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The impact of antidepressants on human neurodevelopment: Brain organoids as experimental tools.
The use of antidepressants during pregnancy benefits the mother's well-being, but the effects of such substances on neurodevelopment remain poorly understood. Moreover, the consequences of early exposure to antidepressants may not be immediately apparent at birth. In utero exposure to selective serotonin reuptake inhibitors (SSRIs) has been related to developmental abnormalities, including a reduced white matter volume. Several reports have observed an increased incidence of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) after prenatal exposure to SSRIs such as sertraline, the most widely prescribed SSRI. The advent of human-induced pluripotent stem cell (hiPSC) methods and assays now offers appropriate tools to test the consequences of such compounds for neurodevelopment in vitro. In particular, hiPSCs can be used to generate cerebral organoids - self-organized structures that recapitulate the morphology and complex physiology of the developing human brain, overcoming the limitations found in 2D cell culture and experimental animal models for testing drug efficacy and side effects. For example, single-cell RNA sequencing (scRNA-seq) and electrophysiological measurements on organoids can be used to evaluate the impact of antidepressants on the transcriptome and neuronal activity signatures in developing neurons. While the analysis of large-scale transcriptomic data depends on dimensionality reduction methods, electrophysiological recordings rely on temporal data series to discriminate statistical characteristics of neuronal activity, allowing for the rigorous analysis of the effects of antidepressants and other molecules that affect the developing nervous system, especially when applied in combination with relevant human cellular models such as brain organoids.
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Parkinson's Disease Phenotypes in Patient Neuronal Cultures and Brain Organoids Improved by 2-Hydroxypropyl-β-Cyclodextrin Treatment.
BACKGROUND: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD. OBJECTIVES: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds. METHODS: Using two-dimensional and three-dimensional models of patients' derived neurons we recapitulated PD-related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model. RESULTS: PD patient-derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2-hydroxypropyl-β-cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient-specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model. CONCLUSION: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient-derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Single-cell RNA sequencing reveals cadmium-induced cell-type-specific mechanisms of developmental neurotoxicity in human cortical organoids.
Fetal brain development is crucial for lifelong neurological health, yet the impact of environmental heavy metal pollutants such as cadmium (Cd) remains incompletely understood. As a typical environmental heavy metal pollutant, Cd is widely encountered by various populations, including pregnant women, and disrupts zinc and copper ion homeostasis-processes essential for fetal brain development. However, cell-type-specific vulnerabilities underlying Cd-induced developmental neurotoxicity have been underexplored in human models. In this study, we employed cortical organoids with single-cell RNA sequencing (scRNA-seq) to evaluate the Cd-induced cell-type-specific developmental neurotoxicity and elucidate molecular mechanisms. scRNA-seq revealed that Cd exposure induced cell cycle arrest concurrent with zinc and copper ion dyshomeostasis in proliferating neural populations, inducing the loss of inhibitory neurons. Mechanistically, Cd activated ribosome biogenesis and mitochondrial energy metabolism across all cell types, while enhancing Cd2+ detoxification in astrocytes and inducing astrocyte dysfunction. Furthermore, Cd disrupted axon guidance as evidenced by decreased axonal growth length and enhanced aberrant intercellular communication via robust binding Glu and Asp of PSMD2, TUBA1B and CFL1. Finally, MEA assays revealed that Cd exposure impaired neural network activity in cortical organoids. Our study reveals previously unrecognized mechanisms by which environmentally relevant cadmium disrupts human corticogenesis at the single-cell level, providing compelling evidence for the developmental neurotoxicity of this ubiquitous pollutant and supporting efforts to reduce prenatal Cd exposure for lifelong neurological health.
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Subacute sclerosing panencephalitis (SSPE). A clinicopathologic study of the retinal lesions.
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Increased vesicular and vacuolar transport in traumatic human brain edema. A combined electron microscopic study and theoretical approach.
Twelve cortical biopsies obtained from patients with traumatic brain injury, some complicated with subdural or epidural hematoma or hygroma, have been analyzed. The endothelial vacuolar and vesicular transports have been studied in order to establish their probable role in both edema formation and edema resolution. Normal or 'non-activated' and 'activated' capillaries were found. The activated capillaries showed predominantly enhanced abluminally orientated vesicular transport by means of small, medium and large uncoated and coated vesicles as well as the presence of endothelial tubular structures. Activation of the endothelial nuclear zone and vesicles internalizing to the hypertrophic Golgi complex, lysosomes and multivesicular bodies were observed. Vacuolar transport was predominant. In brain injuries of long evolution time, a wide spectrum of endothelial cell mechanisms was observed increasing the vesicular and vacuolar transport. These mechanisms were deep invaginations of luminal surface, large coated vesicles, tubular structures and transient and incomplete transendothelial channels formed either by chained plasmalemmal vesicles or elongated protein-containing vacuoles. Uncoated vesicles were seen surrounding lysosomes. Most endothelial junctions were apparently intact. The present results suggest that vesicular transport might be discriminated between abluminally orientated or transendothelial transport (edema formation) and intraendothelial transport (edema resolution) directed towards cell organelles. The transendothelial passage via large vacuoles seems to be caused by macromolecular transport.
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Human iPSC-Derived Neurons and Cerebral Organoids Establish Differential Effects of Germline NF1 Gene Mutations.
Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.
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The Brazilian Zika virus strain causes birth defects in experimental models.
Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys. Until the twentieth century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash, arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKV(BR)) strain causes birth defects remains absent. Here we demonstrate that ZIKV(BR) infects fetuses, causing intrauterine growth restriction, including signs of microcephaly, in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. We also report that the infection of human brain organoids results in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKV(BR) crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, and impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKV(BR) outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human neurodevelopment.
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Primary culture of capillary endothelium from rat brain.
To provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features.
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In vivo model of HIV infection of the human brain.
Approximately one quarter of AIDS patients develop neurologic symptoms attributable to HIV infection within the brain. Previous studies suggest that HIV associated neurologic damage may be mediated by immune factors secreted by activated/infected CNS macrophages. We developed an in vivo system in which human embryonic brain tissue can be infected with HIV and the associated pathology monitored. In this model, dissociated human brain tissue is grown in vitro as single cell suspension in serum free medium. Fetal neural cells aggregate and form "brain microspheres" that are then transplanted into SCID mice. Pilot studies suggest that brain microspheres injected in the fat pad of SCID mice differentiate and survive for several months in vivo. Study of these grafts shows presence of functional neural cells and vascular organization suggesting a blood-brain barrier. When brain microspheres are co-cultured in vitro with HIV-infected human macrophages, virus is detected inside the human neural tissue grafts in SCID mice and measurements of viral and immune factors can be performed. To promote physiologic neuronal differentiation within the human grafts, implantation in the brain of SCID mice is being tested at the present time.
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Oropouche virus disrupts neurodevelopment and exhibits congenital infection potential.
Oropouche virus (OROV) historically caused a self-limiting disease, yet recent strains have been clinically linked to congenital infection and neurodevelopmental disease. These observations highlight the need to study OROV as a congenital pathogen. Here, using both a historical and currently circulating strain, we show OROV infects neurons across differentiation states in human forebrain organoids. Compared with the neurotropic congenital pathogen Zika virus (ZIKV), OROV exhibits a heightened capacity for neuroinfection and pathology in both forebrain organoids and neonatal mice. The increased permissiveness of OROV is driven, in part, by a broader neuronal tropism and a relative insensitivity to neuronal type I interferon-mediated antiviral responses. Consistent with clinical observations, neonatal neuroinfection results in rapid and severe neuropathology marked by cerebral hemorrhage. Finally, using a transient type I interferon-blockade model, we demonstrate that OROV can productively infect the murine placenta and cause fetal growth restriction. Together, our complementary models support a unified framework in which placental and fetal barriers limit productive fetal brain infection, yet OROV exhibits marked neurotropism and neuropathogenic potential upon gaining access to developing neural tissues. These findings reinforce the need for vigilant monitoring of OROV as an emerging pathogen associated with adverse pregnancy outcomes and congenital disease.