Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
1.
Ann Neurol ; 90(3): 490-505, 2021 09.
Article in English | MEDLINE | ID: mdl-34288055

ABSTRACT

OBJECTIVE: We utilized human midbrain-like organoids (hMLOs) generated from human pluripotent stem cells carrying glucocerebrosidase gene (GBA1) and α-synuclein (α-syn; SNCA) perturbations to investigate genotype-to-phenotype relationships in Parkinson disease, with the particular aim of recapitulating α-syn- and Lewy body-related pathologies and the process of neurodegeneration in the hMLO model. METHODS: We generated and characterized hMLOs from GBA1-/- and SNCA overexpressing isogenic embryonic stem cells and also generated Lewy body-like inclusions in GBA1/SNCA dual perturbation hMLOs and conduritol-b-epoxide-treated SNCA triplication hMLOs. RESULTS: We identified for the first time that the loss of glucocerebrosidase, coupled with wild-type α-syn overexpression, results in a substantial accumulation of detergent-resistant, ß-sheet-rich α-syn aggregates and Lewy body-like inclusions in hMLOs. These Lewy body-like inclusions exhibit a spherically symmetric morphology with an eosinophilic core, containing α-syn with ubiquitin, and can also be formed in Parkinson disease patient-derived hMLOs. We also demonstrate that impaired glucocerebrosidase function promotes the formation of Lewy body-like inclusions in hMLOs derived from patients carrying the SNCA triplication. INTERPRETATION: Taken together, the data indicate that our hMLOs harboring 2 major risk factors (glucocerebrosidase deficiency and wild-type α-syn overproduction) of Parkinson disease provide a tractable model to further elucidate the underlying mechanisms for progressive Lewy body formation. ANN NEUROL 2021;90:490-505.


Subject(s)
Glucosylceramidase/deficiency , Lewy Bodies/metabolism , Mesencephalon/metabolism , Mutation/physiology , Organoids/metabolism , alpha-Synuclein/biosynthesis , Embryonic Stem Cells/metabolism , Glucosylceramidase/genetics , Humans , Lewy Bodies/genetics , Lewy Bodies/pathology , Mesencephalon/pathology , Organoids/pathology , alpha-Synuclein/genetics
2.
Int J Mol Sci ; 23(9)2022 May 04.
Article in English | MEDLINE | ID: mdl-35563499

ABSTRACT

In vitro organoids derived from human pluripotent stem cells (hPSCs) have been developed as essential tools to study the underlying mechanisms of human development and diseases owing to their structural and physiological similarity to corresponding organs. Despite recent advances, there are a few methodologies for three-dimensional (3D) skeletal muscle differentiation, which focus on the terminal differentiation into myofibers and investigate the potential of modeling neuromuscular disorders and muscular dystrophies. However, these methodologies cannot recapitulate the developmental processes and lack regenerative capacity. In this study, we developed a new method to differentiate hPSCs into a 3D human skeletal muscle organoid (hSkMO). This organoid model could recapitulate the myogenesis process and possesses regenerative capacities of sustainable satellite cells (SCs), which are adult muscle stem/progenitor cells capable of self-renewal and myogenic differentiation. Our 3D model demonstrated myogenesis through the sequential occurrence of multiple myogenic cell types from SCs to myocytes. Notably, we detected quiescent, non-dividing SCs throughout the hSkMO differentiation in long-term culture. They were activated and differentiated to reconstitute muscle tissue upon damage. Thus, hSkMOs can recapitulate human skeletal muscle development and regeneration and may provide a new model for studying human skeletal muscles and related diseases.


Subject(s)
Organoids , Pluripotent Stem Cells , Cell Differentiation/physiology , Humans , Muscle Development/physiology , Muscle, Skeletal/metabolism
3.
Hum Mol Genet ; 28(7): 1100-1116, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30496485

ABSTRACT

Coiled-coil-helix-coiled-coil-helix domain containing protein 2 (CHCHD2) mutations were linked with autosomal dominant Parkinson's disease (PD) and recently, Alzheimer's disease/frontotemporal dementia. In the current study, we generated isogenic human embryonic stem cell (hESC) lines harboring PD-associated CHCHD2 mutation R145Q or Q126X via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) method, aiming to unravel pathophysiologic mechanism and seek potential intervention strategy against CHCHD2 mutant-caused defects. By engaging super-resolution microscopy, we identified a physical proximity and similar distribution pattern of CHCHD2 along mitochondria with mitochondrial contact site and cristae organizing system (MICOS), a large protein complex maintaining mitochondria cristae. Isogenic hESCs and differentiated neural progenitor cells (NPCs) harboring CHCHD2 R145Q or Q126X mutation showed impaired mitochondria function, reduced CHCHD2 and MICOS components and exhibited nearly hollow mitochondria with reduced cristae. Furthermore, PD-linked CHCHD2 mutations lost their interaction with coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10), while transient knockdown of either CHCHD2 or CHCHD10 reduced MICOS and mitochondria cristae. Importantly, a specific mitochondria-targeted peptide, Elamipretide/MTP-131, now tested in phase 3 clinical trials for mitochondrial diseases, was found to enhance CHCHD2 with MICOS and mitochondria oxidative phosphorylation enzymes in isogenic NPCs harboring heterozygous R145Q, suggesting that Elamipretide is able to attenuate CHCHD2 R145Q-induced mitochondria dysfunction. Taken together, our results suggested CHCHD2-CHCHD10 complex may be a novel therapeutic target for PD and related neurodegenerative disorders, and Elamipretide may benefit CHCHD2 mutation-linked PD.


Subject(s)
Mitochondrial Diseases/genetics , Mitochondrial Proteins/genetics , Mitochondrial Proteins/physiology , Transcription Factors/genetics , Animals , Cell Line , DNA-Binding Proteins , Frontotemporal Dementia/metabolism , Genetic Association Studies/methods , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/physiology , Humans , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Mutation/genetics , Neurodegenerative Diseases/metabolism , Oligopeptides/pharmacology , Parkinson Disease/genetics , Parkinson Disease/physiopathology , Transcription Factors/physiology
4.
Neurobiol Dis ; 58: 49-56, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23659897

ABSTRACT

Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The expanded glutamine stretch in the protein is the result of a CAG triplet repeat expansion in the penultimate exon of the ATXN3 gene. Several gene silencing approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower ataxin-3 protein levels, but since this protein is involved in deubiquitination and proteasomal protein degradation, its long-term silencing might not be desirable. Here, we propose a novel protein modification approach to reduce mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while maintaining important wild type functions of the protein. In vitro studies showed that exon skipping did not negatively impact the ubiquitin binding capacity of ataxin-3. Our in vivo studies showed no toxic properties of the novel truncated ataxin-3 protein. These results suggest that exon skipping may be a novel therapeutic approach to reduce polyglutamine-induced toxicity in spinocerebellar ataxia type 3.


Subject(s)
Machado-Joseph Disease/pathology , Mutation/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Trinucleotide Repeats/genetics , Animals , Ataxin-3 , Cells, Cultured , DNA Mutational Analysis , Dose-Response Relationship, Drug , Exons/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Machado-Joseph Disease/drug therapy , Machado-Joseph Disease/genetics , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Oligonucleotides, Antisense/pharmacology , Peptides/metabolism , Protein Binding/drug effects , Protein Binding/genetics , RNA, Messenger/metabolism , Repressor Proteins/genetics , Transfection , Ubiquitin/metabolism
5.
iScience ; 26(11): 108138, 2023 Nov 17.
Article in English | MEDLINE | ID: mdl-37876801

ABSTRACT

SCN2A protein-truncating variants (PTV) can result in neurological disorders such as autism spectrum disorder and intellectual disability, but they are less likely to cause epilepsy in comparison to missense variants. While in vitro studies showed PTV reduce action potential firing, consequences at in vivo network level remain elusive. Here, we generated a mouse model of Scn2a insufficiency using antisense oligonucleotides (Scn2a ASO mice), which recapitulated key clinical feature of SCN2A PTV disorders. Simultaneous two-photon Ca2+ imaging and electrocorticography (ECoG) in awake mice showed that spontaneous Ca2+ transients in somatosensory cortical neurons, as well as their pairwise co-activities were generally decreased in Scn2a ASO mice during spontaneous awake state and induced seizure state. The reduction of neuronal activities and paired co-activity are mechanisms associated with motor, social and cognitive deficits observed in our mouse model of severe Scn2a insufficiency, indicating these are likely mechanisms driving SCN2A PTV pathology.

6.
Stem Cell Res ; 73: 103264, 2023 12.
Article in English | MEDLINE | ID: mdl-38029556

ABSTRACT

Transmembrane protein 119 (TMEM119) is a recently identified microglia marker that is not expressed by other immune cells. Using CRISPR/Cas9 technology, we introduced enhanced green fluorescence protein (EGFP), into the H9 WA-09 human embryonic stem cell line, directly before the TMEM119 stop codon. Sanger sequencing confirmed successful insertion of the EGFP sequence. The newly created cell line expressed a normal morphology and karyotype, several pluripotency markers, and the ability to differentiate into all three germ layers. H9-TMEM119-EGFP can be used to provide a deeper understanding of the role of TMEM119 in microglia by monitoring its expression under different experimental conditions.


Subject(s)
Human Embryonic Stem Cells , Microglia , Humans , Microglia/metabolism , Cell Line , Cell Differentiation , Embryonic Stem Cells/metabolism , CRISPR-Cas Systems/genetics
7.
Science ; 366(6472): 1486-1492, 2019 12 20.
Article in English | MEDLINE | ID: mdl-31857479

ABSTRACT

Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.


Subject(s)
Angelman Syndrome/metabolism , Calcium Channels, N-Type/metabolism , Ubiquitin-Protein Ligases/metabolism , Angelman Syndrome/physiopathology , Animals , Epilepsy/metabolism , Humans , Mice , Models, Neurological , Neurons/drug effects , Neurons/metabolism , Organoids , Potassium Channel Blockers/pharmacology , Potassium Channel Blockers/therapeutic use , Seizures/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination
8.
Cell Rep ; 16(7): 1942-53, 2016 08 16.
Article in English | MEDLINE | ID: mdl-27498872

ABSTRACT

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cerebral Cortex/metabolism , GABAergic Neurons/metabolism , Pluripotent Stem Cells/metabolism , Prosencephalon/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Biomarkers/metabolism , Cell Differentiation , Cell Line , Cerebral Cortex/cytology , Coculture Techniques , GABAergic Neurons/cytology , Gene Expression , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Interneurons/cytology , Interneurons/metabolism , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neuroglia/cytology , Neuroglia/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Patch-Clamp Techniques , Pluripotent Stem Cells/cytology , Primary Cell Culture , Prosencephalon/cytology , Synapses/physiology , Synaptic Transmission/physiology , Thyroid Nuclear Factor 1 , Transcription Factors/genetics , Transcription Factors/metabolism
9.
Cell Stem Cell ; 19(2): 248-257, 2016 08 04.
Article in English | MEDLINE | ID: mdl-27476966

ABSTRACT

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.


Subject(s)
Dopaminergic Neurons/metabolism , Melanins/metabolism , Mesencephalon/cytology , Organoids/cytology , Pluripotent Stem Cells/cytology , Cell Differentiation , Cell Line , Humans , Transcription, Genetic
10.
Nucleic Acid Ther ; 24(1): 4-12, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24380395

ABSTRACT

Huntington's disease (HD) is a progressive autosomal dominant disorder, caused by a CAG repeat expansion in the HTT gene, which results in expansion of a polyglutamine stretch at the N-terminal end of the huntingtin protein. Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis and it is generally accepted that N-terminal huntingtin fragments are more toxic than full-length protein. Important cleavage sites are encoded by exon 12 of HTT. Here we report proof of concept using antisense oligonucleotides to induce skipping of exon 12 in huntingtin pre-mRNA, thereby preventing the formation of a 586 amino acid N-terminal huntingtin fragment implicated in HD toxicity. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein. Cleavage assays showed reduced 586 amino acid N-terminal huntingtin fragments in the treated samples. In vivo studies revealed exon skipping after a single injection of antisense oligonucleotides in the mouse striatum. Recent advances to inhibit the formation of mutant huntingtin using oligonucleotides seem promising therapeutic strategies for HD. Nevertheless, huntingtin is an essential protein and total removal has been shown to result in progressive neurodegeneration in vivo. Our proof of concept shows a completely novel approach to reduce mutant huntingtin toxicity not by reducing its expressing levels, but by modifying the huntingtin protein.


Subject(s)
Huntington Disease/genetics , Huntington Disease/therapy , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/therapeutic use , Targeted Gene Repair/methods , Animals , Caspase 6/metabolism , Cell Line , Exons , Humans , Huntingtin Protein , Huntington Disease/metabolism , Male , Mice , Mice, Inbred C57BL , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Modification, Translational , Serotonin Plasma Membrane Transport Proteins/genetics , Serotonin Plasma Membrane Transport Proteins/metabolism , Trinucleotide Repeat Expansion
SELECTION OF CITATIONS
SEARCH DETAIL