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CASPR2 Autoimmune Antibodies Induce Neuronal Hyperactivity in Human Brain Organoids.
Gestational transfer of brain-reactive antibodies is a risk factor for neurodevelopmental disorders. Contactin-associated protein-like 2 (CASPR2) is a known target for pathogenic maternal autoantibodies which have been proposed to interfere with fetal neurodevelopment. However, the impact of CASPR2 antibodies on human brain development remains largely unknown. Here, to better understand the neurophysiological changes that occur in the presence of these pathogenic autoantibodies, we cultured unguided human neural organoids for a period of 6-months in media containing anti-CASPR2 antibodies. We then performed neurophysiological characterization via whole-cell patch-clamp and calcium imaging in acute organoid slices. Our results reveal that CASPR2 antibody exposure increased spontaneous synaptic activity, enhanced the maximal frequency of action potential firing and of spontaneous network activity. These findings are consistent with a state of neuronal hyperexcitability, a phenotype which is observed in several models of neurodevelopmental disorders. Mechanistically, the alterations observed in action potential waveform are in accordance with a role for CASPR2 in the regulation of voltage-gated potassium channels and a pathological role for CASPR2 autoantibodies in driving neuronal hyperexcitability.
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Microglia cause HIV-induced transcriptional and metabolic changes in human neural organoids.
Human immunodeficiency virus (HIV) can invade the central nervous system during the initial stages of infection and contribute to HIV-associated neurocognitive disorder, affecting up to 50% of people living with HIV (PLWH). To investigate HIV-1-induced immunometabolic changes in the brain, we used a three-dimensional microglia-embedded human neural organoid model. Transcriptomic analysis and genome-scale metabolic modeling revealed that HIV-1 infection led to more pronounced transcriptional changes in the presence of microglia, including upregulation of pro-inflammatory pathways. We identified CCR6, important for HIV-1 permissiveness, to be significantly upregulated upon infection. Metabolic analysis showed increased expression in metabolite transport-related genes, including solute carrier (SLC) genes and altered amino acid metabolism, particularly involving arginine, proline, and tyrosine. These microglia-driven immunometabolic changes may contribute to neuronal dysregulation and, subsequently, neurological complications, which are often observed in PLWH. Early detection of these alterations could support timely therapeutic intervention to improve HIV-related neurologic insult.
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Cerebral organoids: ethical issues and consciousness assessment.
Organoids are three-dimensional biological structures grown in vitro from different kinds of stem cells that self-organise mimicking real organs with organ-specific cell types. Recently, researchers have managed to produce human organoids which have structural and functional properties very similar to those of different organs, such as the retina, the intestines, the kidneys, the pancreas, the liver and the inner ear. Organoids are considered a great resource for biomedical research, as they allow for a detailed study of the development and pathologies of human cells; they also make it possible to test new molecules on human tissue. Furthermore, organoids have helped research take a step forward in the field of personalised medicine and transplants. However, some ethical issues have arisen concerning the origin of the cells that are used to produce organoids (ie, human embryos) and their properties. In particular, there are new, relevant and so-far overlooked ethical questions concerning cerebral organoids. Scientists have created so-called mini-brains as developed as a few-months-old fetus, albeit smaller and with many structural and functional differences. However, cerebral organoids exhibit neural connections and electrical activity, raising the question whether they are or (which is more likely) will one day be somewhat sentient. In principle, this can be measured with some techniques that are already available (the Perturbational Complexity Index, a metric that is directly inspired by the main postulate of the Integrated Information Theory of consciousness), which are used for brain-injured non-communicating patients. If brain organoids were to show a glimpse of sensibility, an ethical discussion on their use in clinical research and practice would be necessary.
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Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells.
Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.
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Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids.
Human cerebral malaria (HCM), a severe encephalopathy associated with Plasmodium falciparum infection, has a 20-30% mortality rate and predominantly affects African children. The mechanisms mediating HCM-associated brain injury are difficult to study in human subjects, highlighting the urgent need for non-invasive ex vivo human models. HCM elevates the systemic levels of free heme, which damages the blood-brain barrier and neurons in distinct regions of the brain. We determined the effects of heme on induced pluripotent stem cells (iPSCs) and a three-dimensional cortical organoid system and assessed apoptosis and differentiation. We evaluated biomarkers associated with heme-induced brain injury, including a pro-inflammatory chemokine, CXCL-10, and its receptor, CXCR3, brain-derived neurotrophic factor (BDNF) and a receptor tyrosine-protein kinase, ERBB4, in the organoids. We then tested the neuroprotective effect of neuregulin-1 (NRG-1) against heme treatment in organoids. Neural stem and mature cells differentially expressed CXCL-10, CXCR3, BDNF and ERBB4 in the developing organoids and in response to heme-induced neuronal injury. The organoids underwent apoptosis and structural changes that were attenuated by NRG-1. Thus, cortical organoids can be used to model heme-induced cortical brain injury associated with HCM pathogenesis as well as for testing agents that reduce brain injury and neurological sequelae.
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Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.
The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.
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Rotenone, an environmental toxin, causes abnormal methylation of the mouse brain organoid's genome and ferroptosis.
More and more reports have pointed out that rotenone, as an insecticide, has high neurotoxicity and reproductive toxicity to livestock and mammals. As a highly physiological correlation system of internal organs, quasi-organs have great potential in the fields of drug toxicity and efficacy test, toxicology research, developmental biology and so on. In this study, brain organs (mBOs) derived from mouse neural stem cells were used to investigate the effects of rotenone on the physiological activity and epigenetic modification of mBOs. At the same time, Rotenone could significantly stimulate the increase of the concentration of LPO, lactic acid and hydroxyl radical in mBOs, and inhibit the expression of neuronal marker Tuj1, CHAT, PAX6 and so on. Further analysis showed that Rotenonem could induce mitochondrial damage in mBOs. The results of qPCR and Western blot showed that Rotenone could up-regulate the expressions of ferroptosis promoting protein p53, Cox2 and so on, while inhibit the expressions of negative regulatory protein of ferroptosis GPX4, FTH1, SLC7A11. In addition, the results of RRBS-Seq sequencing showed that the methylation modification at DMR level in Rotenone-treated mBOs group was significantly higher than that in Ctrl group. The results of KEGG analysis showed that compared with Ctrl, the genes with hypermethylation of promoter and Genebody in Rotenone-treated mBOs were mainly located in the Neuro active ligand-receptor interaction signal transduction pathway. In summary, rotenone can significantly lead to abnormal methylation of mouse brain organs, and lead to the loss of normal physiological function of neurons by inducing ferroptosis.
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Human SPG11 cerebral organoids reveal cortical neurogenesis impairment.
Spastic paraplegia gene 11(SPG11)-linked hereditary spastic paraplegia is a complex monogenic neurodegenerative disease that in addition to spastic paraplegia is characterized by childhood onset cognitive impairment, thin corpus callosum and enlarged ventricles. We have previously shown impaired proliferation of SPG11 neural progenitor cells (NPCs). For the delineation of potential defect in SPG11 brain development we employ 2D culture systems and 3D human brain organoids derived from SPG11 patients' iPSC and controls. We reveal that an increased rate of asymmetric divisions of NPCs leads to proliferation defect, causing premature neurogenesis. Correspondingly, SPG11 organoids appeared smaller than controls and had larger ventricles as well as thinner germinal wall. Premature neurogenesis and organoid size were rescued by GSK3 inhibititors including the Food and Drug Administration-approved tideglusib. These findings shed light on the neurodevelopmental mechanisms underlying disease pathology.
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Constitutive activity of a G protein-coupled receptor, DRD1, contributes to human cerebral organoid formation.
The intricate balance of neural stem cell (NSC) amplification and neurogenesis is central to nervous system development. Dopamine D1 receptor (DRD1) is a typical G protein-coupled receptor (GPCR) mainly expressed in neurogenic area, with high constitutive activity. The receptor appears in the embryonic period before the formation of mature synaptic contacts, which indicates that dopamine receptor and its constitutive activity play crucial roles in the embryonic brain development. Here, we found that DRD1 was enriched in human NSCs. Inhibition of the receptor activity by its inverse agonists promoted human NSCs proliferation and impeded its differentiation. These results were also mimicked by genetic knockdown of DRD1, which also blocked the effects of inverse agonists, suggesting a receptor-dependent manner. More interestingly, knock-in A229T mutant with reduced DRD1 constitutive activity by CRISPR-Cas9 genome editing technology resulted into increased endogenous human NSCs proliferation. These results were well reproduced in human cerebral organoids, and inhibition of the DRD1 constitutive activity by its inverse agonists induced the expansion and folding of human cerebral organoids. The anatomic analysis uncovered that decreasing the constitutive activity of DRD1 by its inverse agonists promoted the NSCs proliferation and maintenance that led to hindered cortical neurogenesis. Further mechanistic studies revealed that the PKC-CBP pathway was involved in the regulation by DRD1. Thus, our findings indicate that the constitutive activity of DRD1 and possibly other GPCRs plays an important role in the development of human nervous system.
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Organoids from pituitary as a novel research model toward pituitary stem cell exploration.
The pituitary is the master endocrine gland, harboring stem cells of which the phenotype and role remain poorly characterized. Here, we established organoids from mouse pituitary with the aim to generate a novel research model to study pituitary stem cell biology. The organoids originated from the pituitary cells expressing the stem cell marker SOX2 were long-term expandable, displayed a stemness phenotype during expansive culture and showed specific hormonal differentiation ability, although limited, after subrenal transplantation. Application of the protocol to transgenically injured pituitary harboring an activated stem cell population, resulted in more numerous organoids. Intriguingly, these organoids presented with a cystic morphology, whereas the organoids from undamaged gland were predominantly dense and appeared more limited in expandability. Transcriptomic analysis revealed distinct epithelial phenotypes and showed that cystic organoids more resembled the pituitary phenotype, at least to an immature state, and displayed in vitro differentiation, although yet moderate. Organoid characterization further exposed facets of regulatory pathways of the putative stem cells of the pituitary and advanced new injury-activated markers. Taken together, we established a novel organoid research model revealing new insights into the identity and regulation of the putative pituitary stem cells. This organoid model may eventually lead to an interesting tool to decipher pituitary stem cell biology in both healthy and diseased gland.