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1.
Front Psychiatry ; 13: 924956, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36405918

RESUMEN

16p13.11 copy number variants (CNVs) have been associated with autism, schizophrenia, psychosis, intellectual disability, and epilepsy. The majority of 16p13.11 deletions or duplications occur within three well-defined intervals, and despite growing knowledge of the functions of individual genes within these intervals, the molecular mechanisms that underlie commonly observed clinical phenotypes remain largely unknown. Patient-derived, induced pluripotent stem cells (iPSCs) provide a platform for investigating the morphological, electrophysiological, and gene-expression changes that result from 16p13.11 CNVs in human-derived neurons. Patient derived iPSCs with varying sizes of 16p13.11 deletions and familial controls were differentiated into cortical neurons for phenotypic analysis. High-content imaging and morphological analysis of patient-derived neurons demonstrated an increase in neurite branching in patients compared with controls. Whole-transcriptome sequencing revealed expression level changes in neuron development and synaptic-related gene families, suggesting a defect in synapse formation. Subsequent quantification of synapse number demonstrated increased numbers of synapses on neurons derived from early-onset patients compared to controls. The identification of common phenotypes among neurons derived from patients with overlapping 16p13.11 deletions will further assist in ascertaining common pathways and targets that could be utilized for screening drug candidates. These studies can help to improve future treatment options and clinical outcomes for 16p13.11 deletion patients.

2.
Stem Cell Res ; 53: 102276, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33714067

RESUMEN

CDKL5 Deficiency Disorder (CDD) is a rare X-linked monogenic developmental encephalopathy that is estimated to affect 1:42,000 live births. CDD is caused by pathogenic variants in the CDKL5 gene and is observed in both male and female patients. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts of six unrelated CDD patients-three males and three females. These patients are clinically diagnosed to present with classic CDD phenotypes, including refractory epilepsy and global developmental delay, and are being followed in a longitudinal clinical study.


Asunto(s)
Síndromes Epilépticos , Células Madre Pluripotentes Inducidas , Espasmos Infantiles , Femenino , Humanos , Masculino , Proteínas Serina-Treonina Quinasas/genética , Espasmos Infantiles/genética
3.
Hum Mol Genet ; 29(2): 320-334, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31915823

RESUMEN

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.


Asunto(s)
Complejo 4 de Proteína Adaptadora/genética , Complejo 4 de Proteína Adaptadora/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/genética , Paraplejía Espástica Hereditaria/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Red trans-Golgi/metabolismo , Complejo 4 de Proteína Adaptadora/deficiencia , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Adolescente , Autofagosomas/metabolismo , Autofagia/genética , Línea Celular , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Hierro/metabolismo , Mutación con Pérdida de Función , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Paraplejía Espástica Hereditaria/genética , Red trans-Golgi/genética
4.
Mol Biol Cell ; 31(7): 511-519, 2020 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-31774722

RESUMEN

Retrograde membrane trafficking from plasma membrane to Golgi and endoplasmic reticulum typifies one of the key sorting steps emerging from the early endosome that affects cell surface and intracellular protein dynamics underlying cell function. While some cell surface proteins and lipids are known to sort retrograde, there are few effective methods to quantitatively measure the extent or kinetics of these events. Here we took advantage of the well-known retrograde trafficking of cholera toxin and newly defined split fluorescent protein technology to develop a quantitative, sensitive, and effectively real-time single-cell flow cytometry assay for retrograde membrane transport. The approach can be applied in high throughput to elucidate the underlying biology of membrane traffic and how endosomes adapt to the physiologic needs of different cell types and cell states.


Asunto(s)
Bioensayo/métodos , Membrana Celular/metabolismo , Análisis de la Célula Individual/métodos , Transporte Biológico , Toxina del Cólera/metabolismo , Enfermedad , Retículo Endoplásmico/metabolismo , Fluorescencia , Células HEK293 , Humanos , Células K562
5.
J Neurosci ; 39(47): 9294-9305, 2019 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-31591157

RESUMEN

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 Patients frequently have epilepsy, autism spectrum disorder, and/or intellectual disability, as well as other systemic manifestations. In this study, we differentiated human induced pluripotent stem cells (iPSCs) from a female patient with TSC with one or two mutations in TSC2 into neurons using induced expression of NGN2 to examine neuronal dysregulation associated with the neurological symptoms in TSC. Using this method, neuronal differentiation was comparable between the three genotypes of iPSCs. We observed that TSC2+/- neurons show mTOR complex 1 (mTORC1) hyperactivation and associated increased cell body size and process outgrowth, as well as exacerbation of the abnormalities by loss of the second allele of TSC2 in TSC2-/- neurons. Interestingly, iPSC-derived neurons with either a single or biallelic mutation in TSC2 demonstrated hypersynchrony and downregulation of FMRP targets. However, only neurons with biallelic mutations of TSC2 demonstrated hyperactivity and transcriptional dysregulation observed in cortical tubers. These data demonstrate that loss of one allele of TSC2 is sufficient to cause some morphological and physiological changes in human neurons but that biallelic mutations in TSC2 are necessary to induce gene expression dysregulation present in cortical tubers. Finally, we found that treatment of iPSC-derived neurons with rapamycin reduced neuronal activity and partially reversed gene expression abnormalities, demonstrating that mTOR dysregulation contributes to both phenotypes. Therefore, biallelic mutations in TSC2 and associated molecular dysfunction, including mTOR hyperactivation, may play a role in the development of cortical tubers.SIGNIFICANCE STATEMENT In this study, we examined neurons derived from induced pluripotent stem cells with two, one, or no functional TSC2 (tuberous sclerosis complex 2) alleles and found that loss of one or both alleles of TSC2 results in mTORC1 hyperactivation and specific neuronal abnormalities. However, only biallelic mutations in TSC2 resulted in elevated neuronal activity and upregulation of cell adhesion genes that is also observed in cortical tubers. These data suggest that loss of heterozygosity of TSC1 or TSC2 may play an important role in the development of cortical tubers, and potentially epilepsy, in patients with TSC.


Asunto(s)
Alelos , Células Madre Pluripotentes Inducidas/fisiología , Mutación/genética , Neuronas/fisiología , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Esclerosis Tuberosa/genética , Células Cultivadas , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Neuronas/patología , Esclerosis Tuberosa/patología
6.
Stem Cell Res ; 40: 101575, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31525725

RESUMEN

Bi-allelic variants in the subunits of the adaptor protein complex 4 lead to childhood-onset, complex hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1), and SPG52 (AP4S1). Here, we describe the generation of induced pluripotent stem cells (iPSCs) from three AP-4-HSP patients with compound-heterozygous, loss-of-function variants in AP4B1 and sex-matched parents. Fibroblasts were reprogrammed using non-integrating Sendai virus. iPSCs were characterized according to standard protocols including karyotyping, embryoid body formation, pluripotency marker expression and STR profiling. These first iPSC lines for SPG47 provide a valuable resource for studying this rare disease and related forms of hereditary spastic paraplegia.


Asunto(s)
Complejo 4 de Proteína Adaptadora/genética , Línea Celular/citología , Células Madre Pluripotentes Inducidas/metabolismo , Paraplejía Espástica Hereditaria/genética , Complejo 4 de Proteína Adaptadora/metabolismo , Adulto , Alelos , Diferenciación Celular , Línea Celular/metabolismo , Células Cultivadas , Preescolar , Femenino , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Paraplejía Espástica Hereditaria/metabolismo
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