Isolation and Propagation of Primary Human and Rodent Embryonic Neural Progenitor Cells and Cortical Neurons

Darbinyan, Armine; Kaminski, Rafal; White, Martyn K.; Pozniak, Paul D.; Darbinian, Nune; Khalili, Kamel

doi:10.1007/978-1-0716-1437-2_5

Cited by 4 publications

(4 citation statements)

References 11 publications

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“…The effects of EtOH and SSRI are similar, but their effects on the 5-HT pathway are different from their effects on the DA pathway. Previous data in primary cultures [ 75 , 97 , 98 , 99 ], fetal brain tissues, and FB-Es from maternal blood [ 61 , 62 , 63 , 65 ] demonstrated an association between EtOH exposure and several neuronal and OL molecular markers. The literature and the present data suggest that abnormal expression of the 5-HT and DA receptors (5-HT-Rs; D1, D2, β1, β2) and transporters (SERT, DAT) may play a role in the mechanism of EtOH-mediated neurotoxicity and potentially in the affective dysregulation of FASD.…”

Section: Discussionmentioning

confidence: 99%

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH

Darbinian,

Merabova,

Tatevosian

et al. 2023

Cells

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Introduction: Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation is associated with the effects of EtOH exposure on the development of fetal serotonergic (5-HT) and/or dopaminergic (DA) pathways, i.e., pathways that in postnatal life are believed to regulate mood. Many women who use alcohol (ethanol, EtOH) during pregnancy suffer from depression and take selective serotonin reuptake inhibitors (SSRIs), which might influence these monoaminergic pathways in the fetus. Alternatively, monoaminergic pathway abnormalities might reflect a direct effect of EtOH on the fetal brain. To distinguish between these possibilities, we measured their expressions in fetal brains and in fetal brain-derived exosomes (FB-Es) isolated from the mothers’ blood. We hypothesized that maternal use of EtOH and/or SSRIs during pregnancy would be associated with impaired fetal neural development, detectable as abnormal levels of monoaminergic and apoptotic biomarkers in FB-Es. Methods: Fetal brain tissues and maternal blood were collected at 9–23 weeks of pregnancy. EtOH groups were compared with unexposed controls matched for gestational age (GA). The expression of 84 genes associated with the DA and 5-HT pathways was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on microarrays. FB-Es also were assayed for serotonin transporter protein (SERT) and brain-derived neurotrophic factor (BDNF) by enzyme-linked immunosorbent assay (ELISA). Results: Six EtOH-exposed human fetal brain samples were compared to SSRI- or polydrug-exposed samples and to unexposed controls. EtOH exposure was associated with significant upregulation of DA receptor D3 and 5-HT receptor HTR2C, while HTR3A was downregulated. Monoamine oxidase A (MAOA), MAOB, the serine/threonine kinase AKT3, and caspase-3 were upregulated, while mitogen-activated protein kinase 1 (MAPK1) and AKT2 were downregulated. ETOH was associated with significant upregulation of the DA transporter gene, while SERT was downregulated. There were significant correlations between EtOH exposure and (a) caspase-3 activation, (b) reduced SERT protein levels, and (c) reduced BDNF levels. SSRI exposure independently increased caspase-3 activity and downregulated SERT and BDNF. Early exposure to EtOH and SSRI together was associated synergistically with a significant upregulation of caspase-3 and a significant downregulation of SERT and BDNF. Reduced SERT and BDNF levels were strongly correlated with a reduction in eye diameter, a somatic manifestation of FASD. Conclusions: Maternal use of EtOH and SSRI during pregnancy each was associated with changes in fetal brain monoamine pathways, consistent with potential mechanisms for the affective dysregulation associated with FASD.

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Section: Discussionmentioning

confidence: 99%

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH

Darbinian,

Merabova,

Tatevosian

et al. 2023

Cells

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“…We previously established primary cultures of neurons and OLs ( Amini et al, 2009 ; Darbinyan et al, 2013a , b , 2021 ; Darbinian et al, 2021a , b ), and showed that in these cultures, and in FB-E from EtOH-exposed maternal blood, there was an association between EtOH exposure and several brain cell markers ( Goetzl et al, 2016 , 2019a ; Darbinian et al, 2021a , 2023 ). These studies have now been extended to demonstrate that FB-Es from EtOH-exposed fetuses can carry additional markers for abnormalities of mtDNA and mitochondrial function pathways (e.g., low levels of mtDNA, OGG1 mRNA, and low levels of IGF-1), and thus may be a source of additional molecular markers for FASD.…”

Section: Discussionmentioning

confidence: 99%

In utero ethanol exposure induces mitochondrial DNA damage and inhibits mtDNA repair in developing brain

et al. 2023

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IntroductionMitochondrial dysfunction is postulated to be a central event in fetal alcohol spectrum disorders (FASD). People with the most severe form of FASD, fetal alcohol syndrome (FAS) are estimated to live only 34 years (95% confidence interval, 31 to 37 years), and adults who were born with any form of FASD often develop early aging. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, hallmarks of aging, are postulated central events in FASD. Ethanol (EtOH) can cause mtDNA damage, consequent increased oxidative stress, and changes in the mtDNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1). Studies of molecular mechanisms are limited by the absence of suitable human models and non-invasive tools.MethodsWe compared human and rat EtOH-exposed fetal brain tissues and neuronal cultures, and fetal brain-derived exosomes (FB-Es) from maternal blood. Rat FASD was induced by administering a 6.7% alcohol liquid diet to pregnant dams. Human fetal (11–21 weeks) brain tissue was collected and characterized by maternal self-reported EtOH use. mtDNA was amplified by qPCR. OGG1 and Insulin-like growth factor 1 (IGF-1) mRNAs were assayed by qRT-PCR. Exosomal OGG1 was measured by ddPCR.ResultsMaternal EtOH exposure increased mtDNA damage in fetal brain tissue and FB-Es. The damaged mtDNA in FB-Es correlated highly with small eye diameter, an anatomical hallmark of FASD. OGG1-mediated mtDNA repair was inhibited in EtOH-exposed fetal brain tissues. IGF-1 rescued neurons from EtOH-mediated mtDNA damage and OGG1 inhibition.ConclusionThe correlation between mtDNA damage and small eye size suggests that the amount of damaged mtDNA in FB-E may serve as a marker to predict which at risk fetuses will be born with FASD. Moreover, IGF-1 might reduce EtOH-caused mtDNA damage and neuronal apoptosis.

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“…The earliest attempts at separation of individual viable neurons was by extrusion of explanted tissue through a nylon mesh, supplemented by protease treatment and repeated aspiration in media through a Pasteur pipet tip [ 98 , 155 , 165 ]. Decades later, the method still relies on manual trituration of enzyme-treated brain tissue, sometimes using volume-adjustable laboratory pipets, and with the residual-cell-cluster problem being hidden by filtration-based removal [ 26 , 27 ].…”

Section: Discussionmentioning

confidence: 99%

Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery

Tello,

Jiang,

Zohar

et al. 2023

Neural Dev

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Background CASK-related neurodevelopmental disorders are untreatable. Affected children show variable severity, with microcephaly, intellectual disability (ID), and short stature as common features. X-linked human CASK shows dosage sensitivity with haploinsufficiency in females. CASK protein has multiple domains, binding partners, and proposed functions at synapses and in the nucleus. Human and Drosophila CASK show high amino-acid-sequence similarity in all functional domains. Flies homozygous for a hypomorphic CASK mutation (∆18) have motor and cognitive deficits. A Drosophila genetic model of CASK-related disorders could have great scientific and translational value. Methods We assessed the effects of CASK loss of function on morphological phenotypes in Drosophila using established genetic, histological, and primary neuronal culture approaches. NeuronMetrics software was used to quantify neurite-arbor morphology. Standard nonparametric statistics methods were supplemented by linear mixed effects modeling in some cases. Microfluidic devices of varied dimensions were fabricated and numerous fluid-flow parameters were used to induce oscillatory stress fields on CNS tissue. Dissociation into viable neurons and neurite outgrowth in vitro were assessed. Results We demonstrated that ∆18 homozygous flies have small brains, small heads, and short bodies. When neurons from developing CASK-mutant CNS were cultured in vitro, they grew small neurite arbors with a distinctive, quantifiable “bushy” morphology that was significantly rescued by transgenic CASK+. As in humans, the bushy phenotype showed dosage-sensitive severity. To overcome the limitations of manual tissue trituration for neuronal culture, we optimized the design and operation of a microfluidic system for standardized, automated dissociation of CNS tissue into individual viable neurons. Neurons from CASK-mutant CNS dissociated in the microfluidic system recapitulate the bushy morphology. Moreover, for any given genotype, device-dissociated neurons grew larger arbors than did manually dissociated neurons. This automated dissociation method is also effective for rodent CNS. Conclusions These biological and engineering advances set the stage for drug discovery using the Drosophila model of CASK-related disorders. The bushy phenotype provides a cell-based assay for compound screening. Nearly a dozen genes encoding CASK-binding proteins or transcriptional targets also have brain-development mutant phenotypes, including ID. Hence, drugs that improve CASK phenotypes might also benefit children with disorders due to mutant CASK partners.

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Isolation and Propagation of Primary Human and Rodent Embryonic Neural Progenitor Cells and Cortical Neurons

Cited by 4 publications

References 11 publications

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH

In utero ethanol exposure induces mitochondrial DNA damage and inhibits mtDNA repair in developing brain

Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery

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