Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.

Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.

Exposure to the Amino Acids Histidine, Lysine, and Threonine Reduces mTOR Activity and Affects Neurodevelopment in a Human Cerebral Organoid Model.

Evidence of the impact of nutrition on human brain development is compelling. Previous in vitro and in vivo results show that three specific amino acids, histidine, lysine, and threonine, synergistically inhibit mTOR activity and behavior. Therefore, the prenatal availability of these amino acids could be important for human neurodevelopment. However, methods to study the underlying mechanisms in a human model of neurodevelopment are limited. Here, we pioneer the use of human cerebral organoids to investigate the impact of amino acid supplementation on neurodevelopment. In this study, cerebral organoids were exposed to 10 mM and 50 mM of the amino acids threonine, histidine, and lysine. The impact was determined by measuring mTOR activity using Western blots, general cerebral organoid size, and gene expression by RNA sequencing. Exposure to threonine, histidine, and lysine led to decreased mTOR activity and markedly reduced organoid size, supporting findings in rodent studies. RNA sequencing identified comprehensive changes in gene expression, with enrichment in genes related to specific biological processes (among which are mTOR signaling and immune function) and to specific cell types, including proliferative precursor cells, microglia, and astrocytes. Altogether, cerebral organoids are responsive to nutritional exposure by increasing specific amino acid concentrations and reflect findings from previous rodent studies. Threonine, histidine, and lysine exposure impacts the early development of human cerebral organoids, illustrated by the inhibition of mTOR activity, reduced size, and altered gene expression.

Characterization of HIV-1 Infection in Microglia-Containing Human Cerebral Organoids.

The achievement of an HIV cure is dependent on the eradication or permanent silencing of HIV-latent viral reservoirs, including the understudied central nervous system (CNS) reservoir. This requires a deep understanding of the molecular mechanisms of HIV's entry into the CNS, latency establishment, persistence, and reversal. Therefore, representative CNS culture models that reflect the intercellular dynamics and pathophysiology of the human brain are urgently needed in order to study the CNS viral reservoir and HIV-induced neuropathogenesis. In this study, we characterized a human cerebral organoid model in which microglia grow intrinsically as a CNS culture model to study HIV infection in the CNS. We demonstrated that both cerebral organoids and isolated organoid-derived microglia (oMG), infected with replication-competent HIVbal reporter viruses, support productive HIV infection via the CCR5 co-receptor. Productive HIV infection was only observed in microglial cells. Fluorescence analysis revealed microglia as the only HIV target cell. Susceptibility to HIV infection was dependent on the co-expression of microglia-specific markers and the CD4 and CCR5 HIV receptors. Altogether, this model will be a valuable tool within the HIV research community to study HIV-CNS interactions, the underlying mechanisms of HIV-associated neurological disorders (HAND), and the efficacy of new therapeutic and curative strategies on the CNS viral reservoir.

DNA methylation differences in cortical grey and white matter in schizophrenia.

Aim: Identify grey- and white-matter-specific DNA-methylation differences between schizophrenia (SCZ) patients and controls in postmortem brain cortical tissue. Materials & methods: Grey and white matter were separated from postmortem brain tissue of the superior temporal and medial frontal gyrus from SCZ (n = 10) and control (n = 11) cases. Genome-wide DNA-methylation analysis was performed using the Infinium EPIC Methylation Array (Illumina, CA, USA). Results: Four differentially methylated regions associated with SCZ status and tissue type (grey vs white matter) were identified within or near KLF9, SFXN1, SPRED2 and ALS2CL genes. Gene-expression analysis showed differential expression of KLF9 and SFXN1 in SCZ. Conclusion: Our data show distinct differences in DNA methylation between grey and white matter that are unique to SCZ, providing new leads to unravel the pathogenesis of SCZ.

Lay abstract This study investigated the way gene activity is regulated in brain cells of patients with schizophrenia (SCZ; a severe mental illness characterized by psychosis) compared with unaffected controls. The study focuses on the differences between parts of the brain with many cell bodies (grey matter) in contrast to those parts with mainly conducting fibers (white matter). For that purpose, grey and white matter were separated from brain tissue of ten individuals with SCZ and 11 without. All brains were obtained after the patients died and donated their brains to science. Array technology was used to analyze 800,000 sections of the DNA at once. The study identified regions on four genes that can turn the genes on and off differently in schizophrenic patients compared with controls, these genes were also turned on or off depending on their location either in grey or white matter. Two of these genes showed different activation in schizophrenic patients compared with controls. Overall this study identified distinct differences between grey and white matter that are unique to SCZ, providing new leads to unravel the biology of SCZ.

A loss of mature microglial markers without immune activation in schizophrenia.

Microglia, the immune cells of the brain, are important for neurodevelopment and have been hypothesized to play a role in the pathogenesis of schizophrenia (SCZ). Although previous postmortem studies pointed toward presence of microglial activation, this view has been challenged by more recent hypothesis-driven and hypothesis-free analyses. The aim of the present study is to further understand the observed microglial changes in SCZ. We first performed a detailed meta-analysis on studies that analyzed microglial cell density, microglial morphology, and expression of microglial-specific markers. We then further explored findings from the temporal cortex by performing immunostainings and qPCRs on an additional dataset. A random effect meta-analysis showed that the density of microglial cells was unaltered in SCZ (ES: 0.144 95% CI: 0.102 to 0.390, p = .250), and clear changes in microglial morphology were also absent. The expression of several microglial specific genes, such as CX3CR1, CSF1R, IRF8, OLR1, and TMEM119 was decreased in SCZ (ES: -0.417 95% CI: -0.417 to -0.546, p < .0001), consistent with genome-wide transcriptome meta-analysis results. These results indicate a change in microglial phenotype rather than density, which was validated with the use of TMEM119/Iba1 immunostainings on temporal cortex of a separate cohort. Changes in microglial gene expression were overlapping between SCZ and other psychiatric disorders, but largely opposite from changes reported in Alzheimer's disease. This distinct microglial phenotype provides a crucial molecular hallmark for future research into the role of microglia in SCZ and other psychiatric disorders.

Synapse Pathology in Schizophrenia: A Meta-analysis of Postsynaptic Elements in Postmortem Brain Studies.

Changed synapse density has been suggested to be involved in the altered brain connectivity underlying schizophrenia (SCZ) pathology. However, postmortem studies addressing this topic are heterogeneous and it is not known whether changes are restricted to specific brain regions. Using meta-analysis, we systematically and quantitatively reviewed literature on the density of postsynaptic elements in postmortem brain tissue of patients with SCZ compared to healthy controls. We included 3 outcome measurements for postsynaptic elements: dendritic spine density (DSD), postsynaptic density (PSD) number, and PSD protein expression levels. Random-effects meta-analysis (31 studies) revealed an overall decrease in density of postsynaptic elements in SCZ (Hedges's g: -0.33; 95% CI: -0.60 to -0.05; P = .020). Subgroup analyses showed reduction of postsynaptic elements in cortical but not subcortical tissues (Hedges's g: -0.44; 95% CI: -0.76 to -0.12; P = .008, Hedges's g: -0.11; 95% CI: -0.54 to 0.35; P = .671) and specifically a decrease for the outcome measure DSD (Hedges's g: -0.81; 95% CI: -1.37 to -0.26; P = .004). Further exploratory analyses showed a significant decrease of postsynaptic elements in the prefrontal cortex and cortical layer 3. In all analyses, substantial heterogeneity was present. Meta-regression analyses showed no influence of age, sex, postmortem interval, or brain bank on the effect size. This meta-analysis shows a region-specific decrease in the density of postsynaptic elements in SCZ. This phenotype provides an important cellular hallmark for future preclinical and neuropathological research in order to increase our understanding of brain dysconnectivity in SCZ.

Microglia in post-mortem brain tissue of patients with bipolar disorder are not immune activated.

Genetic, epidemiological, and biomarker studies suggest that the immune system is involved in the pathogenesis of bipolar disorder (BD). It has therefore been hypothesized that immune activation of microglia, the resident immune cells of the brain, is associated with the disease. Only a few studies have addressed the involvement of microglia in BD so far and a more detailed immune profiling of microglial activation is lacking. Here, we applied a multi-level approach to determine the activation state of microglia in BD post-mortem brain tissue. We did not find differences in microglial density, and mRNA expression of microglial markers in the medial frontal gyrus (MFG) of patients with BD. Furthermore, we performed in-depth characterization of human primary microglia isolated from fresh brain tissue of the MFG, superior temporal gyrus (STG), and thalamus (THA). Similarly, these ex vivo isolated microglia did not show elevated expression of inflammatory markers. Finally, challenging the isolated microglia with LPS did not result in an increased immune response in patients with BD compared to controls. In conclusion, our study shows that microglia in post-mortem brain tissue of patients with BD are not immune activated.

Microglia innately develop within cerebral organoids.

Cerebral organoids are 3D stem cell-derived models that can be utilized to study the human brain. The current consensus is that cerebral organoids consist of cells derived from the neuroectodermal lineage. This limits their value and applicability, as mesodermal-derived microglia are important players in neural development and disease. Remarkably, here we show that microglia can innately develop within a cerebral organoid model and display their characteristic ramified morphology. The transcriptome and response to inflammatory stimulation of these organoid-grown microglia closely mimic the transcriptome and response of adult microglia acutely isolated from post mortem human brain tissue. In addition, organoid-grown microglia mediate phagocytosis and synaptic material is detected inside them. In all, our study characterizes a microglia-containing organoid model that represents a valuable tool for studying the interplay between microglia, macroglia, and neurons in human brain development and disease.