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The Alzheimer's disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons.
Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.
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A Catalogue of 59,732 Human-Specific Regulatory Sequences Reveals Unique-to-Human Regulatory Patterns Associated with Virus-Interacting Proteins, Pluripotency, and Brain Development.
Extensive searches for genomic regions harboring various types of candidate human-specific regulatory sequences (HSRS) identified thousands' HSRS using high-resolution next-generation sequencing technologies and methodologically diverse comparative analyses of human and nonhuman primates' (NHPs) reference genomes. In this study, a comprehensive catalogue of 59,732 genomic loci harboring candidate HSRS has been assembled to facilitate the systematic analyses of genomic sequences that were either inherited from extinct common ancestors (ECAs) or created de novo in human genomes. These analyses identified thousands of candidate HSRS and HSRS-harboring loci that appear inherited from ECAs, yet absent in genomes of our closest evolutionary relatives, chimpanzee and bonobo, presumably due to the incomplete lineage sorting and/or species-specific loss or regulatory DNA. This pattern is particularly prominent for HSRS-harboring loci that have been putatively associated with human-specific gene expression changes in cerebral organoid models. A prominent majority of regions harboring human-specific mutations associated with human-specific expression changes during brain development is highly conserved in chimpanzee, bonobo, and gorilla genomes. Among NHPs, dominant fractions of HSRS-harboring loci associated with human-specific gene expression in both excitatory neurons (347 loci; 67%) and radial glia (683 loci; 72%) are highly conserved in the gorilla genome. Analysis of 4433 genes encoding virus-interacting proteins (VIPs) revealed that 95.9% of human VIPs are components of human-specific regulatory networks that appear to operate in distinct types of human cells from preimplantation embryos to adult dorsolateral prefrontal cortex. These analyses demonstrate that modern humans captured unique genome-wide combinations of regulatory sequences, divergent subsets of which are highly conserved in distinct species of six NHP separated by 30 million years of evolution. Concurrently, this unique-to-human mosaic of genomic regulatory patterns inherited from ECAs was supplemented with 12,486 created de novo HSRS. Genes encoding VIPs appear to represent a principal genomic target during evolution of human-specific regulatory networks, which contribute to fitness of Homo sapiens and affect a functionally diverse spectrum of biological and cellular processes controlled by VIP-containing liquid-liquid phase-separated condensates.
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Cortical spheroid on perfusable microvascular network in a microfluidic device.
Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic footprint of human brain development. However, hiPSC-derived 3D models such as spheroid and organoids does not have a perfusable microvascular network, which plays a vital role in maintaining homeostasis in vivo. With the critical balance of positive and negative angiogenic modulators, 3D microvascular network can be achieved by angiogenesis. This paper reports on a microfluidic-based three-dimensional, cortical spheroid grafted on the vascular-network. Vascular network was formed by inducing angiogenic sprouting using concentration gradient-driven angiogenic factors in the microfluidic device. We investigate critical factors for angiogenic vascular network formation with spheroid placement, including 1) a PKCα activator, phorbol-12-myristate-13-acetate (PMA); 2) orientation of endothelial cells under perfusion and permeability of vascular network; 3) effect of extracellular matrix (ECM) types and their densities on angiogenesis; and 4) integration with cortical spheroid on vascular network. This paper demonstrates proof of concept for the potential utility of a membrane-free in vitro cortical spheroid tissue construct with perfusable microvascular network that can be scaled up to a high throughput platform. It can provide a cost-effective alternative platform to animal testing by modeling brain diseases and disorders, and screening drugs.
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Astrocytic lipid droplets contain MHCII and may act as cogs in the antigen presentation machinery.
Lipid droplets (LDs) are crucial for energy homeostasis, but are also involved in a wide spectrum of other cellular processes. Accumulating data identifies LDs as an important player in inflammation. However, the underlying mechanisms and the impact of LDs on neuroinflammation remain unclear. Here, we describe a novel function of LDs in human astrocytes, in the context of Alzheimer's disease (AD). Although, the overall lipid profile was unchanged in astrocytes with AD pathology, our data show a clear effect on LD metabolism and specific fatty acids involved in neuroinflammation. Importantly, we found astrocytes to be in close contact with infiltrating CD4 + T cells in the AD brain. Moreover, PLIN3 + LDs in astrocytes co-localize with major histocompatibility complex II (MHCII), indicating a role of LDs in adaptive immunity. Comprehensive analysis of human induced pluripotent stem cell (hiPSC)-derived astrocytes revealed that MHCII is in fact loaded within PLIN3 + LDs and forwarded to neighboring cells via tunneling nanotubes and secretion. Notably, the MHCII molecules are cleaved into its active form prior to packing, indicating an alternative route of MHCII shuttling through LDs, transporting functional immune complexes between cells. Quantification of PLIN3 + LDs in astrocytic cultures, human brain tissue and cerebral organoids indicates that AD pathology initially stimulates PLIN3 + LD formation, but in the long-run results in PLIN3 + LD consumption, which may have consequences on the astrocytes' MHCII distribution capacity. Taken together, our findings present a novel function of PLIN3 + LDs that can be of relevance for AD and other inflammatory conditions.
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A single-cell transcriptome atlas of glial diversity in the human hippocampus across the postnatal lifespan.
The molecular diversity of glia in the human hippocampus and their temporal dynamics over the lifespan remain largely unknown. Here, we performed single-nucleus RNA sequencing to generate a transcriptome atlas of the human hippocampus across the postnatal lifespan. Detailed analyses of astrocytes, oligodendrocyte lineages, and microglia identified subpopulations with distinct molecular signatures and revealed their association with specific physiological functions, age-dependent changes in abundance, and disease relevance. We further characterized spatiotemporal heterogeneity of GFAP-enriched astrocyte subpopulations in the hippocampal formation using immunohistology. Leveraging glial subpopulation classifications as a reference map, we revealed the diversity of glia differentiated from human pluripotent stem cells and identified dysregulated genes and pathological processes in specific glial subpopulations in Alzheimer's disease (AD). Together, our study significantly extends our understanding of human glial diversity, population dynamics across the postnatal lifespan, and dysregulation in AD and provides a reference atlas for stem-cell-based glial differentiation.
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Dysfunction of the blood-brain barrier and neurovascular unit in amyotrophic lateral sclerosis - From advanced human models to treatments.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, characterized by the degeneration of motor neurons, resulting in progressive and eventually complete loss of motor function. The phenotypical heterogeneity of ALS poses a challenge to early diagnosis and treatment, with currently available therapeutics only capable of relieving symptoms and slowing progression. While ALS is classified as a motor neuron disease (MND), research has identified significant impairment to the blood-brain barrier (BBB) and neurovascular unit (NVU) of ALS patients. The BBB along with supporting cells of the NVU selectively control the entry of substances into the central nervous system (CNS) and maintain brain homeostasis, with its many cellular components demonstrating impaired function, specifically relating to inflammation, oxidative stress and TDP-43 proteinopathy. The advent of more advanced in vitro techniques as well as use of patient-derived BBB cells has presented a promising alternative to recreate the in vivo structure of the BBB/NVU in a highly controlled platform. Such approaches have ranged from simple 2-dimensional (2D) cell culture to intricate 3-dimensional (3D) co-culture systems, as well as microfluidic systems and organoids, with a potential to investigate ALS associated BBB/NVU dysfunction and drug discovery. This review provides a comprehensive assessment of known BBB/NVU dysfunction in ALS and methods of investigating this using in vitro and in vivo platforms with a focus on inflammation, oxidative stress and pathological TDP-43 expression. This review also presents a future perspective to overcoming barriers to treatment of ALS using new patient-derived BBB/NVU model systems.
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Molecular Insights into SARS-CoV2-Induced Alterations of the Gut/Brain Axis.
For a yet unknown reason, a substantial share of patients suffering from COVID-19 develop long-lasting neuropsychiatric symptoms ranging from cognitive deficits to mood disorders and/or an extreme fatigue. We previously reported that in non-neural cells, angiotensin-1 converting enzyme 2 (ACE2), the gene coding for the SARS-CoV2 host receptor, harbors tight co-expression links with dopa-decarboxylase (DDC), an enzyme involved in the metabolism of dopamine. Here, we mined and integrated data from distinct human expression atlases and found that, among a wide range of tissues and cells, enterocytes of the small intestine express the highest expression levels of ACE2, DDC and several key genes supporting the metabolism of neurotransmitters. Based on these results, we performed co-expression analyses on a recently published set of RNA-seq data obtained from SARS-CoV2-infected human intestinal organoids. We observed that in SARS-CoV2-infected enterocytes, ACE2 co-regulates not only with DDC but also with a specific group of genes involved in (i) the dopamine/trace amines metabolic pathway, (ii) the absorption of microbiota-derived L-DOPA and (iii) the absorption of neutral amino acids serving as precursors to neurotransmitters. We conclude that in patients with long COVID, a chronic infection and inflammation of small intestine enterocytes might be indirectly responsible for prolonged brain alterations.
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Compound heterozygous mutations in the SSPOP gene lead to epilepsy and developmental disorders.
The SSPOP gene, currently classified as a pseudogene in the human genome, encodes the SCO-spondin protein, which plays an important role in human neurodevelopment, although its function remains poorly understood. In this study, we used trio-based whole exome sequencing to identify compound heterozygous SSPOP variants in four children from three unrelated families, including one pair of dizygotic twins. These children exhibited variable phenotypes, including variation in age of onset, seizure semiology, and response to antiseizure medications, along with neurodevelopmental disorders. We demonstrated that SSPOP is a functional gene by confirming its expression at both the transcriptional and protein levels. We analysed 10 brain tissue samples from seven paediatric patients and brain organoids derived from human induced pluripotent stem cells to confirm its expression via qRT-PCR, immunofluorescence and western blotting, supporting its biological function during both pre- and post-natal stages of brain development. In addition, CRISPR-mediated sspo knockout zebrafish demonstrated abnormal neurodevelopment and epileptic discharges in vivo. Together, these findings suggest that SSPOP is a functional gene and a potential contributor to neurodevelopmental disorders and epilepsy.
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Breaking barriers and beyond: Mechanisms and pathological implications of Zika virus traversal across blood-tissue interfaces.
Blood-tissue barriers are specialized interfaces that safeguard organ homeostasis by restricting pathogen dissemination. Zika virus (ZIKV), an emerging flavivirus of global concern, exhibits an exceptional ability to breach multiple barriers-including the blood-brain, blood-placental, blood-testis, and blood-retinal barriers-enabling neuroinvasion, vertical and sexual transmission, and ocular disease. ZIKV employs diverse strategies to cross these barriers: receptor-mediated entry, disruption of tight junctions, and hijacking immune cells or extracellular vesicles as viral carriers. Adaptive mutations further refine tissue tropism and enhance barrier traversal efficiency. Insights from cell culture, organoid, animal, and ex vivo tissue models reveal not only the conserved and tissue-specific mechanisms of barrier penetration but also the downstream pathological consequences in the affected organs. Understanding how ZIKV breaches these interfaces and induces organ-specific pathology deepens our knowledge of host-pathogen interactions and provides a framework for designing barrier-protective and disease-mitigating strategies against ZIKV and other pathogens that breach blood-tissue barriers.
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Human pluripotent stem cell-derived neural constructs for predicting neural toxicity.
Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.