RESUMEN
Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington's disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD.
Asunto(s)
Encéfalo , Proteínas de Unión al ADN , Proteína Huntingtina , Enfermedad de Huntington , Células Madre Pluripotentes Inducidas , Mitocondrias , Proteínas Mitocondriales , Organoides , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Organoides/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Encéfalo/metabolismo , Encéfalo/patología , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mitocondrias/metabolismo , Mutación , Dinámicas Mitocondriales/genéticaRESUMEN
The documentation, preservation and rescue of biological diversity increasingly uses living biological samples. Persistent associations between species, biosamples, such as tissues and cell lines, and the accompanying data are indispensable for using, exchanging and benefiting from these valuable materials. Explicit authentication of such biosamples by assigning unique and robust identifiers is therefore required to allow for unambiguous referencing, avoid identification conflicts and maintain reproducibility in research. A predefined nomenclature based on uniform rules would facilitate this process. However, such a nomenclature is currently lacking for animal biological material. We here present a first, standardized, human-readable nomenclature design, which is sufficient to generate unique and stable identifying names for animal cellular material with a focus on wildlife species. A species-specific human- and machine-readable syntax is included in the proposed standard naming scheme, allowing for the traceability of donated material and cultured cells, as well as data FAIRification. Only when it is consistently applied in the public domain, as publications and inter-institutional samples and data are exchanged, distributed and stored centrally, can the risks of misidentification and loss of traceability be mitigated. This innovative globally applicable identification system provides a standard for a sustainable structure for the long-term storage of animal bio-samples in cryobanks and hence facilitates current as well as future species conservation and biomedical research.
RESUMEN
Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.
Asunto(s)
Diferenciación Celular , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Diferenciación Celular/genética , Animales , Ratones , Desarrollo Embrionario/genética , Empalme Alternativo , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/citología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , HumanosRESUMEN
Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.
Asunto(s)
Respiración de la Célula , Mitocondrias , Receptor Muscarínico M2 , Animales , Humanos , Ratones , Proliferación Celular , Células HEK293 , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M2/genética , Estrés FisiológicoRESUMEN
The complex neuromuscular network that controls body movements is the target of severe diseases that result in paralysis and death. Here, we report the development of a robust and efficient self-organizing neuromuscular junction (soNMJ) model from human pluripotent stem cells that can be maintained long-term in simple adherent conditions. The timely application of specific patterning signals instructs the simultaneous development and differentiation of position-specific brachial spinal neurons, skeletal muscles, and terminal Schwann cells. High-content imaging reveals self-organized bundles of aligned muscle fibers surrounded by innervating motor neurons that form functional neuromuscular junctions. Optogenetic activation and pharmacological interventions show that the spinal neurons actively instruct the synchronous skeletal muscle contraction. The generation of a soNMJ model from spinal muscular atrophy patient-specific iPSCs reveals that the number of NMJs and muscle contraction is severely affected, resembling the patient's pathology. In the future, the soNMJ model could be used for high-throughput studies in disease modeling and drug development. Thus, this model will allow us to address unmet needs in the neuromuscular disease field.
Asunto(s)
Células Madre Pluripotentes Inducidas , Atrofia Muscular Espinal , Humanos , Unión Neuromuscular/patología , Neuronas Motoras/fisiología , Fibras Musculares Esqueléticas/patología , Atrofia Muscular Espinal/patología , Músculo Esquelético/patología , Células Madre Pluripotentes Inducidas/patologíaRESUMEN
The production of gametes from pluripotent stem cells in culture, also known as invitro gametogenesis, will make an important contribution to reproductive biology and regenerative medicine, both as a unique tool for understanding germ cell development and as an alternative source of gametes for reproduction. Invitro gametogenesis was developed using mouse pluripotent stem cells but is increasingly being applied in other mammalian species, including humans. In principle, the entire process of germ cell development is nearly reconstitutable in culture using mouse pluripotent stem cells, although the fidelity of differentiation processes and the quality of resultant gametes remain to be refined. The methodology in the mouse system is only partially applicable to other species, and thus it must be optimised for each species. In this review, we update the current status of invitro gametogenesis in mice, humans and other animals, and discuss challenges for further development of this technology.