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Ex vivo study of neuroinvasive and neurotropic viruses: what is current and what is next.
Numerous pathogens, including viruses, enter the central nervous system and cause neurological disorders, such as encephalitis. Viruses are the main etiologic agents of such neurological diseases, and some of them cause a high death toll worldwide. Our knowledge about neuroinvasive and encephalitogenic virus infections is still limited due to the relative inaccessibility of the brain. To mitigate this shortcoming, neural ex vivo models have been developed and turned out to be of paramount importance for understanding neuroinvasive and neurotropic viruses. In this review, we describe the major ex vivo models for the central nervous system, including neural cultures, brain organoids, and organotypic brain cultures. We highlight the key findings from these models and illustrate how these models inform on viral processes, including neurotropism, neuroinvasion, and neurovirulence. We discuss the limitations of ex vivo models, highlight ongoing progress, and outline next-generation ex vivo models for virus research at the interface of neuroscience and infectious diseases.
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EGR1 Regulates SHANK3 Transcription at Different Stages of Brain Development.
The expression levels of SHANK3 are associated with autism spectrum disorder (ASD). The dynamic changes in SHANK3 expression during different stages of brain development may impact the progression of ASD. However, no studies or detailed analyses exploring the upstream mechanisms that regulate SHANK3 expression have been reported. In this study, we employed immunofluorescence to examine the expression of SHANK3 in brain organoids at various stages. Our results revealed elevated levels of SHANK3 expression in brain-like organoids at Day 60. Additionally, we utilized bioinformatics software to predict and analyze the SHANK3 gene's transcription start site. Through the dual luciferase reporter gene technique, we identified core transcription elements within the SHANK3 promoter. Site-directed mutations were used to identify specific transcription sites of SHANK3. To determine the physical binding of potential transcription factors to the SHANK3 promoter, we employed electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Our findings demonstrated that the transcription factor EGR1 regulates SHANK3 expression by binding to the transcription site of the SHANK3 promoter. Although this study did not investigate the pathological phenotypes of human brain organoids or animal model brains with EGR1 deficiency, which could potentially substantiate the findings observed for SHANK3 mutants, our findings provide valuable insights into the relationship between the transcription factor, EGR1, and SHANK3. This study contributes to the molecular understanding of ASD and offers potential foundations for precise targeted therapy.
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Uniform neural tissue models produced on synthetic hydrogels using standard culture techniques.
The aim of the present study was to test sample reproducibility for model neural tissues formed on synthetic hydrogels. Human embryonic stem (ES) cell-derived precursor cells were cultured on synthetic poly(ethylene glycol) (PEG) hydrogels to promote differentiation and self-organization into model neural tissue constructs. Neural progenitor, vascular, and microglial precursor cells were combined on PEG hydrogels to mimic developmental timing, which produced multicomponent neural constructs with 3D neuronal and glial organization, organized vascular networks, and microglia with ramified morphologies. Spearman's rank correlation analysis of global gene expression profiles and a comparison of coefficient of variation for expressed genes demonstrated that replicate neural constructs were highly uniform to at least day 21 for samples from independent experiments. We also demonstrate that model neural tissues formed on PEG hydrogels using a simplified neural differentiation protocol correlated more strongly to in vivo brain development than samples cultured on tissue culture polystyrene surfaces alone. These results provide a proof-of-concept demonstration that 3D cellular models that mimic aspects of human brain development can be produced from human pluripotent stem cells with high sample uniformity between experiments by using standard culture techniques, cryopreserved cell stocks, and a synthetic extracellular matrix. Impact statement Pluripotent stem (PS) cells have been characterized by an inherent ability to self-organize into 3D "organoids" resembling stomach, intestine, liver, kidney, and brain tissues, offering a potentially powerful tool for modeling human development and disease. However, organoid formation must be quantitatively reproducible for applications such as drug and toxicity screening. Here, we report a strategy to produce uniform neural tissue constructs with reproducible global gene expression profiles for replicate samples from multiple experiments.
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Organoid models of breathing disorders reveal patterning defect of hindbrain neurons caused by PHOX2B-PARMs.
Retrotrapezoid nucleus (RTN) neurons in the brainstem regulate the ventilatory response to hypercarbia. It is unclear how PHOX2B-polyalanine repeat mutations (PHOX2B-PARMs) alter the function of PHOX2B and perturb the formation of RTN neurons. Here, we generated human brainstem organoids (HBSOs) with RTN-like neurons from human pluripotent stem cells. Single-cell transcriptomics revealed that expression of PHOX2B+7Ala PARM alters the differentiation trajectories of the hindbrain neurons and hampers the formation of the RTN-like neurons in HBSOs. With the unguided cerebral organoids (HCOs), PHOX2B+7Ala PARM interrupted the patterning of PHOX2B+ neurons with dysregulation of Hedgehog pathway and HOX genes. With complementary use of HBSOs and HCOs with a patient and two mutant induced pluripotent stem cell lines carrying different polyalanine repetition in PHOX2B, we further defined the association between the length of polyalanine repetition and malformation of RTN-respiratory center and demonstrated the potential toxic gain of function of PHOX2B-PARMs, highlighting the uniqueness of these organoid models for disease modeling.
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CLARITY in Alzheimer's Research: Merging Tissue Transparency with Next-Gen Neurotechnologies.
CLARITY is a technique that makes tissues optically transparent, enabling the clear visualization of complex cellular and subcellular structures with relative ease. Traditionally, this technique has been used to visualize the pathologies of certain diseases. In the case of Alzheimer's disease (AD), the CLARITY technique of clearing lipids from tissues has enabled precise visualization of amyloid-beta (Aβ) and tau pathologies with a temporal analysis of the extent of protein aggregation associated with disease progression. This information has been invaluable for determining the progression of AD. Moreover, the structural characteristics of Aβ plaques and neurofibrillary tangles provide details about aggregation patterns. Here, we highlight critical insights into the potential applications of the CLARITY technique in conjunction with other modalities. We elucidated microscopy techniques, namely confocal microscopy and light-sheet microscopy, which are often used to detect fluorescently labeled compounds within cells, aiding in the qualitative and quantitative estimation of target proteins. Additionally, we discuss the application of super-resolution expansion microscopy (SREM) to increase the resolution of tissue images without compromising molecular specificity. We also expounded on the application of CLARITY-based imaging in improving optogenetics and organoid development. Furthermore, we highlight the potential of CLARITY in assisting the formulation and validation of machine learning algorithms and conducting hypothesis testing.
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Glioblastoma heterogeneity and the tumour microenvironment: implications for preclinical research and development of new treatments.
Glioblastoma is the deadliest form of brain cancer. Aside from inadequate treatment options, one of the main reasons glioblastoma is so lethal is the rapid growth of tumour cells coupled with continuous cell invasion into surrounding healthy brain tissue. Significant intra- and inter-tumour heterogeneity associated with differences in the corresponding tumour microenvironments contributes greatly to glioblastoma progression. Within this tumour microenvironment, the extracellular matrix profoundly influences the way cancer cells become invasive, and changes to extracellular (pH and oxygen levels) and metabolic (glucose and lactate) components support glioblastoma growth. Furthermore, studies on clinical samples have revealed that the tumour microenvironment is highly immunosuppressive which contributes to failure in immunotherapy treatments. Although technically possible, many components of the tumour microenvironment have not yet been the focus of glioblastoma therapies, despite growing evidence of its importance to glioblastoma malignancy. Here, we review recent progress in the characterisation of the glioblastoma tumour microenvironment and the sources of tumour heterogeneity in human clinical material. We also discuss the latest advances in technologies for personalised and in vitro preclinical studies using brain organoid models to better model glioblastoma and its interactions with the surrounding healthy brain tissue, which may play an essential role in developing new and more personalised treatments for this aggressive type of cancer.
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Herpes simplex virus type 1 infection leads to neurodevelopmental disorder-associated neuropathological changes.
Neonatal herpes simplex virus type 1 (HSV-1) infections contribute to various neurodevelopmental disabilities and the subsequent long-term neurological sequelae into the adulthood. However, further understanding of fetal brain development and the potential neuropathological effects of the HSV-1 infection are hampered by the limitations of existing neurodevelopmental models due to the dramatic differences between humans and other mammalians. Here we generated in vitro neurodevelopmental disorder models including human induced pluripotent stem cell (hiPSC)-based monolayer neuronal differentiation, three-dimensional (3D) neuroepithelial bud, and 3D cerebral organoid to study fetal brain development and the potential neuropathological effects induced by the HSV-1 infections. Our results revealed that the HSV-1-infected neural stem cells (NSCs) exhibited impaired neural differentiation. HSV-1 infection led to dysregulated neurogenesis in the fetal neurodevelopment. The HSV-1-infected brain organoids modelled the pathological features of the neurodevelopmental disorders in the human fetal brain, including the impaired neuronal differentiation, and the dysregulated cortical layer and brain regionalization. Furthermore, the 3D cerebral organoid model showed that HSV-1 infection promoted the abnormal microglial activation, accompanied by the induction of inflammatory factors, such as TNF-α, IL-6, IL-10, and IL-4. Overall, our in vitro neurodevelopmental disorder models reconstituted the neuropathological features associated with HSV-1 infection in human fetal brain development, providing the causal relationships that link HSV biology with the neurodevelopmental disorder pathogen hypothesis.
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An overview of human single-cell RNA sequencing studies in neurobiological disease.
Neurobiological disorders are highly prevalent medical conditions that contribute to significant morbidity and mortality. Single-cell RNA sequencing (scRNA-seq) is a technique that measures gene expression in individual cells. In this review, we survey scRNA-seq studies of tissues from patients suffering from neurobiological disease. This includes postmortem human brains and organoids derived from peripheral cells. We highlight a range of conditions, including epilepsy, cognitive disorders, substance use disorders, and mood disorders. These findings provide new insights into neurobiological disease in multiple ways, including discovering novel cell types or subtypes involved in disease, proposing new pathophysiological mechanisms, uncovering novel drug targets, or identifying potential biomarkers. We discuss the quality of these findings and suggest potential future directions and areas open for additional research, including studies of non-cortical brain regions and additional conditions such as anxiety disorders, mood disorders, and sleeping disorders. We argue that additional scRNA-seq of tissues from patients suffering from neurobiological disease could advance our understanding and treatment of these conditions.
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CYP3A5 Mediates Effects of Cocaine on Human Neocorticogenesis: Studies using an In Vitro 3D Self-Organized hPSC Model with a Single Cortex-Like Unit.
Because of unavoidable confounding variables in the direct study of human subjects, it has been difficult to unravel the effects of prenatal cocaine exposure on the human fetal brain, as well as the cellular and biochemical mechanisms involved. Here, we propose a novel approach using a human pluripotent stem cell (hPSC)-based 3D neocortical organoid model. This model retains essential features of human neocortical development by encompassing a single self-organized neocortical structure, without including an animal-derived gelatinous matrix. We reported previously that prenatal cocaine exposure to rats during the most active period of neural progenitor proliferation induces cytoarchitectural changes in the embryonic neocortex. We also identified a role of CYP450 and consequent oxidative ER stress signaling in these effects. However, because of differences between humans and rodents in neocorticogenesis and brain CYP metabolism, translation of the research findings from the rodent model to human brain development is uncertain. Using hPSC 3D neocortical organoids, we demonstrate that the effects of cocaine are mediated through CYP3A5-induced generation of reactive oxygen species, inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neural tissue development. Furthermore, knockdown of CYP3A5 reversed these cocaine-induced pathological phenotypes, suggesting CYP3A5 as a therapeutic target to mitigate the deleterious neurodevelopmental effects of prenatal cocaine exposure in humans. Moreover, 3D organoid methodology provides an innovative platform for identifying adverse effects of abused psychostimulants and pharmaceutical agents, and can be adapted for use in neurodevelopmental disorders with genetic etiologies.
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RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex.
During neocortical development, neural stem cells (NSCs) divide symmetrically to self-renew at the early stage and then divide asymmetrically to generate post-mitotic neurons. The molecular mechanisms regulating the balance between NSC self-renewal and neurogenesis are not fully understood. Using mouse in utero electroporation (IUE) technique and in vitro human NSC differentiation models including cerebral organoids (hCOs), we show here that regulator of cell cycle (RGCC) modulates NSC self-renewal and neuronal differentiation by affecting cell cycle regulation and spindle orientation. RGCC deficiency hampers normal cell cycle process and dysregulates the mitotic spindle, thus driving more cells to divide asymmetrically. These modulations diminish the NSC population and cause NSC pre-differentiation that eventually leads to brain developmental malformation in hCOs. We further show that RGCC might regulate NSC spindle orientation by affecting the organization of centrosome and microtubules. Our results demonstrate that RGCC is essential to maintain the NSC pool during cortical development and suggest that RGCC defects could have etiological roles in human brain malformations.