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Dolutegravir induces FOLR1 expression during brain organoid development.

ABSTRACT: The brain and spinal cord are formed during neurulation, a developmental process that occurs during the first month of pregnancy and can be modified by low serum levels of folic acid (FA), environmental factors, or genetic predispositions. In 2018, data from a surveillance study in Botswana, a country with one of the highest incidences of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs, revealed an increased risk of neural tube defects (NTDs) among newborns whose mothers were taking Dolutegravir (DTG) during the first trimester of pregnancy. Subsequently, the World Health Organization and the U.S. Food and Drug Administration issued recommendations and guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs, we sought to employ a rapid experimental system to test specific hypotheses. Brain organoids are three-dimensional cellular structures that can be used to study early neural development, and to assess the potential neurotoxicity of drugs and other compounds. To understand the development of brain organoids in the presence of DTG, we analyzed gene expression from RNA sequencing together with biomechanical information from Optical Coherence Tomography, Optical Coherence Elastography and Brillouin microscopy. Our sequencing data indicates that DTG induces the expression of folate receptor (FOLR1) and modulates the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness, while reverberant Optical Coherence Elastography measurements indicate decreased organoid volumes and an internal stiffness decrease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263394 | GEO | 2024/04/10

REPOSITORIES: GEO

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Project description:Background: In the early stage of embryonic development, the neural tube (NT) cannot be closed properly due to some complex factors such as, environmental factors, genetic factors and the relationship between various factors, leading to the occurrence of neural tube defects (NTDs). Methods: In this study, we induced the mouse model of NTDs by feeding mice a low-folate diet and injecting 1.5mg/kg methotrexate on E7.5. Fetal mice were achieved at E13.5, then we extracted proteins from brain tissue with trypsin digestion. After enzymatic digestion, peptides were labeled with TMT/iTRAQ and then separated in high-performance liquid chromatography (HPLC) for subsequent LC-MS/MS analysis. To analyze the functional enrichment of differentially expressed proteins(DEPs) in the NTDs mice tissues with low folate induction, we used Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway annotation to analyze proteomic changes. Results: Low folate induced mouse model was successfully constructed. Folate was used as a sensitizing agent, the teratogenicity rate of the NTDs fetal mice was increased to 36.5% when methotrexate was injected to maternal mice at E7.5. 6614 proteins were identified by mass spectrometry, among them, 5656 proteins were quantified. In the following proteomic analysis, GO classification and KEGG pathway enrichment analysis were conducted, and heatmaps were drawn for differentially expressed proteins(DEPs). Moreover, the main pathways associated with NTDs, such as Hedgehog, Wnt, p53, Hippo signaling pathways, and one carbon pool by folate, can be identified through the generation of a protein-protein interaction (PPI) network. It was also found that the regulation of ribosomal proteins has a certain impact on neural tube development, such as RPL13 and RPL14, which are upregulated in NTDs. Conclusions: Our results revealed proteomic changes in the tissues of low folate induced NTDs mice, as well as validation showed that ribosomal proteins play a regulatory role during the development of NTDs, it provides new ideas for the pathogenesis and preventive measures of NTDs.

Project description:For many therapeutic drugs, including antiretroviral drugs used to treat people living with HIV-1 (PLWH), we have little data on the potential effects on the developing human brain due to limited access to tissue and historical constraints on the inclusion of pregnant populations in clinical trials. Human induced pluripotent stem cells (iPSCs) offer a new avenue to gain insight on how drugs may impact human cell types representative of the developing central nervous system. To prevent vertical transmission of HIV and promote the health of pregnant PLWH, antiretroviral therapy must be initiated and/or maintained throughout pregnancy. However, many antiretroviral drugs are approved for widespread use following clinical testing only in non-pregnant populations and there may be limited information on potential teratogenicity until pregnancy outcomes are evaluated. The integrase strand transfer inhibitor dolutegravir (DTG) is a frontline antiretroviral drug that is effective in viral suppression of HIV but was previously reported to be associated with a slight increase in the risk for neural tube defects in one study, although this has not been replicated in other cohorts. To directly investigate the potential impact of DTG on human cortical neurogenesis, we measured the effects of daily drug exposure on the early stages of corticogenesis in a human iPSC-based forebrain organoid model. We observed deficits in organoid structure and impaired neurogenesis in DTG-treated organoids compared to vehicle-treated control organoids after 20 or 40 days in culture. Our highest dose of DTG (10µM) resulted in significantly smaller organoids with a reduced density of neural rosette structures compared to vehicle-treated controls. Mechanistically, RNA-sequencing and immunohistological analysis suggests dysregulated amino acid transport and activation of the integrated stress response in the DTG-treated organoids, and functionally, a small molecule integrated stress response inhibitor (ISRIB) could partially rescue increased expression of proteins related to cell cycle regulation. Together, these results illustrate the potential for human iPSC-based strategies to reveal biological processes during neurogenesis that may be affected by therapeutic drugs and provide complementary data in relevant human cell types to augment preclinical investigations of drug safety during pregnancy.

Project description:Maternal folic acid intake is crucial for the development of the offspring's nervous system, and folic acid metabolism disorders during pregnancy lead to neural tube defects (NTDs) in the fetus. Folic acid and vitamins biosynthesis is a major biochemical feature of gut microbiota. The complex and diverse microbial ecosystem residing within maternal host contributes critically to these intergenerational impacts. However, the mechanisms still require further investigation. In this study, we found that the low folate diets combined MTX-induced changed the structure/composition of the gut microbiota and substantially altered the fecal metabolic phenotype of pregnant mice, including central carbon metabolism in cancer and vitamin digestion & absorption. We demonstrated that the correlation betweent gut microbiota of pregnant mice and the brain metabolic profiles of NTDs fetal mice. According to our data, the Lactobacillales and Bifidobacteriales abundances in pregnant mice gut were positively correlated with the abundances of lipid metabolites in fetal mice brain. The abundances of Enterobacterales and Clostridiales were negatively correlated with those lipid metabolites. Interestingly, the abundance of Inosine, Uridine, L-Carnitine and Glycerophosphocholine were down-regulated synchronously in pregnant feces and NTDs fetal mice brain. This was probably the intergenerational microbial-metabolism biomarkers of NTDs. Our study provides evidence for how perinatal microecological factors shape fetal neural tube development. Feces metabolomics is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS4893' rel='noopener noreferrer' target='_blank'>MTBLS4893</a>.Brain tissue metabolomics is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS4894' rel='noopener noreferrer' target='_blank'>MTBLS4894</a>.

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Dolutegravir induces FOLR1 expression during brain organoid development.

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