RESUMO
BACKGROUND: The family of AP-1 complexes mediates protein sorting in the late secretory pathway and it is essential for the development of mammals. The ubiquitously expressed AP-1A complex consists of four adaptins γ1, ß1, µ1A, and σ1A. AP-1A mediates protein transport between the trans-Golgi network and early endosomes. The polarized epithelia AP-1B complex contains the µ1B-adaptin. AP-1B mediates specific transport of proteins from basolateral recycling endosomes to the basolateral plasma membrane of polarized epithelial cells. RESULTS: Analysis of the zebrafish genome revealed the existence of three µ1-adaptin genes, encoding µ1A, µ1B, and the novel isoform µ1C, which is not found in mammals. µ1C shows 80% sequence identity with µ1A and µ1B. The µ1C expression pattern largely overlaps with that of µ1A, while µ1B is expressed in epithelial cells. By knocking-down the synthesis of µ1A, µ1B and µ1C with antisense morpholino techniques we demonstrate that each of these µ1 adaptins is essential for zebrafish development, with µ1A and µ1C being involved in central nervous system development and µ1B in kidney, gut and liver formation. CONCLUSIONS: Zebrafish is unique in expressing three AP-1 complexes: AP-1A, AP-1B, and AP-1C. Our results demonstrate that they are not redundant and that each of them has specific functions, which cannot be fulfilled by one of the other isoforms. Each of the µ1 adaptins appears to mediate specific molecular mechanisms essential for early developmental processes, which depends on specific intracellular vesicular protein sorting pathways.
Assuntos
Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Desenvolvimento Embrionário/genética , Peixe-Zebra/embriologia , Rede trans-Golgi/metabolismo , Laranja de Acridina , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Animais , Sequência de Bases , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Hibridização In Situ , Dados de Sequência Molecular , Morfolinos/genética , Filogenia , Subunidades Proteicas/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência , Peixe-Zebra/metabolismoRESUMO
Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases.
Assuntos
Sistemas CRISPR-Cas , Neurônios Dopaminérgicos/metabolismo , Edição de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Imagem Molecular , Tirosina 3-Mono-Oxigenase/genética , Cálcio/metabolismo , Diferenciação Celular , Rastreamento de Células , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Citometria de Fluxo/métodos , Imunofluorescência , Expressão Gênica , Genes Reporter , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia , Mesencéfalo/fisiologiaRESUMO
Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells.
Assuntos
Astrócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Doença de Parkinson/fisiopatologia , Astrócitos/metabolismo , Autofagia/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Neuroglia , Doença de Parkinson/metabolismo , Fenótipo , alfa-Sinucleína/metabolismoRESUMO
Patient-specific iPSC are being intensively exploited as experimental disease models. Even for late-onset diseases of complex genetic influence, such as Parkinson's disease (PD), the use of iPSC-based models is beginning to provide important insights into the genetic bases of PD heritability. Here, we present an update on recently reported genetic risk factors associated with PD. We discuss how iPSC technology, combined with targeted edition of the coding or noncoding genome, can be used to address clinical observations such as incomplete penetrance, and variability in phenoconversion or age-at-onset in familial PD. Finally, we also discuss the relevance of advanced iPSC/CRISPR/Cas9 disease models to ascertain causality in genotype-to-phenotype correlation studies of sporadic PD.