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A cell line-derived, immune-competent neurospheroid model to study neuroinflammation and human brain disorders.
INTRODUCTION: Three-dimensional (3D) human brain models have become indispensable tools to investigate neuroimmune interactions and inflammatory processes in the human central nervous system in vitro. Nevertheless, existing models, including brain organoids and other iPSC-derived systems, are often constrained by lengthy differentiation protocols, considerable cost, and substantial batch-to-batch variability, restricting their applicability in translational neuroimmunology. METHODS: We developed a scalable and reproducible 3D human neurospheroid model (tri-hNSPHs) composed of neuronal, astrocytic, and microglial human cell lines, specifically designed to study neuropathogenic mechanisms. Tri-NSPHs were exposed to a defined pro-inflammatory cytokine cocktail (IL-1β, TNFα, and IFNγ) to quantify the secretion of multiple inflammatory mediators. The inflammation was also counteracted using distinct anti-inflammatory pharmacological compounds and cellular adaptations to hypoxic stress were modeled. RESULTS: Tri-hNSPHs rapidly self-assemble while maintaining key neuro-glial interactions and enabling precise analysis of immune responses not attainable with conventional two-dimensional cultures. The stimuli we provided triggered robust and quantifiable inflammatory activation, demonstrating the versatility of the model and its suitability for dissecting neuroinflammatory pathways. Pharmacological modulation effectively attenuated these responses, further validating the platform for mechanistic and therapeutic studies. In addition to modeling neuroinflammation, tri-hNSPHs reliably recapitulated cell reactions to hypoxic stress, a pathological condition tightly intertwined to neuroimmune activation in numerous neurological disorders. DISCUSSION: Together, these findings establish tri-hNSPHs as a scalable, experimentally robust, and translationally relevant 3D neuroimmune model for investigating inflammation-driven brain pathology and evaluating anti-inflammatory strategies in a controlled and reproducible in vitro setting. This platform holds significant promise for advancing neuroimmune research and preclinical screening of immunomodulatory therapies.
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Differential contributions of fetal mononuclear phagocytes to Zika virus neuroinvasion versus neuroprotection during congenital infection.
Fetal immune cell functions during congenital infections are poorly understood. Zika virus (ZIKV) can vertically transmit from mother to fetus, causing nervous system infection and congenital ZIKV syndrome (CZS). We identified differential functional roles for fetal monocyte/macrophage cell types and microglia in ZIKV dissemination versus clearance using mouse models. Trafficking of ZIKV-infected primitive macrophages from the yolk sac allowed initial fetal virus inoculation, while recruited monocytes promoted non-productive neuroinflammation. Conversely, brain-resident differentiated microglia were protective, limiting infection and neuronal death. Single-cell RNA sequencing identified transcriptional profiles linked to the protective versus detrimental contributions of mononuclear phagocyte subsets. In human brain organoids, microglia also promoted neuroprotective transcriptional changes and infection clearance. Thus, microglia are protective before birth, contrasting with the disease-enhancing roles of primitive macrophages and monocytes. Differential modulation of myeloid cell phenotypes by genetically divergent ZIKVs underscores the potential of immune cells to regulate diverse outcomes during fetal infections.
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A spectrum of AKT3 activating mutations cause focal malformations of cortical development (FMCDs) in cortical organoids.
Focal malformations of cortical development (FMCDs) are brain disorders mainly caused by hyperactive mTOR signaling due to both inactivating and activating mutations of genes in the PI3K-AKT-mTOR pathway. Among them, mosaic and somatic activating mutations of the mTOR pathway activators are more frequently linked to severe form of FMCDs. A human stem cell-based FMCDs model to study these activating mutations is still lacking. Herein, we genetically engineer human embryonic stem cell lines carrying these activating mutations to generate cortical organoids. Mosaic and somatic expression of AKT3 activating mutations in cortical organoids mimicking the disease presentation with overproliferation and the formation of dysmorphic neurons. In parallel comparison of various AKT3 activating mutations reveals that stronger mutation is associated with more severe neuronal migratory and overgrowth defects. Together, we have established a feasible human stem cell-based model for FMCDs that could help to better understand pathogenic mechanism and develop novel therapeutic strategy.
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Alcohol exposure alters pre-mRNA splicing of antiapoptotic Mcl-1L isoform and induces apoptosis in neural progenitors and immature neurons.
Alternative splicing and expression of splice variants of genes in the brain may lead to the modulation of protein functions, which may ultimately influence behaviors associated with alcohol dependence and neurotoxicity. We recently showed that ethanol exposure can lead to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by downregulating the expression levels of serine/arginine rich splicing factor 1 (SRSF1). Little is known about the physiological expression of these isoforms in neuronal cells and their role in toxicity induced by alcohol exposure during the developmental period. In order to investigate the impact of alcohol exposure on alternative splicing of Mcl-1 pre-mRNA and its role in neurotoxicity, we developed a unique primary human neuronal culture model where neurospheres (hNSPs), neural progenitors (hNPCs), immature neurons, and mature neurons were cultured from the matching donor fetal brain tissues. Our data suggest that neural progenitors and immature neurons are highly sensitive to the toxic effects of ethanol, while mature neuron cultures showed resistance to ethanol exposure. Further analysis of Mcl-1 pre-mRNA alternative splicing by semi-quantitative and quantitative analysis revealed that ethanol exposure causes a significant decrease in Mcl-1L/Mcl-1S ratio in a dose and time dependent manner in neural progenitors. Interestingly, ectopic expression of Mcl-1L isoform in neural progenitors was able to recover the viability loss and apoptosis induced by alcohol exposure. Altogether, these observations suggest that alternative splicing of Mcl-1 may play a crucial role in neurotoxicity associated with alcohol exposure in the developing fetal brain.
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A Primate-Specific Isoform of PLEKHG6 Regulates Neurogenesis and Neuronal Migration.
The mammalian neocortex has undergone remarkable changes through evolution. A consequence of such rapid evolutionary events could be a trade-off that has rendered the brain susceptible to certain neurodevelopmental and neuropsychiatric conditions. We analyzed the exomes of 65 patients with the structural brain malformation periventricular nodular heterotopia (PH). De novo coding variants were observed in excess in genes defining a transcriptomic signature of basal radial glia, a cell type linked to brain evolution. In addition, we located two variants in human isoforms of two genes that have no ortholog in mice. Modulating the levels of one of these isoforms for the gene PLEKHG6 demonstrated its role in regulating neuroprogenitor differentiation and neuronal migration via RhoA, with phenotypic recapitulation of PH in human cerebral organoids. This suggests that this PLEKHG6 isoform is an example of a primate-specific genomic element supporting brain development.
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Triethyl tin does not induce intramyelinic vacuoles in the cns of the quaking mouse.
Triethyl tin (TET), when injected intraperitoneally, failed to produce the typical intramyelinic edema in the spinal cord of quaking mice with two different genetic backgrounds (B6C3H-qk and BTBRTF/Nev-qk), while control littermates and normal C57BL/6J mice were susceptible, as expected. The only prominent change in the quaking mice was the presence of spherical vacuoles containing floccular electron-dense materials, some of which were clearly within the oligodendroglial perikarya and the inner and outer tongues. They are likely to represent degenerative responses. Consistent with the lack of edema, no increase in the water content was found in the quaking spinal cord following TET injection. Although the presence of numerous interlamellar tight junctions in quaking CNS myelin may mechanically restrict formation of the intralamellar vacuoles, the unique changes in the oligodendroglia and the lack of edema fluid accumulation suggest more fundamental metabolic abnormality that renders the quaking CNS resistant to the triethyl tin-induced edema.
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[Ultrastructural aspects of experimental infection by lymphocytic choriomeningitis virus].
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P2X purinergic receptors are required for correct cortical development in human brain organoids.
The human neocortex represents a crucial evolutionary advance, the formation of which requires the tight and precise orchestration of both intracellular and extracellular signals. Structures grown in three-dimensional cultures, specifically human-induced pluripotent stem cells (hIPSCs)-derived cerebral organoids (COs), have been fundamental to study the signals that regulate the formation of the cortex, overcoming the limitations of 2D cultures. Amongst these, purinergic signaling driven by extracellular ATP and other nucleotides may encode crucial intercellular communications that govern central nervous system (CNS) development. The ATP that accumulates in the extracellular milieu can interact with both ionotropic P2X and metabotropic P2Y receptors on cells to exert its modulating effects. Although widely studied in different animal models, little is known about the expression and function of this signaling system in the human cortex. Thus, here we analyzed the expression of P2X receptor subunits comprehensively throughout the entire process of CO development, confirming that P2X receptors are functional in ventricular structures of the human cortex. Specifically, we detected the expression of P2X1, P2X4, and P2X6 in CO, showing distinct distributions in Nestin+ radial glial cells and/or DCX+ newborn neurons. Significantly, we also show how prolonged pharmacological inhibition of P2X activity affects CO development, resulting in smaller organoids with fewer and less well-organized cortical ventricles. Altogether, our findings point to a relevant role of purinergic signaling during the formation of the human cerebral cortex.
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Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.
A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.
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Glioblastoma and cerebral organoids: development and analysis of an in vitro model for glioblastoma migration.
It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.