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BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.
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Células Madre Pluripotentes Inducidas , Mucosa Respiratoria , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/citología , Diferenciación Celular/fisiología , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Organoides/metabolismoRESUMEN
Stroke is an important disease with unmet clinical need. To uncover novel paths for treatment, it is of critical importance to develop relevant laboratory models that may help to shed light on the pathophysiological mechanisms of stroke. Induced pluripotent stem cells (iPSCs) technology has enormous potential to advance our knowledge into stroke by creating novel human models for research and therapeutic testing. iPSCs models generated from patients with specific stroke types and specific genetic predisposition in combination with other state of art technologies including genome editing, multi-omics, 3D system, libraries screening, offer the opportunity to investigate disease-related pathways and identify potential novel therapeutic targets that can then be tested in these models. Thus, iPSCs offer an unprecedented opportunity to make rapid progress in the field of stroke and vascular dementia research leading to clinical translation. This review paper summarizes some of the key areas in which patient-derived iPSCs technology has been applied to disease modelling and discusses the ongoing challenges and the future directions for the application of this technology in the field of stroke research.
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Células Madre Pluripotentes Inducidas , Accidente Cerebrovascular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Edición Génica , Medicina de Precisión , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/terapia , GenómicaRESUMEN
Cerebrovascular disorders pose a global health concern. Advances in basic and clinical research, including induced pluripotent stem cell models and multi-omic approaches, have improved our understanding and management of these disorders. However, gaps in our knowledge remain. BMC Cardiovascular Disorders invites authors to submit articles investigating what drives and affects Cerebrovascular disorders to improve patient care.
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Enfermedades Cardiovasculares , Trastornos Cerebrovasculares , Células Madre Pluripotentes Inducidas , Humanos , Trastornos Cerebrovasculares/terapiaRESUMEN
Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.
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Enfermedades de los Pequeños Vasos Cerebrales/etiología , Investigación Biomédica Traslacional , Animales , HumanosRESUMEN
DYT1 dystonia is caused by an autosomal dominant mutation that leads to a glutamic acid deletion in torsinA (TA), a member of the AAA+ ATPase superfamily. In this study, we identified a novel-binding partner of TA, the subunit 4 (CSN4) of CSN signalosome. TA binds CSN4 and the synaptic regulator snapin in neuroblastoma cells and in brain synaptosomes. CSN4 and TA are required for the stability of both snapin and the synaptotagmin-specific endocytic adaptor stonin 2, as downregulation of CSN4 or TA reduces the levels of both proteins. Snapin is phosphorylated by the CSN-associated kinase protein kinase D (PKD) and its expression is decreased upon PKD inhibition. In contrast, the stability of stonin 2 is regulated by neddylation, another CSN-associated activity. Overexpression of the pathological TA mutant (ΔE-TA) reduces stonin 2 expression, causing the accumulation of the calcium sensor synaptotagmin 1 on the cell surface. Retrieval of surface-stranded synaptotagmin 1 is restored by overexpression of stonin 2 in ΔE-TA-expressing cells, suggesting that the DYT1 mutation compromises the role of TA in protein stabilisation and synaptic vesicle recycling.
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Proteínas Portadoras/metabolismo , Chaperonas Moleculares/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Encéfalo/metabolismo , Complejo del Señalosoma COP9 , Proteínas Portadoras/genética , Línea Celular Tumoral , Distonía/genética , Distonía/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Chaperonas Moleculares/genética , Mutación , Unión Proteica , Procesamiento Proteico-Postraduccional , Sinaptotagmina I/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Vascular smooth muscle cells (SMCs) arise from multiple origins during development, raising the possibility that differences in embryological origins between SMCs could contribute to site-specific localization of vascular diseases. In this review, we first examine the developmental pathways and embryological origins of vascular SMCs and then discuss in vitro strategies for deriving SMCs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We then review in detail the potential for vascular disease modeling using iPSC-derived SMCs and consider the pathological implications of heterogeneous embryonic origins. Finally, we touch upon the role of human ESC-derived SMCs in therapeutic revascularization and the challenges remaining before regenerative medicine using ESC- or iPSC-derived cells comes of age.
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Células Madre Embrionarias/fisiología , Modelos Biológicos , Músculo Liso Vascular/embriología , Miocitos del Músculo Liso/fisiología , Medicina Regenerativa , Animales , Diferenciación Celular , Células Cultivadas , Humanos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Medicina Regenerativa/métodos , Medicina Regenerativa/tendenciasRESUMEN
BACKGROUND: Genetic variants in COL4A1 and COL4A2 (encoding collagen IV alpha chain 1/2) occur in genetic and sporadic forms of cerebral small vessel disease (CSVD), a leading cause of stroke, dementia and intracerebral haemorrhage (ICH). However, the molecular mechanisms of CSVD with ICH and COL4A1/COL4A2 variants remain obscure. METHODS: Vascular function and molecular investigations in mice with a Col4a1 missense mutation and heterozygous Col4a2 knock-out mice were combined with analysis of human brain endothelial cells harboring COL4A1/COL4A2 mutations, and brain tissue of patients with sporadic CSVD with ICH. FINDINGS: Col4a1 missense mutations cause early-onset CSVD independent of hypertension, with enhanced vasodilation of small arteries due to endothelial dysfunction, vascular wall thickening and reduced stiffness. Mechanistically, the early-onset dysregulated endothelium-dependent hyperpolarization (EDH) is due to reduced collagen IV levels with elevated activity and levels of endothelial Ca2+-sensitive K+ channels. This results in vasodilation via the Na/K pump in vascular smooth muscle cells. Our data support this endothelial dysfunction preceding development of CSVD-associated ICH is due to increased cytoplasmic Ca2+ levels in endothelial cells. Moreover, cerebral blood vessels of patients with sporadic CSVD show genotype-dependent mechanisms with wall thickening and lower collagen IV levels in those harboring common non-coding COL4A1/COL4A2 risk alleles. INTERPRETATION: COL4A1/COL4A2 variants act in genetic and sporadic CSVD with ICH via dysregulated EDH, and altered vascular wall thickness and biomechanics due to lower collagen IV levels and/or mutant collagen IV secretion. These data highlight EDH and collagen IV levels as potential treatment targets. FUNDING: MRC, Wellcome Trust, BHF.
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Marfan syndrome (MFS) is a rare connective tissue disorder caused by mutations in FBN1. Patients with MFS notably suffer from aortic aneurysm and dissection. Despite considerable effort, animal models have proven to be poorly predictive for therapeutic intervention in human aortic disease. Patient-derived induced pluripotent stem cells can be differentiated into vascular smooth muscle cells (VSMCs) and recapitulate major features of MFS. We have screened 1,022 small molecules in our in vitro model, exploiting the highly proteolytic nature of MFS VSMCs, and identified 36 effective compounds. Further analysis identified GSK3ß as a recurring target in the compound screen. GSK3ß inhibition/knockdown did not ameliorate the proliferation defect in MFS-VSMCs but improved MFS-VSMC proteolysis and apoptosis and partially rescued fibrillin-1 deposition. To conclude, we have identified GSK3ß as a novel target for MFS, forming the foundation for future work in MFS and other aortic diseases.
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Células Madre Pluripotentes Inducidas , Síndrome de Marfan , Animales , Humanos , Síndrome de Marfan/genética , Músculo Liso Vascular , Aorta , Glucógeno Sintasa Quinasa 3 betaRESUMEN
Cerebral small vessel disease (SVD) affects the small vessels in the brain and is a leading cause of stroke and dementia. Emerging evidence supports a role of the extracellular matrix (ECM), at the interface between blood and brain, in the progression of SVD pathology, but this remains poorly characterized. To address ECM role in SVD, we developed a co-culture model of mural and endothelial cells using human induced pluripotent stem cells from patients with COL4A1/A2 SVD-related mutations. This model revealed that these mutations induce apoptosis, migration defects, ECM remodeling, and transcriptome changes in mural cells. Importantly, these mural cell defects exert a detrimental effect on endothelial cell tight junctions through paracrine actions. COL4A1/A2 models also express high levels of matrix metalloproteinases (MMPs), and inhibiting MMP activity partially rescues the ECM abnormalities and mural cell phenotypic changes. These data provide a basis for targeting MMP as a therapeutic opportunity in SVD.
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Células Madre Pluripotentes Inducidas , Accidente Cerebrovascular , Humanos , Células Endoteliales , Encéfalo/patología , Accidente Cerebrovascular/patología , Matriz Extracelular , Metaloproteinasas de la Matriz/genética , Colágeno Tipo IV/genéticaRESUMEN
Although dementia research has been dominated by Alzheimer's disease (AD), most dementia in older people is now recognised to be due to mixed pathologies, usually combining vascular and AD brain pathology. Vascular cognitive impairment (VCI), which encompasses vascular dementia (VaD) is the second most common type of dementia. Models of VCI have been delayed by limited understanding of the underlying aetiology and pathogenesis. This review by a multidisciplinary, diverse (in terms of sex, geography and career stage), cross-institute team provides a perspective on limitations to current VCI models and recommendations for improving translation and reproducibility. We discuss reproducibility, clinical features of VCI and corresponding assessments in models, human pathology, bioinformatics approaches, and data sharing. We offer recommendations for future research, particularly focusing on small vessel disease as a main underpinning disorder.
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Human induced pluripotent stem cells (hiPSC) offer a tractable system to model the blood-brain barrier (BBB). Here we detail the assembly of a triple co-culture hiPSC-BBB model, using hiPSC-derived brain microvascular endothelial cells (BMEC), astrocytes, and mural cells (MC). Transendothelial electrical resistance (TEER) and sodium fluorescein (NaFl) permeability can be used to test the barrier properties. The model has applications in studying BBB-related pathology and for drug screening.
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Células Madre Pluripotentes Inducidas , Astrocitos , Barrera Hematoencefálica , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales , HumanosRESUMEN
A common variant in the Histone Deacetylase 9 (HDAC9) gene is the strongest genetic risk for large-vessel stroke, and HDAC9 offers a novel target for therapeutic modulation. However, the mechanisms linking the HDAC9 variant with increased stroke risk is still unclear due to the lack of relevant models to study the underlying molecular mechanisms. We generated vascular smooth muscle cells using human induced pluripotent stem cells with the HDAC9 stroke risk variant to assess HDAC9-mediated phenotypic changes in a relevant cells model and test the efficacy of HDAC inhibitors for potential therapeutic strategies. Our human induced pluripotent stem cells derived vascular smooth muscle cells show enhanced HDAC9 expression and allow us to assess HDAC9-mediated effects on promoting smooth muscle cell dysfunction, including proliferation, migration, apoptosis and response to inflammation. These phenotypes could be reverted by treatment with HDAC inhibitors, including sodium valproate and small molecules inhibitors. By demonstrating the relevance of the model and the efficacy of HDAC inhibitors, our model provides a robust phenotypic screening platform, which could be applied to other stroke-associated genetic variants.
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The extracellular matrix (ECM) is a key interface between the cerebrovasculature and adjacent brain tissues. Deregulation of the ECM contributes to a broad range of neurological disorders. However, despite this importance, our understanding of the ECM composition remains very limited mainly due to difficulties in its isolation. To address this, we developed an approach to extract the cerebrovascular ECM from mouse and human post-mortem normal brain tissues. We then used mass spectrometry with off-line high-pH reversed-phase fractionation to increase the protein detection. This identified more than 1000 proteins in the ECM-enriched fraction, with > 66% of the proteins being common between the species. We report 147 core ECM proteins of the human brain vascular matrisome, including collagens, laminins, fibronectin and nidogens. We next used network analysis to identify the connection between the brain ECM proteins and cerebrovascular diseases. We found that genes related to cerebrovascular diseases, such as COL4A1, COL4A2, VCAN and APOE were significantly enriched in the cerebrovascular ECM network. This provides unique mechanistic insight into cerebrovascular disease and potential drug targets. Overall, we provide a powerful resource to study the functions of brain ECM and highlight a specific role for brain vascular ECM in cerebral vascular disease.
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Trastornos Cerebrovasculares/fisiopatología , Matriz Extracelular/fisiología , Proteómica/métodos , Adulto , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , RatonesRESUMEN
A GAG deletion in the DYT1 gene is responsible for the autosomal dominant movement disorder, early onset primary torsion dystonia, which is characterised by involuntary sustained muscle contractions and abnormal posturing of the limbs. The mutation leads to deletion of a single glutamate residue in the C-terminus of the protein torsinA, a member of the AAA+ ATPase family of proteins with multiple functions. Since no evidence of neurodegeneration has been found in DYT1 patients, the dystonic phenotype is likely to be the result of neuronal functional defect(s), the nature of which is only partially understood. Biochemical, structural and cell biological studies have been performed in order to characterise torsinA. These studies, together with the generation of several animal models, have contributed to identify cellular compartments and pathways, including the cytoskeleton and the nuclear envelope, the secretory pathway and the synaptic vesicle machinery where torsinA function may be crucial. However, the role of torsinA and the correlation between the dysfunction caused by the mutation and the dystonic phenotype remain unclear. This review provides an overview of the findings of the last ten years of research on torsinA, a critical evaluation of the different models proposed and insights towards future avenues of research.
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Distonía/metabolismo , Chaperonas Moleculares/metabolismo , Sinapsis/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Distonía/genética , Distonía/patología , Distonía/fisiopatología , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Sinapsis/patologíaRESUMEN
The neural crest (NC) is a transient multipotent cell population present during embryonic development. The NC can give rise to multiple cell types and is involved in a number of different diseases. Therefore, the development of new strategies to model NC in vitro enables investigations into the mechanisms involved in NC development and disease. In this study, we report a simple and efficient protocol to differentiate human pluripotent stem cells (HPSC) into NC using a chemically defined media, with basic fibroblast growth factor 2 (FGF2) and the transforming growth factor-ß inhibitor SB-431542. The cell population generated expresses a range of NC markers, including P75, TWIST1, SOX10, and TFAP2A. NC purification was achieved in vitro through serial passaging of the population, recreating the developmental stages of NC differentiation. The generated NC cells are highly proliferative, capable of differentiating to their derivatives in vitro and engraft in vivo to NC specific locations. In addition, these cells could be frozen for storage and thawed with no loss of NC properties, nor the ability to generate cellular derivatives. We assessed the potential of the derived NC population to model the neurocristopathy, Treacher Collins Syndrome (TCS), using small interfering RNA (siRNA) knockdown of TCOF1 and by creating different TCOF1+/- HPSC lines through CRISPR/Cas9 technology. The NC cells derived from TCOF1+/- HPSC recapitulate the phenotype of the reported TCS murine model. We also report for the first time an impairment of migration in TCOF1+/- NC and mesenchymal stem cells. In conclusion, the developed protocol permits the generation of the large number of NC cells required for developmental studies, disease modeling, and for drug discovery platforms in vitro.
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Diferenciación Celular , Técnicas de Reprogramación Celular/métodos , Disostosis Mandibulofacial/genética , Cresta Neural/citología , Células Madre Pluripotentes/citología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Benzamidas/farmacología , Muerte Celular , Movimiento Celular , Embrión de Pollo , Dioxoles/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Disostosis Mandibulofacial/patología , Cresta Neural/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in FBN1, which encodes the extracellular matrix protein fibrillin-1. To investigate the pathogenesis of aortic aneurysms in MFS, we generated a vascular model derived from human induced pluripotent stem cells (MFS-hiPSCs). Our MFS-hiPSC-derived smooth muscle cells (SMCs) recapitulated the pathology seen in Marfan aortas, including defects in fibrillin-1 accumulation, extracellular matrix degradation, transforming growth factor-ß (TGF-ß) signaling, contraction and apoptosis; abnormalities were corrected by CRISPR-based editing of the FBN1 mutation. TGF-ß inhibition rescued abnormalities in fibrillin-1 accumulation and matrix metalloproteinase expression. However, only the noncanonical p38 pathway regulated SMC apoptosis, a pathological mechanism also governed by Krüppel-like factor 4 (KLF4). This model has enabled us to dissect the molecular mechanisms of MFS, identify novel targets for treatment (such as p38 and KLF4) and provided an innovative human platform for the testing of new drugs.
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Aneurisma de la Aorta/patología , Apoptosis , Células Madre Pluripotentes Inducidas/patología , Síndrome de Marfan/patología , Modelos Biológicos , Músculo Liso Vascular/patología , Aneurisma de la Aorta/metabolismo , Fibrilina-1/metabolismo , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Síndrome de Marfan/metabolismo , Músculo Liso Vascular/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Avian left-right (L/R) axis determination involves the establishment of asymmetric gene expression in Hensen's node, resulting in two discrete signalling pathways on the left and right sides of the embryo. The extracellular signalling molecule Sonic Hedgehog (SHH) is known to be an important left-side determinant. Transcription of Shh is initially bilateral in Hensen's node (stage 4), but is restricted to the left side by stage 5. The Gli genes (Gli1, Gli2 and Gli3) are the main transcriptional mediators of the Hedgehog pathway in vertebrates. GLI1 and GLI2 are primarily transcriptional activators of Hedgehog target genes, while GLI3 is primarily a transcriptional repressor of Hedgehog targets. In order to gain insight into the mechanisms of asymmetrical Hedgehog signal transduction in the node, we have analysed the expression patterns of the Gli genes in Hensen's node from stage 4 to stage 8. Here, we reveal that the Gli genes are asymmetrically expressed in Hensen's node: Gli1 and Gli2, are expressed on the left side, while Gli3 is expressed on the right side.
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Regulación del Desarrollo de la Expresión Génica , Proteínas Oncogénicas/genética , Organizadores Embrionarios/fisiología , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo , Embrión de Pollo , Lateralidad Funcional , Gástrula/fisiología , Morfogénesis , Transducción de Señal , Transactivadores , Proteína con Dedos de Zinc GLI1RESUMEN
Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8-9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner.
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Linaje de la Célula , Células Madre Embrionarias/citología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Células Madre Pluripotentes/citología , Receptores Notch/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Células Madre Embrionarias/metabolismo , Humanos , Ratones , Músculo Liso Vascular/crecimiento & desarrollo , Miocitos del Músculo Liso/metabolismo , Células Madre Pluripotentes/metabolismo , Receptor Notch3 , Receptores Notch/genéticaRESUMEN
The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.
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Endocitosis/fisiología , Chaperonas Moleculares/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Dopamina/genética , Dopamina/metabolismo , Distonía Muscular Deformante/genética , Distonía Muscular Deformante/metabolismo , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Células PC12 , Mutación Puntual , ARN Interferente Pequeño/genética , Ratas , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Opitz G/BBB syndrome (OS) is a congenital defect characterized by hypertelorism and hypospadias, but additional midline malformations are also common in OS patients. X-linked OS is caused by mutations in the ubiquitin ligase MID1. In chick, MID1 is involved in left-right determination: a mutually repressive relationship between Shh and cMid1 in Hensen's node plays a key role in establishing the avian left-right axis. We have utilized our existing knowledge of the molecular basis of avian L/R determination to investigate the possible existence of functional redundancy between MID1 and its close homologue MID2. The expression of cMid2 overlaps with that of cMid1 in the node, and we demonstrate that MID2 can both mimic MID1 function as a right side determinant and rescue the laterality defects caused by knocking down endogenous MID proteins in the node. Our results show that MID2 is able to compensate for an absence in MID1 during chick left-right determination and may explain why OS patients do not suffer laterality defects despite the association between midline and L/R development. The demonstration of functional redundancy between MID1 and MID2 in the node provides supports for the hypothesis that partial functional redundancy between MID proteins in other developing structures contributes to the wide variability of OS phenotype.