Folic acid ameliorates high glucose–induced neurotoxicity in human forebrain organoids: Insights from proteomics
Haoni Yan, Shujin Chen, and Aynur Abdulla contributed equally.
Abstract
Pregestational diabetes (PGDM) has been associated with an elevated risk of congenital abnormalities, particularly those affecting the nervous system. The efficacy of folic acid (FA) supplementation in reducing the incidence of neurodevelopmental damage caused by PGDM has been well documented. However, the exact mechanism is unclear. Here, a human forebrain organoid model, which replicates the three-dimensional structure of the early fetal neural tissue, was employed to study the neuroprotective effects of FA in PGDM. In this study, the forebrain organoids were cultured at high glucose (HG) concentrations from Days 20 to 40 with or without FA. Immunostaining revealed that the supplementation of FA significantly decreased HG-induced neuron apoptosis. The proteomics examination suggested HG caused an increase in glial fibrillary acidic protein expression, a marker of astrocytes, leading to the upregulation of metallothionein expression and perturbation of mineral absorption, whereas FA reversed this effect. Proteomics analysis further showed that FA reduced HG-induced cell migration. Moreover, Western blot analysis verified that FA mitigated HG-induced apoptosis and cell migration via AMPK/FOXO pathway. Overall, current findings indicate that FA, as a functional food ingredient, has a protective effect on HG-induced abnormal fetal neurodevelopment.
1 INTRODUCTION
The presence of inadequately managed pregestational diabetes (PGDM) before conception and throughout the initial trimester of pregnancy has been linked to neurodevelopmental disorders in the offspring (Reece, 2012). Around 60 million women in the world who are of childbearing age (18–44 years) suffer from diabetes mellitus, either Type 1 or Type 2 (Li et al., 2022), and the incidence of diabetes mellitus among those women has exhibited a consistent upward trend, resulting in an impact on around 1% of pregnancies (Ornoy et al., 2021). Epidemiological studies have indicated that maternal diabetes has a series of side effects on the fetus, ranging from neurobehavioral abnormalities to embryonic death (Eletri & Mitanchez, 2022). Infants born to mothers diagnosed with PGDM exhibit diminished cognitive abilities and reduced attention span (Hod et al., 1999) and are at a significantly higher risk of experiencing linguistic impairment compared to infants born to mothers without diabetes. Previous studies employing in vitro and animal models had discovered that HG had a significant impact on cortex functioning. For example, when gestational diabetes is induced by streptozotocin in maternal Sprague–Dawley rats, the offspring may exhibit cognitive and neurological deficiencies because of the generation of oxidative stress (Huerta-Cervantes et al., 2021). However, the comprehensive understanding of the molecular pathophysiology and neurodevelopmental implications resulting from high-glucose (HG) exposure in utero is currently limited in humans. Furthermore, there is yet no proven method to reduce HG-induced neurodevelopmental damage in utero.
To guarantee the normal development of the embryonic nervous system, ensuring a sufficient and continuous provision of suitable nutrients is imperative. The incorporation of nutraceuticals represents a novel and stimulating subject within the realm of neurodevelopment, which combines pharmaceutical and nutritional therapies (Miller et al., 2019). The utilization of dietary therapy has been suggested as a potentially effective method for mitigating PGDM and associated metabolic diseases. Folic acid (FA), also known as vitamin B9, is a water-soluble vitamin composed of three components: pyridine, p-aminobenzoic acid, and glutamic acid (Wang et al., 2022). FA plays a crucial role in numerous metabolic processes within organisms and exhibits a protective effect against many forms of tissue injury (Akgun et al., 2021). Previous research has also demonstrated that oxidative stress significantly contributes to the development of fetal congenital defects induced by gestational diabetes, and FA has been found to mitigate oxidative stress in embryos during pregnancies affected by diabetes (Wentzel & Eriksson, 2005). These findings suggest that in the development of diabetic embryopathy, FA may function as an antioxidant. Moreover, several experts have suggested that pregnant women with diabetes may consider taking FA supplementation in order to alleviate the potential occurrence of fetal congenital malformations (Hill, 2007). Hence, the objective is to investigate the potential protective impact of FA on embryonic neurodevelopmental abnormalities caused by PGDM.
However, the ability to accurately forecast the therapeutic effects or side effects of medications in humans, particularly fetuses, needs to be improved by several ethical and practical problems (Du et al., 2023). As for animal models, although they play a significant role in analyzing various aspects of cortical development, their limitations arise due to the fundamental differences in the brains of humans and experimental animals, such as the lack of outer radial glial cells (oRGs) and gyrus formation (Cao, 2022). The utilization of three-dimensional (3D) human brain organoids presents clear advantages when compared to two-dimensional cell cultures. First, the brain organoids include the process and characteristics of human cortical development, especially the appearance of an outer subventricular zone containing abundant oRGs, which are the key to improving human cortex complexity and size. Second, in brain organoids, the cell types are more diverse, and neurons and glial cells are more mature, which increases communication among these kinds of cells (Di Lullo & Kriegstein, 2017). Therefore, brain organoid technology has developed rapidly and has arisen as an innovative approach to investigate neurodegenerative disease (such as Alzheimer's diseases [AD] and Huntington's diseases) (Wray, 2021), congenital cerebral malformation (such as microcephaly and autism), neuroinfectious disorders (such as herpes simplex virus 1 infection) (Rybak-Wolf et al., 2023), and neurotoxicity induced by drugs. For example, chronic (2R, 6R)-ketamine exposure was reported to inhibit the differentiation of neural progenitor cells (NPCs) and transform the division mode of apical radial glia cells from a vertical division plane to a horizontal one in brain organoids (Du et al., 2023). Of note, during the period spanning Days 20–40, brain organoids exhibit similar features to those observed during the first trimester of human pregnancy (Gabriel et al., 2023; Kelava & Lancaster, 2016). Therefore, the human brain organoid is a reliable model to reveal the underlying molecular mechanism of FA against HG-induced neurodevelopmental damage in PGDM.
In the present investigation, a structured approach was employed to cultivate forebrain organoids derived from human embryonic stem cells (hESCs) to explore the effect of HG exposure on developmental neurotoxicity, and also to assess the possible preventive effects of FA supplements on these variables. Based on the analysis of proteomic data, the current study demonstrates that the expression levels of many proteins associated with biological processes were altered after HG exposure in brain organoids, showing neuron apoptosis, increased cell migration, disturbed mineral absorption, and an altered AMPK/FOXO pathway. As far as we know, this is the first investigation to mimic the developmental neurotoxicity induced by PGDM by using brain organoids as well as evaluate the protective impact of FA.
2 MATERIALS AND METHODS
2.1 Chemicals
Glucose (CAS: 50-99-7; >99%) was purchased from Sigma-Aldrich and diluted into a 35 mM working concentration for human forebrain organoid treatment. Folic acid (CAS: 59-30-3; >99%) was purchased from Sigma-Aldrich and diluted into a 40 μM working concentration in current experiments.
2.2 The hESC culture
Dr. Zhen-Ge Luo provided us with the H9 hESCs as a gift. The hESCs were cultured in the mTeSR medium (85850; Stemcell Technologies) on a six-well plate (3516, Corning) coated with hESC-qualified Matrigel (354277, Corning), and 2 mL of fresh culture medium was replaced daily. Cells were passaged with ReLeSR (05872; Stemcell Technologies) at 37°C for 3–4 min when the confluency reached 80%.
2.3 Generation of human forebrain organoids
According to a previous study (Gabriel et al., 2021), the human forebrain organoids were generated after some modifications. In brief, when the confluency reached about 80%, Accutase (07920; Stemcell Technologies) was used to dissociate hESCs into single cells at 37°C for 3–4 min. After digestion and centrifugation, cells were resuspended in NIM medium (05835; Stemcell Technologies) containing 10 μM Y27632 (72302; Stemcell Technologies) and then planted at 9000 cells/well in a lipidure-coated (YS-CM5206; NOF CORPRATION) V-bottom 96-well plate (701211; NEST) to form embryonic bodies. In the next 5 days, the NIM medium was half-renewed every day until the formation of neurospheres. Then, after culturing in a 96-well plate for 5 days, the neurospheres were transferred to the neurosphere medium containing 0.1% hESC-qualified Matrigel, 48.4% DMEM/F12 (11330032; Gibco), 48.4% Neurobasal medium (21103049; Gibco), 0.4% N2 supplement (17502048; Gibco), 0.4% B27 supplement without vitamin A (17504044; Gibco), 5 μM β-mercaptoethanol (8057400250; Merck), 2.5 μM SB431542 (S1067; Selleck), 0.2755 μM insulin (I9278; Sigma-Aldrich), 1% GlutaMAX (35050061; Gibco), 0.5% MEM-NEAA (11140050; Gibco), and 1% Antibiotic-Antimycotic (15240112; Gibco) in a 60 mm Petri dish. On Day 8, 2 mL of organoid medium containing 48.4% DMEM/F12, 48.4% neurobasal medium, 0.4% N2 supplement, 0.4% B27 supplement with vitamin A (17504044; Gibco), 5 μM β-mercaptoethanol, 5 μM SB431542, 0.2755 μM insulin, 0.5 μM dorsomorphine (3093, Tocris), 60 nM retinol acetate (CAS: 127-47-9; Sigma-Aldrich), 1% GlutaMAX and 0.5% MEM-NEAA, and 1% Antibiotic-Antimycotic were added for the next 2 days. On Day 10, the neurospheres were transferred to the organoid medium on a shaker for maturation, and the medium was replaced twice a week. On Day 20 of maturation, the forebrain organoids were treated with 30 mM glucose (HG), 40 μM folic acid (FA), and 30 mM glucose + 40 μM folic acid (HG + FA) for 20 days, respectively. Then forebrain organoids were collected for the following experiment on Day 40. The reagents and chemical information are listed in Table S3.
2.4 Immunofluorescence staining
The immunofluorescence staining of the forebrain organoids was performed as per the previously described protocol (Du et al., 2023). Briefly, the forebrain organoids were fixed in 4% para-formaldehyde for 4–6 h at 4°C after washing with DPBS. Then, washed three times with Dulbecco's Phosphate-Buffered Saline (DPBS) again, the organoids were dehydrated with 30% sucrose at 4°C overnight. These dehydrated organoids were embedded in an optimal cutting temperature compound (OCT) and cryo-sectioned into 10 μm slices with a cryostat (Thermo). Before performing immunofluorescence staining, the slides were washed with PBS three times to remove excessive OCT compounds. For the immunostaining procedure, these slides were permeabilized in 0.5% Triton X-100 for 30 min, retrieved with antigen retrieval solution (P0090; Beyotime) for 5 min, washed three times with Phosphate-Buffered Saline (PBS), and blocked with blocking buffer (P0260; Beyotime) for 1 h. Then, the sections were incubated with primary antibodies for 2 h, washed three times with PBS, and incubated with the fluorophore-conjugated secondary antibodies for 2 h. Finally, DAPI (D9564; Sigma-Aldrich) was used to counterstain the nuclei. All images were captured using a Leica SP8 confocal microscope (Leica Microsystems). All primary and secondary antibodies used in the immunostaining procedure are shown in Table S1.
2.5 TUNEL assay
With the In Situ Cell Death Detection Kit (12156792910; Roche), the TUNEL assay was used to label apoptotic cells in forebrain organoids. According to the manufacturer's protocol, after permeabilization with 0.5% Triton, the slices were incubated with the TUNEL reaction mixture containing TdT enzyme and TMR-dUTP at 37°C for 1 h. Images were photographed with a confocal microscope to calculate the ratio of TUNEL+ cells to DAPI+ cells.
2.6 Mass spectrometry and proteomics data analysis
The label-free proteomics of forebrain organoids used in this work was performed according to the previously reported study by Abdulla et al. (2023). Briefly, forebrain organoids were lysed, sonicated, and centrifuged to collect the supernatant. Then, the proteins were precipitated by acetone, reduced by 1 M dithiothreitol, alkylated by 1 M iodoacetamide, and digested by trypsin. By incubating with trypsin at 37°C overnight, the proteins were digested into pipets, and the digestion process was stopped by adding 1% folic acid (FA). The peptides obtained above were desalted by a Macro Spin Column TARGA C18 and then prepared for label-free proteomic analysis.
The liquid chromatography–tandem mass spectrometry (LC–MS/MS) apparatus, which consists of an EASY-nLC 1200 connected to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) through a nano-spray electron ionization source, was used to analyze the peptides. The raw files acquired from MS were subjected to a search against the human database (9606-homo sapiens) using Spectronaut (14.5.200813.47784) for the purpose of performing data-independent acquisition analysis, employing the default parameters. The different expressions of proteins (DEPs) (fold change [FC] >1.5, or FC <−0.667, p < .05) were analyzed with Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). STRING analysis was performed to predict responses of proteins associated with specific pathways and visualized by Cytoscape software.
2.7 Quantitative real-time PCR (qPCR)
The forebrain organoids were used for RNA isolation, reverse transcription, and qPCR, performed as previously described (Du et al., 2023). Briefly, total RNA was purified from forebrain organoids by using the TRIzol kit (15596026; Thermo Fisher). Then, the PrimeScript RT Reagent Kit (RR047A; Takara) was used to reverse transcribe PCR and SYBR Premix Ex Taq II (RR820A, Takara) was used to determine the transcription level. The mRNA levels of forebrain organoids were calculated using the standard curve method of Ct value and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was used for standardization in forebrain organoids. The primer information is listed in Table S2.
2.8 Western blotting
Protein preparation and Western blotting were conducted as described previously (Cao et al., 2023). In short, forebrain organoids were homogenized, lysed, and centrifuged to extract the supernatant. Then, the total protein concentration was measured by a BCA Assay kit (20201ES76; Yeasen). The denatured proteins (30 μg) were placed in 10% SDS–PAGE, separated by electrophoresis, and then transferred electrically to a polyvinylidene fluoride membrane. After the nonspecific binding was blocked, the primary antibody was reacted, and then the secondary antibody was linked, and the protein bands were visualized with an ECL kit (ECL kit; Yeasen). The following antigens, GAPHD, glial fibrillary acidic protein (GFAP), FOXO3, AMPK, and its phosphorylated proteins (pAMPK) were detected. The protein density was calculated by ImageJ software and standardized to GAPDH, the housekeeping protein. The primary and secondary antibody information is listed in Table S1.
2.9 Cell migration assay
Cell migration experiments were performed as follows. In short, forebrain organoids collected from Days 35 to 40 were washed with DPBS and triturated into small tissues by a 200 μL Pasteur pipette. Then, these small neurosphere tissues were plated on coverslips coated with Matrigel and cultured with organoid medium for 72 h. After the neurospheres were attached, neurons were migrated and evaluated with brightfield images and immunofluorescence staining images. The migration distance of neurons was assessed by manually measuring the four radii of the migrating area, and the migration number was the definite number of cells in the migration areas.
2.10 Statistical analysis
All data are shown in the mean ± standard error of the mean and analyzed by the GraphPad Prism 9 software (GraphPad). Data were analyzed by one-way analysis of variance and Tukey's multiple comparisons to compare the differences among different groups. p < .05 was considered statistically significant in this study.
3 RESULTS
3.1 Generation and identification of hESC-derived forebrain organoids
To investigate the toxicity of long-term HG exposure on fetal brain development and whether FA has a protective effect, hESC-derived human forebrain organoids based on a previous protocol were generated and differentiated (Gabriel et al., 2021) (Figure 1a,b). To further validate the feasibility of this 3D model, the H9 hESC line was stained for the proliferation markers, KI67 and phospho-histone H3 (PH3), and the stem cell markers, nestin and SRY-box transcription factor 2 (SOX2) (Figure 1c), and the forebrain organoids were stained with a number of markers before being treated with HG and FA. At Day 40, apoptosis markers (TUNEL), proliferation markers (KI67), stem cell markers (SOX2 and PAX6), neuronal markers (TUJ1 and MAP2B), preplate markers (TBR1), and cortical layer markers (CTIP2) were all found in our forebrain organoids (Figure 1d), which confirmed the viability and feasibility of the 3D model.
3.2 Effects of FA on cell proliferation, apoptosis, and neuron differentiation in HG-exposed forebrain organoids
First, entire forebrain organoids were examined for signs of cell proliferation and apoptosis in neural rosettes. KI67 expression was used to find changes in proliferation, whereas TUNEL was used to detect apoptosis in this study. Compared with control group, the number of TUNEL+ cells increased, and there were no significant changes in the number of KI67+ cells in the HG-treated group, whereas the number of KI67+ cells increased in the FA-treated group. However, after HG + FA administration, the number of KI67+ cells remained unchanged, and the number of TUNEL+ cells decreased compared to the HG-treated group (Figure 2a,b). Meanwhile, the results of qPCR were consistent with fluorescence staining (Figure S1). To further explore the target cell types for HG exposure in forebrain organoids, SOX2 and TUNEL were co-immunostained in forebrain organoids. The TUNEL staining revealed that there were more apoptotic cells in the cortical plate (CP) areas of organoids in the HG group, whereas after HG + FA administration, the number of TUNEL+ cells in the CP areas decreased to a similar level compared to the control group (Figure 2c). These results indicated that FA reduced HG-induced neuronal apoptosis and that FA can promote NPC proliferation. In addition, the thickness of the ventricular zone (VZ)/subventricular zone (SVZ) of neural rosettes was significantly decreased in the HG exposure group; however, HG + FA administration significantly increased the thickness of the VZ/SVZ (Figure 2d). Furthermore, the entire forebrain was examined for signs of neuron differentiation. Compared with the control group, the mean fluorescence intensity of TUJ1 increased in both HG- and FA-treated groups (Figure S2), which indicated both HG and FA could promote neuron differentiation in forebrain organoids. Meanwhile, the number of SOX2+ cells was unchanged in all groups (Figure S2).
3.3 The proteome profiles in forebrain organoids of HG and FA treatments
To explore the hazardous effects of HG exposure on forebrain organoids as well as the protective effect of FA, proteomics and bioinformatics analyses were conducted (Figure 3a). When the FC is higher than 1.5 or lower than .667, p-values <.05 were used to identify DEPs. The volcano plots showed that 35 mM HG significantly upregulated the expression of 303 proteins and downregulated the expression of 107 proteins, and 40 μM FA significantly upregulated the expression of 427 proteins and downregulated the expression of 369 proteins, compared to the control group. In contrast, HG + FA treatment significantly upregulated the expression of 300 proteins and downregulated the expression of 203 proteins, compared with the HG group (Figure 3b–d and Figure S3). Heat map plots showed the top 15 up/downregulated DEPs identified in HG-treated groups (Figure S3). A total of 57 DEPs were identified, of which 13 were upregulated, and 6 were downregulated simultaneously in the HG and HG + FA treatment groups, along with 21 that were upregulated after HG exposure and then downregulated with FA treatment and 17 that were first downregulated after HG administration and then upregulated after FA treatment (Figure 3e,f). To validate the accuracy of proteome data, the expression of the key DEPs was evaluated by qPCR. The mRNA levels of GPC6, BCL2L11, IRS2, and MT1F were consistent with the proteomic results (Figure 3g).
3.4 The effects of FA on cell migration in HG-exposed forebrain organoids
Then, GO analysis was performed to further explore molecular mechanisms of the FA protective effect on HG-induced forebrain organoids. Functional enrichment analysis of DEPs revealed that the main changed terms included “regulation of apoptosis progress,” “cell proliferation,” “cell migration,” and “cellular response to cadmium ion” under this model in HG-treated group versus control group and HG + FA-treated group versus HG-treated group (Figure 4a,b). The effects of HG on the apoptosis and proliferation of forebrain organoids, as well as the protective effect of FA, have been performed in previous sections.
The heat map showed that a major group of DEPs belonged to the migration family, for example, INSR, IRS2, IGF1R, and MMP3, upregulated in HG-treated group but downregulated in HG + FA-treated group (Figure 4c). The protein abundance of MMP3, a matrix metalloproteinase (MMP), increased in the HG-treated group compared with the control group, whereas it decreased in the HG + FA-treated groups compared with the HG-treated group. In addition, the mRNA level of MMP3 was consistent with the proteomic results (Figure S5). To further verify the effects of FA on cell migration in HG-exposed human forebrain organoids, adherent neurospheres from forebrain organoids were treated with HG and HG + FA for 3 days and analyzed with TUJ1 staining. More neurons migrated from the HG exposure neurospheres than the control neurospheres, whereas after being treated with HG + FA, the migration of neurons returned to the level of the control neurospheres (Figure 4d). Quantitatively, the HG exposure groups showed a significant increase in the migration distance from the center of neurospheres. However, after HG + FA administration, the distance of cell migration was notably decreased (Figure 4e). Then, TUJ1 labeling and counting were used to analyze cell migration in order to verify the changed migration. Consistently, the migrated TUJ1+ cells were statistically increased in the HG exposure groups compared with the control group, whereas it decreased in the HG + FA exposure groups compared with the HG exposure group (Figure 4f,g). These findings suggest that FA can reverse the cortical neuron migration disorder caused by HG in forebrain organoid models.
3.5 Effects of FA on mineral absorption and ion homeostasis in HG-exposed forebrain organoids
According to the GO enrichment analyses, HG exposure disrupted the cellular responses to cadmium, copper, zinc, and ion homeostasis (Figure 5a). The heat map showed that a major group of DEPs belonged to the metallothionein (MT) family, for example, the proteins, including MT1F, MT3, SUMO1, and SLC39A4, were upregulated in HG-treated group, whereas they were downregulated in HG + FA-treated group (Figure 5b). To validate the accuracy of proteome data, the expression of the key DEPs was evaluated by qPCR. The mRNA levels of MT1F, MT2A, ATP1A3, and SLC39A4 were significantly upregulated after HG treatment but downregulated after HG + FA treatment compared to HG-treated group (Figure 5c). It was reported that astrocytes could maintain ion homeostasis to reduce neuronal injury in HG environments (Kelleher et al., 1993), and MT-I/II were particularly localized to astrocytes, which had neuroprotective effects (Aschner, 1997). Therefore, UC1MT was stained by immunofluorescence staining, and GFAP expression was evaluated by qPCR and Western blotting in forebrain organoids. After treated with HG, Western blotting and qPCR for GFAP revealed upregulation (Figure 5d–f), and immunostaining for UC1MT showed a significant rise in forebrain organoids (Figure 5g and Figure S4). In conclusion, these findings indicated that the HG environment stimulated the activation of astrocytes to produce MTs and indirectly protected the forebrain organoids, and FA supplementation can reverse this effect.
3.6 The effects of FA on AMPK/FOXO pathway in HG-exposed forebrain organoids
The proteomic analysis indicated that the pathways were associated with the “AMPK signaling pathway” and the “FOXO signaling pathway” in KEGG pathway enrichment analysis in HG-treated group versus the control group and HG + FA-treated group versus the HG-treated group (Figure S6, Figure 6a,b). Meanwhile, the protein level of FOXO3a, a critical molecule in the FOXO signaling pathway, and its upstream phosphorylation of AMPK at the Thr172 location were verified. In Figure 6c,d, the ratio of P-AMPK/AMPK and the level of FOXO3a were remarkably increased after treatment with HG, but the ratio of P-AMPK/AMPK and the level of FOXO3a were recovered in the HG + FA-treated group. These findings support the results of the KEGG bioinformatics analysis, which suggested that FA mitigated HG-induced cortical developmental damage via the AMPK/FOXO pathway.
Next, the protein–protein interaction analysis of the core proteins associated with the AMPK signaling pathway, FOXO signaling pathway, apoptosis, and cell migration was performed by STRING. The overwhelming majority of protein interactions within the network were either substantiated by curated databases or established through empirical investigation. These DEPs have been found to interact with each other, which illustrated a close connection among AMPK signaling pathway, FOXO signaling pathway, apoptosis, and cell migration underlying HG and FA treatment (Figure 6e,f).
4 DISCUSSION
As a significant public health issue, PGDM has been drawing attention to its detrimental impact on the nervous system. However, insufficient data exist to elucidate the underlying mechanisms responsible for the heightened risks associated with PGDM, and limited research has been conducted to evaluate prospective neuroprotective therapies aimed at mitigating these adverse neurodevelopmental consequences caused by PGDM (Bonnier, 2008). Herein, the forebrain organoid model was utilized to examine the impact of HG exposure on the development of the cortex in this study. Exposure to HG was found to increase the occurrence of apoptosis in forebrain organoids. Meanwhile, neurons exhibited greater susceptibility to apoptosis compared to NPCs. Then proteomics analysis was applied to identify potential alterations in protein expression levels, and then the cellular mechanism of HG-induced neurotoxicity was confirmed. The findings from bioinformatics analyses and immunofluorescence staining indicated that the changes of proteins induced by HG are linked not only to mineral absorption but also to cell migration. Importantly, we have discovered the impaired AMPK/FOXO signaling as a potential mechanism for the HG-induced neurotoxicity in forebrain organoids. Subsequently, we provide a novel solution, a forebrain organoid model, to mitigate HG-induced neurotoxicity and report that FA can reverse these changes caused by HG and ultimately reduce the neurodevelopmental abnormalities. As far as we know, this is the first investigation to mimic the developmental neurotoxicity induced by PGDM in forebrain organoids as well as evaluate the protective impact of FA.
In this study, we adopted the original protocol generated by Gabriel et al. (2023), which is mainly based on the scRNA-seq data. On Day 20, the forebrain organoids with primitive eye fields were formed. On Day 40, the organoids demonstrate a diverse array of cellular phenotypes, and this particular time point, Day 40, marks a significant milestone in the progression of CP development (Gabriel et al., 2023). So, Days 20–40 were chosen as the exposure time. The hESC line was characterized through the utilization of immunofluorescence staining targeting the stem cell markers, NESTIN and SOX2, as well as the proliferative markers, KI67 and PH3 (Figure 1b). Immunofluorescence staining with multiple markers identified the characteristics of cerebral organoids. Moreover, immunostaining with multiple markers in the organoids on Day 40 indicated the successful establishment of the forebrain organoid model (Figure 1c), which could be effectively employed to investigate the HG-induced developmental neurotoxicity and to evaluate the potential protective effects of FA.
Morphological alteration serves as a visual indicator employed in the evaluation of neurotoxicity on brain development (Yang et al., 2023). In this study, the thickness of VZ/SVZ in neural rosettes was shown to decrease in the HG exposure groups, and FA supplementation can increase the thickness (Figure 2e). Typically, these abnormal structures observed in the forebrain organoids are associated with neurogenesis, cell apoptosis, and cell proliferation. It was reported that the excessive induction of apoptosis by HG has the potential to impact the development of the central nervous system (CNS) (Jiang et al., 2008), which may be associated with the oxidative stress imbalance. However, there is a lack of comprehensive reports and detailed analyses on the susceptibility of various neural cells to apoptosis in response to HG. In the present investigation, it was observed that exposure to HG resulted in apoptosis specifically in neurons, not in NPCs, and FA supplementation can decrease the apoptosis (Figure 2d). Moreover, FA can improve the proliferation of NPCs, which is not affected in the HG exposure groups (Figure 2a). The dimension and structure of the CNS are predominantly influenced by the frequency at which NPCs reenter the cell cycles (Ohnuma & Harris, 2003). According to previous reports, there is evidence suggesting that FA facilitates the proliferation of NPCs through the activation of ERK1/2 and notch signaling pathways (Zhang et al., 2009). All of these findings indicate that FA promotes the proliferation process of NPCs and reduces neuron apoptosis, which could potentially stabilize the dimension and structure of forebrain organoids that are impaired in HG exposure groups.
To explore the dangerous effects of HG exposure on forebrain organoids as well as the protective effect of FA, proteomics and bioinformatics analyses were conducted to examine proteome-wide alterations. A total of 410 proteins were significantly altered in HG-treated groups, and 503 were altered after FA administration compared to HG-treated groups. The results of the GO enrichment analysis indicated that DEGs were predominantly associated with processes, such as apoptosis, cell migration, and ion homeostasis.
Cell migration is of utmost importance in the establishment and preservation of tissue integrity and serves a crucial function in various biological phenomena, such as inflammation, wound healing, embryonic development, and invasiveness of the extracellular matrix (ECM) (Marchant et al., 2022). Neural migration takes place between 10 and 20 weeks of pregnancy and continues throughout embryonic development (Francis & Cappello, 2021). MMPs are a group of proteolytic enzymes that facilitate the remodeling of the pericellular environment through the modulation of ECM proteins, adhesion molecules, receptors, and cytoskeletal proteins (Nagase et al., 2006). In specific, MMP-3 has been associated with cell migration, synaptic plasticity, neuronal development, and learning processes depending on the hippocampus. In focal ischemic brain injury, like stroke, the involvement of MMP-3 in astrocyte migration may play a role in brain remodeling processes, such as scar formation, which subsequently impair neural regeneration (Aerts et al., 2015). Moreover, upregulated levels and enhanced activity of MMP-3 have been observed to facilitate neuroinflammation and apoptosis, two critical processes that significantly contribute to the progression of neurodegeneration (Kim & Hwang, 2011), and it is reported that Tbr1 has been found to have a significant association with neurodegenerative disorders, such as AD and Parkinson's disease, through its involvement in neuronal migration and the development of axon tracts (Huang et al., 2014). In short, current findings provide confirmation that cell migration caused by HG and mediated by MMP-3 may play a role in brain remodeling, ultimately leading to PGDM-induced fetus CNS complications, and FA can reverse this change. However, the exact molecular mechanism remains to be elucidated in the future.
The process of mineral absorption encompasses the participation of proteins such as MTs and SUMO1, which are implicated in the reactive responses to oxidative damage generated by HG exposure. MT is a protein that is commonly activated in response to various stimulants, such as endotoxins, glucocorticoids, and metal ions. This responsiveness is attributed to the promoter regions of MT genes, which contain metal-responsive elements and glucocorticoid-responsive elements (Coyle et al., 2002). Current research showed that HG induced the activation of astrocytes and an increase in MT protein levels, which were good buffer substances to have neuroprotective effects from HG exposure. In addition, FA supplements can reverse this effect. Consistent with previous research, diabetes induces the increase of GFAP-positive astrocytes in the hippocampus and cerebellum, which may take part in MT induction and metal homeostasis (Beltramini et al., 2006). Furthermore, it is noteworthy to remark that the upregulation of MT induces the activation of anti-inflammatory cytokines IL-10 and neurotrophic, and growth factors expressed by astrocytes (Penkowa et al., 2005).
Western blot was employed to elucidate the mechanism of the protective impact of FA on HG-induced developmental neurotoxicity. HG may affect neurodevelopmental processes through the AMPK (AMP-activated protein kinase) signaling pathway and FOXO (Forkhead box O) signaling pathway, as they were the main pathways affected by HG exposure from proteomics analysis. The Western blot findings were in agreement with the proteomics analysis, indicating that phosphorylated AMPK and FOXO increased after HG exposure and decreased after HG and FA administration. AMPK serves as a cellular energy status sensor, effectively controlling the expression of FOXO gene families through phosphorylation (Greer et al., 2007). Phosphorylation AMPK can stimulate the transcriptional activity of FOXO, through which nuclear localization can be maintained and downstream biological effects can be activated. The expression of FOXO transcription factors was observed in several regions of the developing brain, suggesting their potential significance in neurodevelopmental processes as well as apoptosis, cell cycle arrest, and the oxidative stress response (Luo et al., 2007). It is worth mentioning that the insulin/IGF-1-PI3K signaling pathway regulates the function of the DAF-16/FOXO transcription factor, facilitating the anterior migrations of the hermaphrodite-specific neurons in the embryonic stage of Caenorhabditis elegans, which means FOXO plays an important role in the control of neuronal migration in vertebrates (Kennedy et al., 2013). Next, the STRING analysis revealed a strong correlation among the proteins related to apoptosis, migration, AMPK signaling pathway, and FOXO signaling pathway. In short, current data indicate that FA might exhibit a protective effect through AMPK/FOXO signaling pathway against HG-induced neurotoxicity in forebrain organoids.
5 CONCLUSION
In this investigation, current findings suggest that prenatal exposure to HG is associated with apoptosis of neurons, activation of mineral absorption, and promotion of cell migration, potentially mediated by the AMPK/FOXO pathway in forebrain organoids. FA exhibits considerable potential as a fortified food ingredient due to its ability to protect against HG-induced neurotoxicity. Collectively, current results offer a novel and deeper understanding of the molecular mechanisms of maternal exposure to HG, including molecular, cellular, and structural abnormalities that can be observed during the crucial developmental phase of forebrain organoids. Furthermore, the utilization of the forebrain organoid model presents a promising platform for assessing the neurodevelopmental abnormalities induced by PGDM and other pregestational diseases.
AUTHOR CONTRIBUTIONS
Haoni Yan: Conceptualization; writing—original draft; methodology; software; formal analysis and data curation. Shujin Chen: Formal analysis; methodology; software; writing—review and editing. Aynur Abdulla: Formal analysis; software; writing—review and editing. Zhile Li: Software; writing—review and editing. Tsz Yui Zhuang, Manlin Zhang, Leqi Wu, and Yizhi Zhang: Investigation and validation. Xianting Ding and Lai Jiang: Supervision; writing—review and editing.
ACKNOWLEDGMENTS
We gratefully thank the financial support from the National Natural Science Foundation of China (T2122002, 22077079, 82272229), Shanghai Jiao Tong University Projects (YG2019ZDA20, YG2021ZD19), Shanghai Healthcare System Key Supporting Discipline Construction Project (2023ZDFC0202), Shanghai Municipal Science and Technology Projects (23YF1425600, 22Z510202478), and the National Key R&D Program of China (2022YFC2601700, 2022YFF0710202, and 2022YFA1104200). Thanks to AEMD SJTU, Shanghai Jiao Tong University Laboratory Animal Center, for the technical support.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT
All data needed to evaluate the conclusions in this paper are presented in the paper or in the Supporting Information section.
