Brain Cell Types Research Articles

Pathological tau signatures and nuclear alterations in neurons, astrocytes and microglia in Alzheimer's disease, progressive supranuclear palsy, and dementia with Lewy bodies.

Accumulation of pathological tau aggregates is a prominent feature in tauopathies that leads during the course of the diseases to neuronal dysfunction before and cell death after. Microglia and astrocytes have been described as playing important roles in synaptic spreading of toxic tau in several neurodegenerative diseases (NDs). Here, we have investigated the immunological and biochemical properties of aggregated tau species in different brain cell types in tau-induced neurodegenerative diseases such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Additionally, we examined nuclear size, nuclear density, and chromatin compaction in neuronal and glial cells from diseased brain tissues. Microscopic-histological examination was performed using in-house mouse monoclonal antibodies for toxic tau conformers (TTC-M1 and TTC-M2) and tau oligomers (TOMA1-4). By immunohistochemistry and co-immunofluorescence assays using TOMA/TTC-Ms and cell-type specific markers for neurons, astrocytes, and microglia, we observed that TOMA/TTC-Ms were immunoreactive to diverse tau species in different cell types. Analysis of colocalization coefficients indicated an increased pathological tau deposition mainly in the neurons. Western blot analysis of brain homogenates using TOMA/TTC-Ms revealed distinct patterns of tau aggregation in each disease, suggesting that TOMA/TTC-Ms can distinguish between different tau aggregates present in different tauopathies. Additionally, using DAPI staining, we observed that neuronal and astrocytic nuclei had significantly greater nuclear area and increased chromatin compaction in AD cortices compared to non-demented controls. In contrast, reduction in nuclear density/area and more relaxed chromatin was noticed in DLB neurons, astrocytes and microglia and PSP astrocytes and microglia. Cell-type specific tropism of toxic tau species in tauopathies will provide a greater understanding of the involvement of different brain cell types in tau pathology. In this study, we observed that each disease presented cell-type specific nuclear phenotype and tau deposition pattern.

β-Hydroxybutyrate and Medium-Chain Fatty Acids are Metabolized by Different Cell Types in Mouse Cerebral Cortex Slices.

Ketogenic diets and medium-chain triglycerides are gaining attention as treatment of neurological disorders. Their major metabolites, β-hydroxybutyrate (βHB) and the medium-chain fatty acids (MCFAs) octanoic acid (C8) and decanoic acid (C10), are auxiliary brain fuels. To which extent these fuels compete for metabolism in different brain cell types is unknown. Here, we used acutely isolated mouse cerebral cortical slices to (1) compare metabolism of 200µM [U-13C]C8, [U-13C]C10 and [U-13C]βHB and (2) assess potential competition between metabolism of βHB and MCFAs by quantifying metabolite 13C enrichment using gas chromatography-mass spectrometry (GC-MS) analysis. The 13C enrichment in most metabolites was similar with [U-13C]C8 and [U-13C]C10 as substrates, but several fold lower with [U-13C]βHB. The 13C enrichment in glutamate was in a similar range for all three substrates, whereas the 13C enrichments in citrate and glutamine were markedly higher with both [U-13C]C8 and [U-13C]C10 compared with [U-13C]βHB. As citrate and glutamine are indicators of astrocytic metabolism, the results indicate active MCFA metabolism in astrocytes, while βHB is metabolized in a different cellular compartment. In competition experiments, 12C-βHB altered 13C incorporation from [U-13C]C8 and [U-13C]C10 in only a few instances, while 12C-C8and 12C-C10 only further decreased the low [U-13C]βHB-derived 13C incorporation into citrate and glutamine, signifying little competition for oxidative metabolism between βHB and the MCFAs. Overall, the data demonstrate that βHB and MCFAs are supplementary fuels in different cellular compartments in the brain without notable competition. Thus, the use of medium-chain triglycerides in ketogenic diets is likely to be beneficial in conditions with carbon and energy shortages in both astrocytes and neurons, such as GLUT1 deficiency.

P13. Transduction of the rat spinal cord using adenoassociated virus serotype 2.5

<h3>BACKGROUND CONTEXT</h3> Gene therapy is a method by which exogenous genes are introduced to treat disease or deliver drugs of interest. This is commonly done through either direct gene delivery in vivo or ex vivo through transduction of cells that are to be transplanted. There are several vectors for delivering genes, including viral vectors, naked plasmids, electroporation and lipid nanoparticles. Adeno-associated viruses (AAVs) are commonly used and considered to be well tolerated by the brain, muscles, nerves and multiple cell types. Rat Schwann cells are transduced efficiently by AAV1 whereas human Schwann cells are better transduced with AAV2 and 6. Rat nerves segments have demonstrated to be efficiently transduced by AAV1, 5, 7 and 9 whereas human nerves prefer AAV2. Many studies also agree that AAV1, 2, 4, 8 and 9 are optimal serotypes for CNS. We have utilized a novel viral vector, AAV serotype 2.5, that is a chimera of AAV2 vector modified with 5 amino acids from AAV1. AAV2.5 has shared characteristics of AAV1 and AAV2 with enhanced transduction for multiple cell types, such as in the muscle and articular tissue. Since AAV2.5 is not a naturally occurring virus, there is lower cross-reactivity to AAV2 antibodies. However, the ability of AAV2.5 to transduce tissues of the central (CNS) and peripheral nervous system (PNS) is not well understood. <h3>PURPOSE</h3> The purpose of this study is to determine the trophism and transduction efficiency of AAV2.5 for CNS and PNS tissue through in vitro transduction of dorsal root ganglion derived neurons, Schwann cells, and in vivo transduction of spinal cord. We predict that since AAV1 and AAV2 have known tropism for CNS and PNS cells to different extents depending on cell type, that the chimera AAV2.5 would be broadly effective. <h3>STUDY DESIGN/SETTING</h3> Cell culture and animal study. <h3>METHODS</h3> The cDNA encoding green fluorescent protein (GFP) was cloned into the conventional AAV packaging vector pTRUF2. For generation of self-complementing AAV vector plasmids, the cDNAs for GFP were directionally inserted into the SacII, NotI sites of pHpa-trs-SK plasmid. Transcription was driven by the cytomegalovirus promoter. Primary Schwann cells were isolated from the sciatic nerves of postnatal day 5 rat pups and dorsal root ganglion (DRG) neurons from embryonic day 15 rat pups. The cells were plated at a density of 125,000 cells per well and infected with 104 viral genomes per cell. For in vivo transduction of spinal cord tissue, 4 µL of 5 × 106– 5 × 1010vg/mL of AAV2.5-GFP was injected into 4 positions at T9. Immunostaining for markers of different spinal cord cell types colabeled with anti-GFP was done. <h3>RESULTS</h3> AAV2.5 was able to successfully infect DRG neurons and Schwann cells. AAV-2.5 vector was much more efficient than the commonly used AAV2 vector at transducing the Schwann cells. Cell survival and gene product production by AAV2.5 was greater than electroporation. When AAV2.5 -GFP was injected into the spinal cord there was a dose dependent increase in GFP expression by neurons and astrocytes up to 4 mm away from the injection site. <h3>CONCLUSIONS</h3> The study presents a novel vector for broad transduction in vivo or ex vivo of cells in the CNS and PNS. AAV2.5 was more efficient than the more commonly used AAV2 vector or electroporation. Since AAV2.5 is presently in phase 1 trials for osteoarthritis (NCT02790723), this vector type presents an interesting clinical translation opportunity for spinal cord and peripheral nerve injuries/diseases. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Cell-type-specific cis-eQTLs in eight human brain cell types identify novel risk genes for psychiatric and neurological disorders.

To date, most expression quantitative trait loci (eQTL) studies, which investigate how genetic variants contribute to gene expression, have been performed in heterogeneous brain tissues rather than specific cell types. In this study, we performed an eQTL analysis using single-nuclei RNA sequencing from 192 individuals in eight brain cell types derived from the prefrontal cortex, temporal cortex and white matter. We identified 7,607 eGenes, a substantial fraction (46%, 3,537/7,607) of which show cell-type-specific effects, with strongest effects in microglia. Cell-type-level eQTLs affected more constrained genes and had larger effect sizes than tissue-level eQTLs. Integration of brain cell type eQTLs with genome-wide association studies (GWAS) revealed novel relationships between expression and disease risk for neuropsychiatric and neurodegenerative diseases. For most GWAS loci, a single gene co-localized in a single cell type, providing new clues into disease etiology. Our findings demonstrate substantial contrast in genetic regulation of gene expression among brain cell types and reveal potential mechanisms by which disease risk genes influence brain disorders.

Sphingolipids and their role in health and disease in the central nervous system.

Sphingolipids (SLs) are lipids derived from sphingosine, and their metabolism involves a broad and complex network of reactions. Although SLs are widely distributed in the body, it is well known that they are present in high concentrations within the central nervous system (CNS). Under physiological conditions, their abundance and distribution in the CNS depend on brain development and cell type. Consequently, SLs metabolism impairment may have a significant impact on the normal CNS function, and has been associated with several disorders, including sphingolipidoses, Parkinson's, and Alzheimer's. This review summarizes the main SLs characteristics and current knowledge about synthesis, catabolism, regulatory pathways, and their role in physiological and pathological scenarios in the CNS.

Protein Expression Profile of ACE2 in the Normal and COVID-19-Affected Human Brain.

SARS-coronavirus 2 (SARS-CoV-2) that caused the coronavirus disease 2019 (COVID-19) pandemic has posed to be a global challenge. An increasing number of neurological symptoms have been linked to the COVID-19 disease, but the underlying mechanisms of such symptoms and which patients could be at risk are not yet established. The suggested key receptor for host cell entry is angiotensin I converting enzyme 2 (ACE2). Previous studies on limited tissue material have shown no or low protein expression of ACE2 in the normal brain. Here, we used stringently validated antibodies and immunohistochemistry to examine the protein expression of ACE2 in all major regions of the normal brain. The expression pattern was compared with the COVID-19-affected brain of patients with a varying degree of neurological symptoms. In the normal brain, the expression was restricted to the choroid plexus and ependymal cells with no expression in any other brain cell types. Interestingly, in the COVID-19-affected brain, an upregulation of ACE2 was observed in endothelial cells of certain patients, most prominently in the white matter and with the highest expression observed in the patient with the most severe neurological symptoms. The data shows differential expression of ACE2 in the diseased brain and highlights the need to further study the role of endothelial cells in COVID-19 disease in relation to neurological symptoms.

Standard metadata for 3D microscopy

Recent advances in fluorescence microscopy techniques and tissue clearing, labeling, and staining provide unprecedented opportunities to investigate brain structure and function. These experiments’ images make it possible to catalog brain cell types and define their location, morphology, and connectivity in a native context, leading to a better understanding of normal development and disease etiology. Consistent annotation of metadata is needed to provide the context necessary to understand, reuse, and integrate these data. This report describes an effort to establish metadata standards for three-dimensional (3D) microscopy datasets for use by the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative and the neuroscience research community. These standards were built on existing efforts and developed with input from the brain microscopy community to promote adoption. The resulting 3D Microscopy Metadata Standards (3D-MMS) includes 91 fields organized into seven categories: Contributors, Funders, Publication, Instrument, Dataset, Specimen, and Image. Adoption of these metadata standards will ensure that investigators receive credit for their work, promote data reuse, facilitate downstream analysis of shared data, and encourage collaboration.

Role of Vitamin E in Neonatal Neuroprotection: A Comprehensive Narrative Review.

Vitamin E (Vit E) is an essential lipophilic antioxidant and anti-inflammatory agent that has potential as a neuroprotectant in newborn infants with brain injury. Vit E has shown promise in many in vitro studies, but success in translation to in vivo animal studies and the clinical setting has been mixed, with concern of adverse effects at high intravenous doses in preterm infants. However, a recent rise in knowledge of the beneficial effects of fat emulsions containing higher levels of Vit E, along with associated improved outcomes in some neonatal co-morbidities, has led many to reconsider Vit E administration as a potential therapeutic modality to improve neurological outcomes in the setting of neonatal brain injury. This narrative review discusses Vit E’s structure, mechanism(s) of action, evidence in animal models, and association with health outcomes in neonates, including both dietary and supplemental Vit E and their bioavailability and pharmacokinetics as it relates to the brain. Lastly, long-term neurodevelopmental outcomes along with gaps in current knowledge are critiqued, which to date suggests that additional translational studies in larger animal models and assessment of safety profiles of different routes and doses of administration should be explored prior to large clinical trials. Importantly, a greater understanding of the brain region(s) and cell type(s) affected by Vit E may help to target the use of Vit E as a beneficial neuroprotective agent to specific populations or types of injury seen in newborns.

Single-cell network biology characterizes cell type gene regulation for drug repurposing and phenotype prediction in Alzheimer's disease.

Dysregulation of gene expression in Alzheimer's disease (AD) remains elusive, especially at the cell type level. Gene regulatory network, a key molecular mechanism linking transcription factors (TFs) and regulatory elements to govern gene expression, can change across cell types in the human brain and thus serve as a model for studying gene dysregulation in AD. However, AD-induced regulatory changes across brain cell types remains uncharted. To address this, we integrated single-cell multi-omics datasets to predict the gene regulatory networks of four major cell types, excitatory and inhibitory neurons, microglia and oligodendrocytes, in control and AD brains. Importantly, we analyzed and compared the structural and topological features of networks across cell types and examined changes in AD. Our analysis shows that hub TFs are largely common across cell types and AD-related changes are relatively more prominent in some cell types (e.g., microglia). The regulatory logics of enriched network motifs (e.g., feed-forward loops) further uncover cell type-specific TF-TF cooperativities in gene regulation. The cell type networks are also highly modular and several network modules with cell-type-specific expression changes in AD pathology are enriched with AD-risk genes. The further disease-module-drug association analysis suggests cell-type candidate drugs and their potential target genes. Finally, our network-based machine learning analysis systematically prioritized cell type risk genes likely involved in AD. Our strategy is validated using an independent dataset which showed that top ranked genes can predict clinical phenotypes (e.g., cognitive impairment) of AD with reasonable accuracy. Overall, this single-cell network biology analysis provides a comprehensive map linking genes, regulatory networks, cell types and drug targets and reveals cell-type gene dysregulation in AD.

Critical Role of Neuronal Vps35 in Blood Vessel Branching and Maturation in Developing Mouse Brain.

Vps35 (vacuolar protein sorting 35), a key component of retromer, plays a crucial role in selective retrieval of transmembrane proteins from endosomes to trans-Golgi networks. Dysfunctional Vps35/retromer is a risk factor for the development of neurodegenerative diseases. Vps35 is highly expressed in developing pyramidal neurons, both in the mouse neocortex and hippocampus, Although embryonic neuronal Vps35’s function in promoting neuronal terminal differentiation and survival is evident, it remains unclear whether and how neuronal Vps35 communicates with other types of brain cells, such as blood vessels (BVs), which are essential for supplying nutrients to neurons. Dysfunctional BVs contribute to the pathogenesis of various neurodegenerative disorders. Here, we provide evidence for embryonic neuronal Vps35 as critical for BV branching and maturation in the developing mouse brain. Selectively knocking out (KO) Vps35 in mouse embryonic, not postnatal, neurons results in reductions in BV branching and density, arteriole diameter, and BV-associated pericytes and microglia but an increase in BV-associated reactive astrocytes. Deletion of microglia by PLX3397 enhances these BV deficits in mutant mice. These results reveal the function of neuronal Vps35 in neurovascular coupling in the developing mouse brain and implicate BV-associated microglia as underlying this event.

Regulation of Inflammation-Related Genes through Fosl1 Suppression in a Levetiracetam-Treated Pilocarpine-Induced Status Epilepticus Mouse Model

Levetiracetam (LEV) suppresses the upregulation of proinflammatory molecules that occurs during epileptogenesis after status epilepticus (SE). Based on previous studies, LEV likely helps prevent the onset of epilepsy after insults to the brain, unlike other conventional anti-epileptic drugs. Recently, we discovered that the increase in Fosl1 expression that occurs after lipopolysaccharide (LPS) stimulation is suppressed by LEV and that Fosl1 inhibition suppresses inflammation in BV-2 microglial cells. These data indicate that Fosl1 is an important target of LEV and a key factor in preventing epilepsy onset. In this study, we examined the effects of LEV on Fosl1 expression and neuroinflammation in vivo. During epileptogenesis, the post-SE upregulation of hippocampal levels of Fosl1 and many inflammatory factors were suppressed by LEV. Fosl1 expression showed a characteristic pattern different from that of the expression of Fos, an immediate-early gene belonging to the same Fos family. At 2 days after SE, Fosl1 was predominantly expressed in astrocytes but was rarely detected in microglia, whereas Fos expression was distributed in various brain cell types. The expression of A2 astrocyte markers was similar to that of Fosl1 and was significantly suppressed by LEV. These results suggest that LEV may regulate astrocyte reactivity through regulation of Fosl1.

Distinct cell type-specific protein signatures in GRN and MAPT genetic subtypes of frontotemporal dementia

Frontotemporal dementia is characterized by progressive atrophy of frontal and/or temporal cortices at an early age of onset. The disorder shows considerable clinical, pathological, and genetic heterogeneity. Here we investigated the proteomic signatures of frontal and temporal cortex from brains with frontotemporal dementia due to GRN and MAPT mutations to identify the key cell types and molecular pathways in their pathophysiology. We compared patients with mutations in the GRN gene (n = 9) or with mutations in the MAPT gene (n = 13) with non-demented controls (n = 11). Using quantitative proteomic analysis on laser-dissected tissues we identified brain region-specific protein signatures for both genetic subtypes. Using published single cell RNA expression data resources we deduced the involvement of major brain cell types in driving these different protein signatures. Subsequent gene ontology analysis identified distinct genetic subtype- and cell type-specific biological processes. For the GRN subtype, we observed a distinct role for immune processes related to endothelial cells and for mitochondrial dysregulation in neurons. For the MAPT subtype, we observed distinct involvement of dysregulated RNA processing, oligodendrocyte dysfunction, and axonal impairments. Comparison with an in-house protein signature of Alzheimer’s disease brains indicated that the observed alterations in RNA processing and oligodendrocyte function are distinct for the frontotemporal dementia MAPT subtype. Taken together, our results indicate the involvement of different brain cell types and biological mechanisms in genetic subtypes of frontotemporal dementia. Furthermore, we demonstrate that comparison of proteomic profiles of different disease entities can separate general neurodegenerative processes from disease-specific pathways, which may aid the development of disease subtype-specific treatment strategies.

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.

Intersectional mapping of multi-transmitter neurons and other cell types in the brain.

SUMMARYRecent developments in intersectional strategies have greatly advanced our ability to precisely target brain cell types based on unique co-expression patterns. To accelerate the application of intersectional genetics, we perform a brain-wide characterization of 13 Flp and tTA mouse driver lines and selected seven for further analysis based on expression of vesicular neurotransmitter transporters. Using selective Cre driver lines, we created more than 10 Cre/tTA combinational lines for cell type targeting and circuit analysis. We then used VGLUT-Cre/VGAT-Flp combinational lines to identify and map 30 brain regions containing neurons that co-express vesicular glutamate and gamma-aminobutyric acid (GABA) transporters, followed by tracing their projections with intersectional viral vectors. Focusing on the lateral habenula (LHb) as a target, we identified glutamatergic, GABAergic, or co-glutamatergic/GABAergic innervations from ~40 brain regions. These data provide an important resource for the future application of intersectional strategies and expand our understanding of the neuronal subtypes in the brain.

Brain Cell Type-Specific Nuclear Proteomics Is Imperative to Resolve Neurodegenerative Disease Mechanisms.

Neurodegenerative diseases (NDs) involve complex cellular mechanisms that are incompletely understood. Emerging findings have revealed that disruption of nuclear processes play key roles in ND pathogenesis. The nucleus is a nexus for gene regulation and cellular processes that together, may underlie pathomechanisms of NDs. Furthermore, many genetic risk factors for NDs encode proteins that are either present in the nucleus or are involved in nuclear processes (for example, RNA binding proteins, epigenetic regulators, or nuclear-cytoplasmic transport proteins). While recent advances in nuclear transcriptomics have been significant, studies of the nuclear proteome in brain have been relatively limited. We propose that a comprehensive analysis of nuclear proteomic alterations of various brain cell types in NDs may provide novel biological and therapeutic insights. This may be feasible because emerging technical advances allow isolation and investigation of intact nuclei from post-mortem frozen human brain tissue with cell type-specific and single-cell resolution. Accordingly, nuclei of various brain cell types harbor unique protein markers which can be used to isolate cell-type specific nuclei followed by down-stream proteomics by mass spectrometry. Here we review the literature providing a rationale for investigating proteomic changes occurring in nuclei in NDs and then highlight the potential for brain cell type-specific nuclear proteomics to enhance our understanding of distinct cellular mechanisms that drive ND pathogenesis.

Abstract 3906: Characterization of mTORC2 interactome reveals association with microtubules in glioblastoma and neuroblastoma cells

Abstract The Mechanistic Target of Rapamycin Complex 2 (mTORC2) is a multiprotein complex representing one of the key players in the mTOR signaling pathway. This signal transduction is essential for the regulation of various cellular activities, including proper cell growth, proliferation, differentiation, survival, cell motility, and metabolism. Aberration of the mTOR cascade can be found in several types of disorders, including cancers and neurodegenerative diseases. It has been reported to correlate with acquired aggressive characteristics promoting metastasis, drug resistance, and recurrence in cancer cells and more progressive phenotypes in other diseased cells. More specifically, mTORC2 signaling promotes actin cytoskeleton reorganization, cell motility, cell survival, and has been recently reported to be responsible for DNA damage control. Unlike mTORC1 that has been more thoroughly studied, molecular mechanisms of mTORC2 are yet to be elaborated. Therefore, characterization of the mTORC2 signaling pathway will provide further information needed for better understandings of the biology of diseases. Previously, we have identified multiple cytoskeleton regulators, including actin-binding and microtubule-associated proteins as binding partners of mTORC2 in glioblastoma cells by affinity purification-mass spectrometry (AP-MS). In this work, we provided extensive characterization of mTORC2 interactome in both neuronal and non-neuronal cells focusing on microtubule-associated proteins. Interestingly, apart from many MAP proteins found in non-neuronal cells (glioblastoma), we identified Tau isoforms and their regulatory proteins such as Tau kinases from affinity-purified mTORC2 in neuroblastoma cells. The quantitative proteomic analyses of mTORC2-interacting proteins in various cellular conditions were performed to help determine the dynamics of the mTORC2-associated microtubule network. These novel protein-protein interactions acquired from this study suggested additional mTORC2 functions in different types of brain cells, which might also be beneficial to the understandings of neurodegenerative diseases in addition to cancers. Citation Format: Narawit Pacharakullanon, Piriya Wongkongkathep, Fuyuhiko Tamanoi, Joseph A. Loo, Trairak Pisitkun, Naphat Chantaravisoot. Characterization of mTORC2 interactome reveals association with microtubules in glioblastoma and neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3906.

Chimeric cerebral organoids reveal the essentials of neuronal and astrocytic APOE4 for Alzheimer’s tau pathology

The apolipoprotein E4 (APOE4) genotype is one of the strongest genetic risk factors for Alzheimer’s disease (AD), and is generally believed to cause widespread pathological alterations in various types of brain cells. Here, we developed a novel engineering method of creating the chimeric human cerebral organoids (chCOs) to assess the differential roles of APOE4 in neurons and astrocytes. First, the astrogenic factors NFIB and SOX9 were introduced into induced pluripotent stem cells (iPSCs) to accelerate the induction of astrocytes. Then the above induced iPSCs were mixed and cocultured with noninfected iPSCs under the standard culturing condition of cerebral organoids. As anticipated, the functional astrocytes were detected as early as 45 days, and it helped more neurons matured in chCOs in comparation of the control human cerebral organoids (hCOs). More interestingly, this method enabled us to generate chCOs containing neurons and astrocytes with different genotypes, namely APOE3 or APOE4. Then, it was found in chCOs that astrocytic APOE4 already significantly promoted lipid droplet formation and cholesterol accumulation in neurons while both astrocytic and neuronal APOE4 contributed to the maximum effect. Most notably, we observed that the co-occurrence of astrocytic and neuronal APOE4 were required to elevate neuronal phosphorylated tau levels in chCOs while Aβ levels were increased in chCOs with neuronal APOE4. Altogether, our results not only revealed the essence of both neuronal and astrocytic APOE4 for tau pathology, but also suggested chCOs as a valuable pathological model for AD research and drug discovery.

Neural network learning defines glioblastoma features to be of neural crest perivascular or radial glia lineages.

Glioblastoma is believed to originate from nervous system cells; however, a putative origin from vessel-associated progenitor cells has not been considered. We deeply single-cell RNA–sequenced glioblastoma progenitor cells of 18 patients and integrated 710 bulk tumors and 73,495 glioma single cells of 100 patients to determine the relation of glioblastoma cells to normal brain cell types. A novel neural network–based projection of the developmental trajectory of normal brain cells uncovered two principal cell-lineage features of glioblastoma, neural crest perivascular and radial glia, carrying defining methylation patterns and survival differences. Consistently, introducing tumorigenic alterations in naïve human brain perivascular cells resulted in brain tumors. Thus, our results suggest that glioblastoma can arise from the brains’ vasculature, and patients with such glioblastoma have a significantly poorer outcome.

Novel Therapeutic Strategies for Ischemic Stroke: Recent Insights into Autophagy.

Stroke is one of the leading causes of death and disability worldwide. Autophagy is a conserved cellular catabolic pathway that maintains cellular homeostasis by removal of damaged proteins and organelles, which is critical for the maintenance of energy and function homeostasis of cells. Accumulating evidence demonstrates that autophagy plays important roles in pathophysiological mechanisms under ischemic stroke. Previous investigations show that autophagy serves as a “double-edged sword” in ischemic stroke as it can either promote the survival of neuronal cells or induce cell death in special conditions. Following ischemic stroke, autophagy is activated or inhibited in several cell types in brain, including neurons, astrocytes, and microglia, as well as microvascular endothelial cells, which involves in inflammatory activation, modulation of microglial phenotypes, and blood-brain barrier permeability. However, the exact mechanisms of underlying the role of autophagy in ischemic stroke are not fully understood. This review focuses on the recent advances regarding potential molecular mechanisms of autophagy in different cell types. The focus is also on discussing the “double-edged sword” effect of autophagy in ischemic stroke and its possible underlying mechanisms. In addition, potential therapeutic strategies for ischemic stroke targeting autophagy are also reviewed.

Ultra-rare and common genetic variant analysis converge to implicate negative selection and neuronal processes in the aetiology of schizophrenia

Both common and rare genetic variants (minor allele frequency >1% and <0.1% respectively) have been implicated in the aetiology of schizophrenia. In this study, we integrate single-cell gene expression data with publicly available Genome-Wide Association Study (GWAS) and exome sequenced data in order to investigate in parallel, the enrichment of common and (ultra-)rare variants related to schizophrenia in several functionally relevant gene-sets. Four types of gene-sets were constructed 1) protein-truncating variant (PTV)-intolerant (PI) genes 2) genes expressed in brain cell types and neurons ascertained from mouse and human brain tissue 3) genes defined by synaptic function and location and 4) intersection genes, i.e., PI genes that are expressed in the human and mouse brain cell gene-sets. We show that common as well as ultra-rare schizophrenia-associated variants are overrepresented in PI genes, in excitatory neurons from the prefrontal cortex and hippocampus, medium spiny neurons, and genes enriched for synaptic processes. We also observed stronger enrichment in the intersection genes. Our findings suggest that across the allele frequency spectrum, genes and genetic variants likely to be under stringent selection, and those expressed in particular brain cell types, are involved in the same biological pathways influencing the risk for schizophrenia.