isoforms is perturbed by small interfering RNAs, a reversal of the splicing choices is observed. The activity of CDK12 on splicing is counteracted by SF2/ASF and SC35, but not by SRp40, SRp55, and SRp75. Together, our findings indicate that CDK12 and cyclin L1/L2 are cyclin-dependent kinase and cyclin partners and regulate alternative splicing.