Demyelination and impaired oligodendrogenesis in the corpus callosum following lead exposure

Abstract

The corpus callosum is an oligodendrocyte-enriched brain region, replenished by newborn oligodendrocytes from oligodendrocyte progenitor cells (OPCs) in subventricular zone (SVZ). Lead (Pb) exposure has been associated with multiple sclerosis, a disease characterized by the loss of oligodendrocytes. This study aimed to investigate the effects of Pb exposure on oligodendrogenesis in SVZ and myelination in the corpus callosum. Adult female mice were used for a disproportionately higher prevalence of multiple sclerosis in females. Acute Pb exposure (one ip-injection of 27 mg Pb/kg as PbAc₂ 24 hr before sampling) caused mild Pb accumulation in the corpus callosum. Ex vivo assay using isolated SVZ tissues collected from acute Pb-exposed brains showed a diminished oligodendrogenesis in SVZ-derived neurospheres compared with controls. In vivo subchronic Pb exposure (13.5 mg Pb/kg by daily oral gavage 4 wk) revealed significantly decreased newborn BrdU⁺/MBP⁺ oligodendrocytes in the corpus callosum, suggesting demyelination. Mechanistic investigations indicated decreased Rictor in SVZ OPCs, defective self-defense pathways, and reactive gliosis in the corpus callosum. Given the interwined pathologies between multiple sclerosis and Alzheimer’s disease, the effect of Pb on myelination was evaluated in AD-modeled APP/PS1 mice. Myelin MRI on mice following chronic exposure (1,000 ppm Pb in drinking water as PbAc₂ for 20 wk) revealed a profound demyelination in the corpus callosum compared with controls. Immunostaining of the choroid plexus showed diminished signaling molecule (Klotho, OTX2) expressions in Pb-treated animals. These observations suggest that Pb caused demyelination in the corpus callosum, likely by disrupting oligodendrogenesis from SVZ OPCs. Pb-induced demyelination represents a crucial pathogenic pathway in Pb neurotoxicity, including multiple sclerosis.

Multiple sclerosis is a neurological disorder characterized primarily with demyelination caused by the loss of oligodendrocytes—the cell type critical to the insulation of neuronal axons with a myelin sheath (Frischer et al. 2009). Oligodendrocytes are particularly abundant in the corpus callosum (located underneath the longitudinal fissure and cerebral cortex), which is vital to nerve signal transmission. In the United States, approximately one million individuals are affected by multiple sclerosis (Wallin et al. 2019); the patients experience symptoms such as blurred vision, muscle weakness, and numbness that significantly impact life quality (Ysrraelit et al. 2017). Although genetic factors like HLA-DRB1 mutations are linked to familial multiple sclerosis (Patsopoulos 2018), the majority of cases are sporadic (Kahana 2000), highlighting the potential influence of environmental risk factors such as exposure to toxic substances including lead (Pb).

Studies in literature have indeed established a strong association between Pb exposure and multiple sclerosis through case–control studies (Cone 1934; Napier et al. 2016; Dehghanifiroozabadi et al. 2019), twin cohort occupational analysis (Juntunen et al. 1989), and an ecological study (Tsai and Lee 2013). Since Pb is an indispensable metal in modern industry, its persistent presence in the environment due to mining, refining, and industrial application remains a major global public health challenge, notably in less industrialized nations. The risk is magnified by the prolonged biological half-life of Pb in humans (Rabinowitz 1991; Collin et al. 2022). In addition, Pb is a known risk factor for many types of neurodegenerative diseases, particularly Alzheimer’s disease (AD) (Bakulski et al. 2012). Given the overlapping symptoms and likely contributions of demyelination in neurodegenerative alterations (Depp et al. 2023), it becomes indispensable to investigate whether Pb exposure causes multiple sclerosis-like pathologies and explore the underlying mechanisms.

The corpus callosum in the brain functions to facilitate connectivity between the brain’s hemispheres; it houses a dense population of oligodendrocytes that are essential for maintaining this interhemispheric communication. In adult brain, the oligodendrocyte population in the corpus callosum continues to be replenished with newborn oligodendrocytes derived from oligodendrocyte progenitor cells (OPCs) in the subventricular zone (SVZ) (Menn et al. 2006; Delgado et al. 2021); a dysfunctional SVZ has been linked to demyelinating alterations in the corpus callosum (Tepavčević et al. 2011; Butti et al. 2019). Literature data has suggested a detrimental effect of Pb on parenchymal OPCs (Deng et al. 2001; Ma et al. 2015; Bakulski et al. 2020); yet, it is unclear if exposure to Pb may impair SVZ OPCs and the resultant myelination in the corpus callosum.

The proliferation, migration, and differentiation of OPCs are regulated by both intrinsic programs and extrinsic signals (Bergles and Richardson 2016; Cayre et al. 2021; Nishiyama et al. 2021). For example, Rictor, a subunit of the mammalian target of rapamycin complex 2 (mTORC2) that regulates cell proliferation and survival, was a known regulatory protein for oligodendrogenesis and myelination in the corpus callosum (Dahl et al. 2022). Additionally, OPCs are influenced by signals from other cells and tissues, such as FGF-17 secreted by a subset of cortical neurons to the cerebrospinal fluid (CSF) to promote the proliferation and differentiation of hippocampal OPCs (Iram et al. 2022). Given the proximity of SVZ OPCs to the CSF, it is plausible that CSF-associated cells and tissues, such as the choroid plexus (CP), may modulate OPCs through secreted cues. The CP, as a barrier at the interface of blood and CSF, is known to sequester large amounts of Pb from blood circulation upon exposure (Zheng et al. 1991). Since the CP naturally contains antioxidant molecules (Saudrais et al. 2018) and secretes signaling proteins to support neurodevelopment (Lun et al. 2015; Silva-Vargas et al. 2016; Planques et al. 2019), it became necessary to investigate the mechanisms by which Pb affects SVZ OPCs through Pb interference of choroidal function.

This study aimed to define the effects of Pb exposure on myelination in the corpus callosum as well as the underlying molecular mechanisms. Data from this study will likely provide novel evidence regarding the Pb toxicity toward the oligodendrocyte lineage in the brain.

Materials and methods

Materials

Chemical reagents utilized in this study were acquired from the following suppliers: Lead acetate trihydrate (PbAc₂, molecular weight: 379.33 g/mol, purity >99.99%), sodium acetate trihydrate (NaAc, molecular weight: 136.08 g/mol, purity >99%), 5-bromo-2′-deoxyuridine (Bromodeoxyuridine, BrdU), heparin sodium, and paraformaldehyde (PFA) from Sigma Aldrich (St Louis, MO); Nunclo Sphera 96-well U-shaped-bottom microplate, neurobasal plus medium, B-27 plus supplement, GlutaMAX supplement, gentamicin (50 mg/ml), trypsin-EDTA (0.05%), defined trypsin inhibitor, DNAse I, epidermal growth factor (EGF), fibroblast growth factors (FGF), Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L), Alexa Fluor 568-conjugated goat anti-chicken IgY (H + L), Cy5-conjugated goat anti-rat IgG (H + L) from Thermo Scientific (Waltham, MA); normal goat serum (NGS) from Jackson ImmunoResearch (West Grove, PA); 24-well plate with #1.5 glass-like polymer coverslip bottom from Cellvis (Mountain View, CA); Cultrex ready-to-use poly-L-ornithine (PLO) solution from R&D Systems (Minneapolis, MN); Triton X-100 from Bio-Rad (Hercules, CA); and permeable Transwell insert 3470 from Corning (Glendale, AZ). Detailed information regarding primary antibodies and their dilutions can be found in Table S1. All reagents were procured at the highest available quality standards, being either analytical grade, HPLC grade, or of the highest pharmaceutical grade.

Animals

Three-month-old female C57BL/6 mice were selected for this gender’s higher predisposition in developing multiple sclerosis, a condition approximately three times more prevalent in females than males (Harbo et al. 2013). Mice were procured from Envigo Inc. (Indianapolis, IN). Upon arrival, the mice were settled in a regulated environment with a consistent 12-hr light/dark cycle to acclimatize for 1 wk before any experimental procedures commenced. They had continuous access to distilled-deionized water and a semipurified diet from Purina Mills TestDiet (Richmond, IN). Similarly, female APP/PS1 mice, obtained from Jackson Laboratory (Stock No. 005864, Bar Harbor, Maine), were housed under identical conditions, with the exception being their chronic exposure to Pb via the drinking water, as outlined in the study design. All experimental protocols were performed in accordance with established ethical guidelines for animal care and were duly authorized by the Purdue University Animal Care and Use Committee (PACUC No. 1112000526).

Experimental design

The primary objective of this study was to understand the toxic effects and mechanisms associated with Pb-induced alterations in oligodendrocyte/myelination within the corpus callosum. To achieve this goal, we employed a range of experimental approaches following acute, subchronic, or chronic exposure of animals to Pb. These approaches included ex vivo neurosphere assays, newborn oligodendrocyte tracing, myelination evaluation by immunostaining and MRI, mechanistic immunohistochemical staining, and utilizing a CP-neurosphere co-culture system, each tailored for specific research goals.

For procedures conducted exclusively under one type of Pb exposure, we have provided detailed descriptions in the respective subsections described below. Common techniques and procedures used, such as atomic absorption spectrometry (AAS) and immunostaining, have been described in detail in the sections titled “Atomic absorption spectrometry” through “Data analysis”.

Experiment 1: assessing the impact of acute Pb exposure on oligodendrocyte differentiation in vitro

In this experiment, we adopted an ex vivo approach to investigate whether acute Pb exposure disrupted the process of oligodendrogenesis. Our objective was to characterize oligodendrocyte differentiation using neurospheres cultured from the SVZ tissues of animals acutely exposed to Pb, as illustrated in Fig. 1a.

Fig. 1.

Experimental designs to study the effects of Pb exposure on myelination and oligodendrogenesis in adult brains. The workflows describe key timepoints for critical procedures and data acquisition. (a) Ex vivo experiments to probe the effects of in vivo acute Pb exposure on oligodendrogenesis in vitro using SVZ-derived neurosphere model. Briefly, mice received one i.p. injection of Pb at 27 mg Pb/kg b.w. as PbAc₂ and were sacrificed 24 hr later for SVZ sampling and neurosphere formation. Following neurosphere differentiation, immunostaining was performed to evaluate oligodendrogenesis as affected by the in vivo Pb exposure or the presence of choroid plexus. In this experiment, the whole blood and brain regional tissues were also collected for Pb measurements by AAS. (b) In vivo subchronic Pb exposure to evaluate the myelination and oligodendrogenesis in the corpus callosum and cortex and to study the molecular mechanisms. To label newborn oligodendrocytes, BrdU was i.p. administered daily for 3 d (50 mg/kg b.w. with an interval of 24 hr) prior to Pb treatment. For Pb exposure, two groups of mice, i.e. controls and Pb-exposed ones (n = 4/each group), received NaAc and 13.5 mg Pb/kg b.w. as PbAc₂, respectively, through oral gavage 5 d/wk for 4 wk. Animals were sacrificed on day 28 for whole blood and brain sampling. The brains were dissected with caution to preserve leptomeninges for microtome slicing and immunostaining. (c) In vivo chronic Pb exposure for quantitative myelin MRI in neurodegenerative APP/PS1 AD brains. APP/PS1 mice at 8 wk old were exposed to Pb by drinking water containing 1,000 ppm Pb for 20 wk (controls with drinking water containing NaAc at equivalent molar concentration). Using ultrashort echo time (UTE) and 3D Rosette k-Space pattern in MRI, myelin content was quantitatively mapped by extracting the ultrashort T₂ signals by dual-exponential complex model fitting. Myelination was evaluated in both the corpus callosum and the cortical regions as affected by chronic Pb exposure. (d) In vitro follow-up experiments to investigate the role of choroid plexus in Pb-inhibited oligodendrogenesis observed in (a). Briefly, neurospheres (lower chamber) formed from acutely exposed mouse SVZ tissues were co-cultured with choroid plexus tissues (upper chamber) from nonexposed mice; the controls in this experiment had no choroid plexus tissues in the upper chamber. Differentiation outcomes were evaluated by immunostaining to elucidate the influence of choroid plexus in oligodendrogenesis.

To achieve this, adult C57BL/6 mice were administered a single intraperitoneal (i.p.) injection of Pb (27 mg Pb/kg b.w. as PbAc₂) as previously described (Gu et al. 2011) to model Pb exposure level by elevating the whole-blood Pb concentrations to the level reported among the occupational populations (Lee 1982; Shah et al. 2012). Saline injections served as controls. After a 24-hr period, a microdissection of the brain SVZ tissues was performed for neurosphere assays as previously described in this lab (Liu et al. 2022). Briefly, under deep anesthesia induced by an i.p. injection of ketamine (75 mg/kg b.w.) and xylazine (10 mg/kg b.w.), SVZ tissues from mice perfused with PBS were isolated. The isolated SVZ was mechanically dissociated and then digested in 2 ml of prewarmed 0.05% trypsin-EDTA in a 37 °C water bath for 7 min. Trypsin inhibitor was used to halt the trypsin dissociation process, and the cell suspension was centrifuged at 300×g for 5 min. After removing the supernatant, the cell pellet was gently resuspended in 1 ml of neurobasal background medium (Neurobasal plus medium supplemented with B-27 Plus, 0.5 ml GlutaMAX, and Gentamicin at 50 µg/ml; referred to as Medium-1). An additional 4 ml of Medium-1 was then added, and the suspension was filtered through a 40-μm sterile cell strainer to remove undissociated tissue chunks. The strained suspension was centrifuged at 300×g for 5 min, followed by resuspension in 12 ml of proliferative Medium-2 (Medium-1 supplemented with heparin at 2 µg/ml, EGF at 20 ng/ml, and FGF at 20 ng/ml). The 12-ml suspension was seeded in a 12-well culture dish (1 ml per well), and the culture was maintained at 37 °C with 5% CO₂ for 6 d. Early neurosphere formation on day 5 and primary neurospheres ready for passage on day 8 were observed under a light microscope (Fig. 1a).

On day 8, primary neurospheres derived from SVZ tissues of both control and Pb-treated mice were dissociated into single cells by incubation with trypsin-EDTA at 37 °C for 2 min. These single cells were then resuspended in proliferative Medium-2, and trypan blue staining was performed for counting viable cells. The single-cell suspension was reseeded at the density of 2,500 cells/well in a Nunclo Sphera 96-well U-shaped-bottom microplate. Secondary neurospheres were formed on day 10, and their diameters were recorded under the light microscope.

The secondary neurospheres from both control and Pb-treated mice were collected, resuspended in Medium-1, and plated on a PLO-coated Cellvis 24-well plate. These neurospheres were allowed to attach, grow, migrate, and differentiate for 7 d, with fresh Medium-1 supplementation on day 13. On day 17, the neurospheres were fixed with 4% PFA in PBS for 10 min and stored in PBS at 4 °C for subsequent immunocytochemistry staining, which was completed within 1 wk.

In a parallel study using another set of animals and following the same acute Pb dosing procedure, we collected whole blood samples and microdissected specific brain regions of interest (CP, meninges, SVZ, corpus callosum, and hippocampus) following transcardial PBS perfusion. These samples were subjected to Pb concentration measurements using AAS.

Experiment 2: assessing the impact of subchronic Pb exposure on myelination and newborn oligodendrocytes in the corpus callosum

OPCs in the SVZ contribute newborn oligodendrocyte to the corpus callosum (Menn et al. 2006). To trace these newborn oligodendrocyte, adult C57BL/6 mice were administered three daily doses of BrdU (50 mg/kg b.w.) by i.p. injections prior to Pb treatment, with a 24-hr interval between doses (Fig. 1b). The mice were then subsequently exposed to Pb at a dose of 13.5 mg Pb/kg b.w. (as PbAc₂) by oral gavage, 5 d/wk, for 28 d. Controls received an equivalent amount of NaAc. At the conclusion of the subchronic Pb exposure, deeply anesthetized animals underwent whole blood collection, PBS perfusion, and 4% PFA perfusion.

It is noteworthy that the extraction of brains was conducted in a manner consistent with a previous protocol (Louveau et al. 2018) to preserve the leptomeninges covering the parenchyma for subsequent immunohistochemistry staining. To quantify newborn oligodendrocytes in the corpus callosum and cortex, microtome-cut brain slices were stained with primary antibodies targeting myelin basic protein (MBP, an oligodendrocyte marker) and BrdU. This allowed us to identify and quantify MBP⁺/BrdU⁺ cells using confocal microscopy. Additionally, immunohistochemistry staining of MBP was employed to assess myelination by quantifying MBP signal intensities. Detailed procedures for brain slice preparation and immunostaining can be found in the sections titled “Brain slice preparation” and “Immunocytochemistry, immunohistochemistry, and confocal microscopy”, respectively.

Noticeably, brain slices collected from this subchronic Pb exposure experiment served as the basis for the following investigations through immunohistochemistry staining. (i) Rictor and Sp1 characterization: Two critical signaling molecules involved in OPC differentiation to oligodendrocytes for myelination (Deng et al. 2001; Grier et al. 2017; Dahl et al. 2022) were characterized in SVZ OPCs following Pb exposure. (ii) Copper Chaperone for Superoxide Dismutase (SOD4) and Metallothionein-3 (MT3): These two representative self-defense proteins against chemical toxicities (Coyle et al. 2002; Johnson and Giulivi 2005) were characterized in SVZ OPCs, CP, and meninges. (iii) Reactive astrogliosis and microgliosis: These two biological processes, known to have detrimental effects on oligodendrogenesis (Kwon and Koh 2020), were studied by quantifying astroglial and microglial density with specific phenotypes (resting/reactive) in the corpus callosum. (iv) Klotho and OTX2 expression: Signaling molecules naturally enriched in the CP to modulate SVZ activities (Zhu et al. 2018; Planques et al. 2019) were probed for their expressions as affected by Pb exposure. These planned immunohistochemistry analyses were designed to provide mechanistic insights into the alterations observed in the corpus callosum following Pb exposure.

Experiment 3: MRI of myelin in APP/PS1 mice following chronic Pb exposure

In this experiment, we employed APP/PS1 mice, a model for early-onset Alzheimer’s disease (AD) (Jankowsky et al. 2004), to investigate the effects of Pb exposure on myelination in a neurodegenerative context of AD (Fig. 1c). Starting at 8 wk of age, APP/PS1 mice were subjected to a chronic exposure regimen, receiving 1,000 ppm Pb in drinking water for a duration of 20 wk. Control APP/PS1 mice received the drinking water containing an equivalent molar concentration of NaAc. At the end of 20-wk exposure, live mice were examined by whole-brain MRI to evaluate brain myelination alterations with or without Pb treatment. All MRI experiments were conducted at the Small Animal MRI Facility within the Bindley Bioscience Center of Purdue University.

The MRI procedure involved the following steps. Under isoflurane anesthesia with continuous vital sign monitoring, mice were positioned for scanning in a 7-tesla horizontal-bore small animal MRI system (BioSpec 70/30, Bruker, Billerica, MA). This system was equipped with a gradient insert (maximum gradient: 660 mT/m; maximum slew rate: 4,570 T/m/s) and a volume transmit/receive 1H RF head coil. We employed multiparametric mapping (MPM) sequences constructed on a multi-gradient echo sequence with radiofrequency (RF) spoiling at 117° (Weiskopf et al. 2013). Echoes were collected starting from 2.41 ms with a spacing of 2.1 ms. Three contrast-generating parameters were weighted: Magnetization transfer (MT), proton density (PD), and T1 (longitudinal relaxation time).

The following settings were applied for these parameters: TR (repetition time)=25/25/18 ms, FA (flip angle)=6°/6°/40°, and the number of echoes = 6/8/6. The matrix size was 256 × 160 × 128 mm³ with a field of view of 30 × 22 × 30 mm³, resulting in an isotropic spatial resolution of 109 x 175 x 175 µm³. A single 4-ms (BW 685 Hz) Gaussian preparation pulse with a 2k Hz off-resonance at 10 µT was applied for MT-based images. To accelerate imaging, zero-filling was performed in the phase encoding directions with an acceleration factor of 1.6. The total scan time for the MPM sequences was approximately 17.76 min. Subsequently, T1 and MT saturation (MTsat) signals were mapped for each animal.

All MRI images were preprocessed using the SPM12 software (University College London, London, United Kingdom) with the SPMMouse extension. The SPMMouse extension was employed to utilize a masking function for mouse gray matter (GM) and white matter (WM), allowing for the quantification of myelination in each scanned animal. Finally, MR signal intensities (myelin water fraction) originating from myelin were transformed into a brain heatmap, facilitating the assessment of myelination across different brain regions (Weiskopf et al. 2013). This comprehensive MRI analysis aimed to shed light on the effects of chronic Pb exposure on myelin content in the brains of APP/PS1 mice, potentially contributing to our understanding of the known Pb-aggravated AD phenotypes.

Experiment 4: investigating the contribution of the CP to Pb-induced aberrant oligodendrogenesis using a two-chamber CP-SVZ neurosphere co-culture system

To explore how the CP contributed to the Pb-induced disruption of oligodendrogenesis, a two-chamber co-culture system with secondary neurospheres derived from SVZ tissues of mice acutely exposed to Pb (as described in the section titled “Experiment 1: assessing the impact of acute Pb exposure on oligodendrocyte differentiation in vitro”) was used. Briefly, after acute Pb exposure, the secondary neurospheres derived from SVZ tissues (Fig. 2f) were seeded onto a PLO-coated surface in the lower chamber. These neurospheres were allowed to settle down on the coated surface for 3 hr. Freshly dissected plexus tissues from two control mouse brains were collected, spun down, and resuspended in 0.6 ml of Medium-1. The plexus tissues were then transferred to an insert in the upper chamber of the co-culture system, where they were co-cultured with the neurospheres in the lower chamber for the initial 24 hr. The control group in this experiment had a culture medium only in the insert. After this 24-hr co-culture period, the insert with or without CP tissues was removed, and the neurospheres in the lower chamber continued to culture for additional 6 d. At the end of the culture, the neurospheres were fixed with 4% PFA, and immunocytochemistry was conducted to assess how the presence of CP tissues influenced migration, differentiation, and interactions between neurons and oligodendrocytes within the neurospheres. This innovative co-culture system allowed us to investigate the potential role of the CP in mediating the effects of Pb exposure on oligodendrogenesis, shedding light on the mechanisms underlying these alterations.

Fig. 2.

SVZ-specific enrichment of proliferative OPCs. (a) A detailed illustration of the SVZ to show the distribution of Ki67⁺/NG2⁺ proliferative OPCs. Five subregions along the lateral ventricle wall—roof, septal wall (SW), dorsal lateral corner (DLC), lateral wall (LW), and ventral corner (VC)—are magnified in panels A1 through A5, respectively. Key anatomical landmarks labeled are the corpus callosum (CC), septum (Sp), striatum (Str), and lateral ventricle (LV). (b) Quantification of Ki67⁺/NG2⁺ proliferative OPCs across LW, DLC, VC, SW, and the roof of the lateral ventricle. Data represent mean±SD; n = 3. (c) A hippocampal section illustrates the presence of NG2⁺ OPCs but no Ki67 signal. (d) A cortical section displays a Ki67⁺ cell without NG2 expression, suggesting a proliferative non-OPC.

Atomic absorption spectrometry

Following the completion of the treatment regimen, mice were deeply anesthetized to facilitate whole blood collection via the inferior vena cava. Subsequently, transcardial perfusions were conducted using ice-cold PBS to thoroughly remove blood from the brain. Once the brains were extracted, samples were obtained from specific brain regions, including the CP, meninges, SVZ, corpus callosum, and hippocampus. These samples were then subjected to volume or weight determination. Subsequent steps involved the digestion of both whole blood and brain tissues using ultrapure nitric acid within the MARSX press microwave-accelerated reaction system. After appropriate dilutions, the prepared samples were loaded onto the autosampler tray of the GTA 120 graphite tube atomizer (Agilent Technologies 200 Series, Santa Clara, CA) for the quantification of Pb concentrations. All diluted samples had Pb concentration reading within the linear range (0 to 20 μg/l) of this AAS method. Throughout the study, Pb standard curves generated using Agilent’s Pb standard solution were regularly plotted and monitored, consistently achieving an R² value exceeding 99.5%. This AAS assay has been used in this lab for the past 25 yr with a detection limit for Pb of 1.35 µg Pb/l (Zheng et al. 1996).

Brain slice preparation

Mice, under deep anesthesia, underwent a sequential transcardial perfusion procedure involving ice-cold PBS followed by 4% PFA in PBS. A specialized dissection method was employed to ensure the preservation of the leptomeninges covering the parenchyma, as previously described (Louveau et al. 2018; Remsik et al. 2021). This meticulous process involved several steps: First, the lower orbits of the jaw on both sides were carefully removed with a sharp-point stainless steel dissection scissors. Second, a clockwise cut was made beneath the post-tympanic hook to excise the lower portion of the skull. Following the removal of the nasal bone, a leptomeninges-preserved brain was carefully separated from the skull and subsequently fixed in 4% PFA overnight. Finally, following dehydration in a 30% sucrose solution for 72 hr, the brains were precision-cut into 40-µm slices using a microtome. To encompass the entire corpus callosum structure, coronal slices spanning approximately from +2.0 mm (anterior) to −4.0 mm (posterior) relative to the bregma were serially collected into a 12-well plate prefilled with cryopreservation medium (30% sucrose, 1% polyvinylpyrrolidone, 30% ethylene glycol in 0.1 M phosphate buffer) and stored at −20 °C. Subsequently, for staining and analysis, each well contained a total of 10 to 12 slices, allowing for the acquisition of specific parameters, such as cell density or protein expression levels, corresponding to the individual brains under investigation.

Immunocytochemistry, immunohistochemistry, and confocal microscopy

For immunostaining and confocal microscopic analysis, the protocol began with the rinsing of paraformaldehyde-fixed neurospheres or cryopreserved brain sections three times in PBS. The samples were then blocked in PBST containing 5% normal goat serum (NGS) and 0.3% Triton X-100 for one hr at room temperature. After blocking, samples were incubated with primary antibodies overnight at 4 °C (antibody details provided in Table S1). After three washes in PBST, samples labeled with primary antibodies were incubated with fluorophore-conjugated secondary antibodies (dilution 1:500) for one hr at room temperature in the dark. Following adequate washes, a DAPI counterstain was applied. Neurospheres were immediately subjected to microscopy, whereas brain slices were mounted on slides, sealed with a coverslip and mounting medium, and allowed to dry overnight before confocal scanning.

For developing BrdU signals, an additional DNA hydrolysis step preceded the NGS blocking: Slices were treated with 2 N HCl at 37 °C for 30 min and subsequently neutralized in a 0.1 M borate buffer (pH 8.5) for another 30 min, followed by thorough PBS washes. The workflow then resumed from serum blocking, as detailed above, to identify BrdU⁺/MBP⁺ newborn oligodendrocytes.

During data collection, a Nikon A1Rsi confocal microscope equipped with the ND acquisition software was employed for extensive image stitching and 3D reconstruction. In cell density assessments, newborn oligodendrocytes were marked by the co-localization of MBP in the cytoplasm and BrdU in the nucleus, verified through z axis manipulation. Cell density was calculated by dividing the total number of BrdU⁺/MBP⁺ cells by the examined area. Furthermore, for quantification of specific proteins like SOD4 or MT3, imaging was performed consistently across various regions of interest (ROIs) using unaltered microscopy settings. ImageJ software (version 1.52q, Bethesda, MD) was then utilized for the relative quantification of protein expression.

Data analysis

The data presented in this report were processed and analyzed using Prism 8 software by GraphPad (San Diego, CA). The results are expressed as the mean±SD. Unpaired t-tests were employed to evaluate the statistical significance between the control and the Pb-treated group. Sample sizes are specified within the results section or corresponding figure legends. A P-value of 0.05 or below was established as the threshold for statistical significance.

Results

Distinct proliferation of OPCs in the SVZ

OPCs are present throughout the brain but typically remain in quiescence (Dawson et al. 2003; Ximerakis et al. 2019). Contrary to parenchymal OPCs, those in the SVZ display a higher proliferation rate, serving a steady supply of newborn oligodendrocytes to the corpus callosum (Nait-Oumesmar et al. 1999; Picard-Riera et al. 2002; Menn et al. 2006). Our investigation, employing Ki67 and NG2 co-staining, aligned well with the literature reports, by revealing a selective concentration of proliferating OPCs (NG2⁺/Ki67⁺) within the SVZ (Fig. 2a). Other brain regions, such as the hippocampus (Fig. 2c), also contained OPCs (NG2⁺ cells); most of these cells, however, appeared to lack the proliferative activity, i.e. Ki67⁻. Additionally, Ki67⁺ cells in areas like the cortex (shown in Fig. 2d) were rarely OPCs due to minimal NG2 co-expression. For instance, the Ki67⁺ cell observed in the cortex (Fig. 2d) was unlikely to represent an active OPC. Quantitative analysis of OPC proliferation in the SVZ further delineated their distribution, highlighting a notable presence in the lateral wall and the dorsal lateral corner compared with the lower density in the ventral corner, the septal wall, or the roof (Fig. 2b). These patterns confirm the presence of SVZ-originated proliferative OPCs, suggesting a critical role of SVZ in the generation of oligodendrocytes for the corpus callosum.

Impact of acute Pb exposure on newborn oligodendrocytes differentiated from SVZ neurospheres ex vivo

In light of the likely proliferative state of SVZ OPCs, we set out to investigate the impact of Pb exposure on SVZ OPCs by using neurosphere assay in a well-established in vivo acute Pb exposure model (Gu et al. 2011). Figure 1a illustrates the experimental workflow and critical timepoints for the specific parameters detailed below.

Our AAS analysis revealed that acute Pb exposure significantly elevated whole-blood Pb concentrations in mice, reaching 37.20 ± 19.30 µg/dl, a level similar to those observed in occupational workers, in contrast to the controls with 2.25 ± 2.60 µg/dl (n = 4; P < 0.05) (Fig. 3a). Further analysis uncovered that acute Pb exposure led to Pb accumulation across brain regions examined, with the highest concentrations found in the CP (8.57 ± 1.32 µg/g) and meninges (6.16 ± 1.40 µg/g). Pb levels in the SVZ, corpus callosum, and hippocampus were also increased as compared with controls; however, the magnitude of Pb accumulation in these regions was considerately lower than those in the CP and meninges; e.g. the Pb levels in the corpus callosum and SVZ were about 40- to 57-folds lower than in the CP (Fig. 3b). The data suggested that despite the high Pb concentrations in the plexus and meninges, only a mild Pb accumulation occurred in the SVZ or corpus callosum in this acute Pb exposure model, suggesting a high sensitivity and susceptibility of SVZ OPCs to Pb toxicity.

Fig. 3.

Effects of acute Pb exposure on newborn oligodendrocytes differentiated from SVZ neurospheres ex vivo. (a) Pb concentration in the whole blood of mice following acute Pb exposure. (b) Regional Pb concentration in the brain following acute Pb exposure: Choroid plexus, meninges, SVZ, corpus callosum, and hippocampus. (c) Day 5: Early neurospheres started to be visible and floated in the culture medium. Early neurospheres were further magnified toward the right. (d) Day 8: Primary neurospheres were formed and floated in the culture medium. (e) Quantification of viable cells by dissociating day 8 primary neurospheres formed per brain SVZ. Data represent mean±SD (n = 3/group). (f) Day 10: Representative images of secondary neurospheres formed by plating 2,500 viable cells dissociated from primary neurospheres in (f). (g) Quantification of secondary neurosphere diameters. Data represent mean±SD (n = 3/group), *P < 0.05. (h) Differentiation and migration of secondary neurospheres from SVZ tissues of control or acutely Pb-treated mice. Neurospheres were stained with Tuj1 (neuronal marker) and MBP (oligodendrocyte marker). (i) Quantification of migration areas of neurospheres. Data represent mean±SD (n = 6 to 7/group), **P < 0.01. (j) Tuj1⁺ neuronal cells per neurosphere were quantified in the control and Pb group. Data represent mean±SD (n = 6/group), *P < 0.05. (k) MBP⁺ oligodendrocytes per neurosphere were quantified in the control and Pb group. Data represent mean±SD (n = 6/group), ***P < 0.001.

Tracking neurosphere formation enabled the assessment of isolated SVZ cell proliferative activity (Soares et al. 2021). On day 5 (72 hr after initial seeding), the formation of early neurospheres did not appear to be affected by acute Pb exposure (Fig. 3c). Progressing to day 8, primary neurospheres in both groups exhibited normal growth, with no evidence of core darkening or delayed formation of larger neurospheres (Fig. 3d). These observations were corroborated by unchanged viable cell counts determined by trypan blue staining after dissociating all primary neurospheres from a single brain (Fig. 3e). Subsequently, dissociated primary neurospheres (as single cells) were reseeded to generate secondary neurospheres for downstream differentiation analysis. Interestingly, despite seeding equal numbers of viable cells dissociated from primary neurospheres, secondary neurospheres in the Pb-exposed group displayed a significantly smaller diameter (245.9 ± 3.91 µm) compared with the controls (268.3 ± 8.86 µm; n = 3; P < 0.05) following 48 hr of seeding on day 10 (Fig. 3f and g). This reduction in secondary neurosphere diameter in the Pb group suggests a detrimental effect of Pb on the SVZ, possibly with an alteration at the differentiation stage.

Following 7 d of differentiation (on day 17), the data by immunocytochemistry on ex vivo oligodendrocyte differentiation revealed that control neurospheres migrated extensively (Fig. 3h) and covered a considerably larger area compared with Pb-treated neurospheres; the neurosphere migration area in Pb group was 65.2 ± 22.6% less than that of controls (n = 6 to 7; P < 0.01) (Fig. 3h and i). Additionally, control neurospheres produced numerous Tuj1⁺ neurons and MBP⁺ oligodendrocytes with distinctive morphologies. Magnified images in Fig. 3h (on the right panel) further revealed that newborn neurons and oligodendrocytes in controls were capable of establishing intercellular interactions, as evident through yellow fluorescence resulting from signal overlap between neurons and oligodendrocytes (Fig. 3h in the middle panel). In contrast, Pb-exposed neurospheres exhibited a poor differentiation. Although the neurites of newborn neurons seemed to develop normally, the morphology of oligodendrocytes was severely affected compared with controls. Moreover, interactions between neurons and oligodendrocytes were limited (Fig. 3h). Quantitative analysis revealed that the number of newborn neurons per neurosphere in the Pb group was significantly reduced by 24.4% following initial Pb exposure (503.7 ± 82.92 in controls versus 380.70 ± 88.02 in Pb-exposed group; n = 6; P < 0.05) (Fig. 3J). Moreover, newborn oligodendrocytes per neurosphere following initial Pb exposure were decreased by 53.4% (12.17 ± 1.72 in controls versus 5.67 ± 1.75 in the Pb group; n = 6; P < 0.001) (Fig. 3k).

Thus, considering the mild Pb accumulation in the SVZ, the data from this ex vivo experiment strongly suggest that SVZ OPCs and oligodendrogenesis are highly susceptible to Pb exposure. These findings prompted us to extend the investigation to in vivo studies with the aim to understand the impact of Pb exposure on oligodendrogenesis, SVZ OPC activities, and myelination in the brain.

Decreased newborn oligodendrocytes and demyelination in the corpus callosum following subchronic Pb exposure in mice

We utilized a well-established in vivo subchronic Pb exposure paradigm developed by this lab (Gu et al. 2020) to investigate the potential Pb effects on newborn oligodendrocytes and myelination. Prior to Pb exposure, mice received three consecutive daily doses of BrdU to label actively proliferating cells, followed by 28-d oral gavage of Pb (Fig. 1b). This subchronic exposure paradigm significantly elevated whole-blood Pb levels to 34.67 ± 7.77 µg/dl compared with controls (0.10 ± 0.16 µg/dl) (Fig. 4a).

Fig. 4.

Subchronic Pb exposure disrupts oligodendrogenesis and myelination in the corpus callosum. (a) Pb concentration in the whole blood following subchronic Pb exposure. Data represent mean±SD, n = 3. **P < 0.01, as compared with controls. (b) Representative images of MBP⁺/BrdU⁺ newborn oligodendrocytes in the corpus callosum. Arrows marked MBP⁺/BrdU⁺ cells. (c) Representative images of MBP⁺/BrdU⁺ newborn oligodendrocytes in the brain cortex. (d) Quantification of MBP⁺/BrdU⁺ newborn oligodendrocytes in the corpus callosum and cortex. Data represent mean±SD (n = 4), *P < 0.05. (e) Representative images of MBP expression and Olig2⁺ cells in the corpus callosum. (f) Representative images of MBP expression and Olig2⁺ cells in the cortex. (g) Quantification of MBP expression in the corpus callosum and cortex. Data represent mean±SD (n = 4), *P < 0.05. (h) Density of Olig2⁺ cells in the corpus callosum and cortex. Data represent mean±SD (n = 4), *P < 0.05. (i) Quantification of Olig2 expression in Olig2⁺ cells in the corpus callosum and cortex. Data represent mean±SD (n = 4/group), *P < 0.05 as compared with controls.

Immunohistochemical staining revealed significant changes in the corpus callosum region. Pb exposure led to a notable 39.5% reduction in BrdU⁺/MBP⁺ newborn oligodendrocyte density from 54.52 ± 13.71/mm² in controls to 32.99 ± 3.27/mm² in Pb-exposed mice (n = 4; P < 0.05) (Fig. 4b). In the brain cortex, although there were fewer BrdU⁺/MBP⁺ cells observed overall (Fig. 4c), Pb treatment did not significantly alter the density of newborn oligodendrocytes (Fig. 4d).

Interestingly, the myelin expression, as indicated by MBP signal intensity, decreased in response to Pb exposure (Fig. 4b). To address concerns related to a harsh acidic treatment involving 2 N HCl during BrdU signal development in the immunohistochemistry procedure, which may have influenced MBP staining, we conducted a separate experiment to confirm this finding. In the absence of HCl treatment in the regular immunohistochemistry procedure (Fig. 4e), immunohistochemistry data showed a 35.7% reduction in MBP expression in the corpus callosum in the Pb-treated animals compared with controls, indicating a Pb-induced demyelination in the corpus callosum (n = 4; P < 0.05) (Fig. 4g). Additionally, the density of Olig2⁺ cells, which define the entire oligodendrocyte lineage, decreased by 31.0% due to Pb exposure (n = 4; P < 0.05) (Fig. 4e and h). Considering the fact that Olig2 expression is crucial for intralineage homeostasis (Mei et al. 2013; Gibson et al. 2014), we further assessed Olig2 expression within Olig2⁺ cells in the corpus callosum; the data revealed a 28.6% reduction in Olig2 expression in the Pb-exposed group (n = 4; P < 0.05) (Fig. 4e and i).

It is worth noting that the cortex appeared to be less susceptible to Pb exposure compared with the corpus callosum (Fig. 4f–i). MBP expression in the cortex remained unchanged despite mild disorganization in myelin arrangement (Fig. 4f and g). Furthermore, neither the density of Olig2⁺ cells nor the Olig2 expression level showed significant changes following Pb exposure in the cortex (Fig. 4f, h, and i).

Thus, the observations from this subchronic study indicate that oligodendrogenesis and myelination in the corpus callosum are susceptible to Pb exposure. Given the importance of SVZ OPCs in the corpus callosum myelination, it became necessary to understand how these SVZ OPCs mediate Pb-induced demyelination in the corpus callosum.

MRI evidence of demyelination in the corpus callosum following chronic Pb exposure in APP/PS1 mice

Pb exposure is recognized as a risk factor for Alzheimer’s disease (AD) (Bakulski et al. 2012). An impaired myelination has been linked to AD pathologies (Chen et al. 2021). Thus, we used an MR imaging technique to examine the demyelination in AD-model APP/PS1 mice (Jankowsky et al. 2004) following chronic Pb exposure (Fig. 1c). APP/PS1 mice exposed to Pb in drinking water for 20 wk exhibited blood Pb levels of 35.58 ± 13.97 µg/dl, whereas the control APP/PS1 mice had blood Pb levels of 0.25 ± 0.50 µg/dl (Fig. 5a).

Fig. 5.

MRI evidence of demyelination in the corpus callosum following chronic Pb exposure in APP/PS1 mice. (a) Pb concentration in the whole blood of mice following chronic Pb exposure via drinking water. Data represent mean±SD, n = 4 to 6. **P < 0.01, as compared with controls. (b) Representative (myelin water fraction) imaging in control and chronically Pb-exposed APP/PS1 mice. (c) Quantification of myelin content in the brain following chronic Pb exposure in APP/PS1 mice. Data represent mean±SD (n = 3 to 5), *P < 0.01 as compared with controls.

Following chronic Pb exposure, we evaluated brain myelination using newly established multiparametric mapping (MPM) MRI method. As anticipated, we observed a higher degree of myelination in the corpus callosum compared with the cortex in control mice (Fig. 5b). However, Pb exposure led to a notable 18.8% reduction in myelin signals in the corpus callosum (P < 0.05; n = 4) (Fig. 5b and c), indicating Pb-induced demyelination in this region. In the cortex, however, myelin intensities did not appear significantly altered. Thus, our MRI data from mice chronically exposed to Pb confirm Pb-induced demyelination, specifically in the corpus callosum.

Disrupted expression of Rictor, but not Sp1, in SVZ OPCs following subchronic Pb exposure

Rictor and Sp1 are the pivotal signaling molecules known to play crucial roles in orchestrating OPC differentiation into oligodendrocytes for myelination (Zawia et al. 1998; Deng et al. 2001; Wei et al. 2003; Grier et al. 2017; Dahl et al. 2022). Pb exposure could disrupt these intracellular signaling pathways leading to perturbed SVZ OPCs. Thus, we examined the expression of Rictor and Sp1 in these cells following subchronic Pb exposure.

The immunohistochemistry data revealed a specific enrichment of Rictor in SVZ OPCs located within the dorsal lateral conner, a subregion known for its robust proliferative OPC activity (Fig. 6a) (referred also to Fig. 2a and b). Pb exposure led to a significant decrease in Rictor expression with a reduction of 50.6% (n = 3; P < 0.05) compared with controls (Fig. 6a and b). This finding, in conjunction with the decreased expression of Olig2 (Fig. 3), another molecule pivotal for OPC differentiation into oligodendrocytes for myelination (Zhou and Anderson 2002), suggested that Pb exerted its toxicity on SVZ OPCs by disrupting the intracellular signaling pathways.

Fig. 6.

Differential effects of subchronic Pb exposure on Rictor and Sp1 expression in SVZ OPCs. (a) Expression of Rictor in SVZ OPCs in control or Pb-exposed mice. A significant enrichment of Rictor in SVZ OPCs was magnified in the lower panels. Please see Fig. 1b for the dose regimen for subchronic Pb exposure in mice. (b) Quantification of Rictor fluorescent intensity in SVZ OPCs in control and Pb-exposed mice. (c) Expression of Sp1 in SVZ OPCs in control and Pb-exposed mice. (d) Quantification of Sp1 fluorescent intensity in SVZ OPCs control and Pb-exposed mice. Data represent mean±SD (n = 4/group), *P < 0.05 compared with controls.

In contrast, Sp1, which is responsible for driving MBP expression and facilitating OPC differentiation into OLs, did not exhibit any noticeable alterations in response to Pb exposure (Fig. 6c and d). Control data showed a consistent, nonspecific expression pattern across brain cells, which remained unchanged in the Pb exposure group. Taken together, these observations suggest that Rictor may serve as a key toxicological target in Pb-induced impairments of SVZ OPCs.

Responses of SOD4 and MT3 in SVZ OPCs, CP, and leptomeninges to subchronic Pb exposure

SOD4 and MT3 are well-recognized “self-defense” proteins for their capabilities in counteracting toxicities caused by chemicals (Coyle et al. 2002; Johnson and Giulivi 2005). This series of experiments aimed to characterize responses of SOD4 and MT3 in SVZ OPCs, CP, and meninges, the regions where significant Pb accumulation occurred (Fig. 2a).

In the SVZ, SOD4 was preferentially expressed in OPCs, with moderate levels in other cell types (Fig. 7a). Interestingly, Pb exposure appeared to enhance overall SOD4 signal intensity in the SVZ, although it did not significantly alter its expression in OPCs (Fig. 7a and d). In the CP, SOD4 was more abundant in blood-facing endothelial cells than in CSF-facing epithelial cells (Fig. 7b). After Pb exposure, an increase in SOD4 was observed in both cell types, with Pb inducing a notable translocation of SOD4 to the blood-contacting side of choroidal endothelia (Fig. 7b and d), suggesting a cellular adaptation to mitigate blood-borne Pb toxicity. In leptomeninges, SOD4 levels were significantly elevated by 159.0% following Pb exposure, suggesting a potential upregulation of defensive mechanisms against Pb toxicity (Fig. 7c and d).

Fig. 7.

Responses of SOD4 and MT3 in SVZ OPCs, choroid plexus, and leptomeninges to subchronic Pb exposure. (a–c) Expressions of SOD4 in SVZ OPCs, choroid plexus, and leptomeninges were shown in (a), (b), and (c), respectively. Please see Fig. 1b for the dose regimen for subchronic Pb exposure in mice. Magnified images in (b) revealed a translocated SOD4 in choroidal endothelia by Pb. (d) Quantification of relative SOD4 expressions in SVZ OPCs, choroid plexus, and leptomeninges as affected by Pb exposure. Data represent mean±SD (n = 4/group), *P < 0.05 compared with controls. (e–g) Expressions of MT3 in SVZ OPCs, choroid plexus, and leptomeninges were shown in (e), (f), and (g), respectively. (h) Quantification of relative MT3 expression levels in SVZ OPCs, choroid plexus, and leptomeninges following Pb exposure. Data represent mean±SD (n = 4/group), *P < 0.05, **P < 0.01.

MT3 was predominantly found in SVZ OPCs and was significantly reduced by Pb exposure (Fig. 7e and h), suggesting a vulnerability of this defense mechanism to Pb. The CP showed only a mild baseline expression of MT3, which remained unchanged after Pb treatment (Fig. 7f and h). In leptomeninges, despite a strong baseline presence of MT3, Pb exposure resulted in a reduction of its expression (Fig. 7g and h).

These findings indicate a complex interplay of responses of these defense molecules to Pb exposure, with SVZ OPCs potentially leveraging intracellular defenses like MT3 and extracellular support from Pb-sequestering tissues such as the CP and leptomeninges to mitigate Pb toxicity.

Reactive astrogliosis and microgliosis in the corpus callosum following subchronic Pb exposure

Newborn oligodendrocytes originated from SVZ OPCs migrate to the corpus callosum as their primary destination (Menn et al. 2006), a process often disrupted in the inflamed milieu of multiple sclerosis (Garg et al. 2015). Given the significance of astrogliosis and microgliosis as hallmarks of neuroinflammation (Kwon and Koh 2020), we examined astrogliosis and microgliosis by differentiating between resting and reactive states of astrocytes and microglia (Sofroniew 2009; Sorci et al. 2010; Jurga et al. 2020) following subchronic Pb exposure in mice.

In the control corpus callosum, a baseline population of GFAP⁺ astrocytes had a minority exhibiting a reactive S100β⁺/GFAP⁺ phenotype (Fig. 8a and b). Pb exposure caused not only a 30.1% increase in GFAP⁺ astrocyte density (P < 0.05), but also a 153.5% rise in the proportion of S100β⁺/GFAP⁺ reactive astrocytes (P < 0.01), accompanied by a 47.5% augmentation in GFAP expression typically associated with neuroinflammatory conditions (Brahmachari et al. 2006) (Fig. 8a–c). These results suggested a shift in astrocytic behavior from the resting to reactive state due to Pb exposure.

Fig. 8.

Reactive astrogliosis and microgliosis in the corpus callosum following subchronic Pb exposure. (a) Representative images showing astrocytes in the corpus callosum. GFAP⁺ astrocytes include S100β⁻/GFAP⁺ resting astrocytes and S100β⁺/GFAP⁺ reactive astrocytes. Please see Fig. 1b for the dose regimen for subchronic Pb exposure in mice. (b) Relative density of resting and reactive astrocytes in the corpus callosum. (c) Quantification of GFAP level in astrocytes in the corpus callosum. Data represent mean±SD (n = 4/group), *P < 0.05, **P < 0.01 compared with controls. (d) Representative images showing microglia in the corpus callosum. Iba-1⁺ microglia are categorized as Iba-1⁺/CD68⁻ resting microglia and Iba-1⁺/CD68⁺ reactive microglia. (e) Relative density of resting and reactive microglia in the corpus callosum. (f) Quantification of Iba-1 level in microglia in the corpus callosum. Data represent mean±SD (n = 4/group), *P < 0.05, ***P < 0.001.

Microglial presence in the control corpus callosum was characterized by a relatively sparse distribution, with minimal Iba-1⁺/CD68⁺ reactivity (Fig. 8d). In contrast, Pb exposure induced a significant 86.6% increase in microglial density and a 77.6% increase in reactive microglia density (P < 0.05) (Fig. 7d and e). An associated increase in Iba-1 expression was also observed, indicating a phenotype linked to inflammatory responses that were detrimental to brain health (Fig. 8d–f) (Ito et al. 2001; Ekdahl et al. 2003). Considering the established impact of inflammation on OPC migration and differentiation (Harrington et al. 2020), the Pb-induced reactive astrocytes and microglia could potentially contribute to the observed attenuation of oligodendrogenesis in the corpus callosum.

Pb exposure decreases OTX2 and klotho in the CP

The CP, known to accumulate Pb upon exposure, is a repository of signaling molecules that govern oligodendrogenesis and neurogenesis, including OTX2 and Klotho (Chen et al. 2013; Marques et al. 2018; Planques et al. 2019). Prior research has indicated that Pb accumulation can suppress the secretion of certain CP-abundant proteins, such as transthyretin (TTR) (Zheng et al. 2001). This led us to hypothesize that the presence of Pb in the CP may interfere with the signaling processes essential for SVZ OPC function, which may be mediated by molecules like Klotho and OTX2.

A high expression of Klotho in the choroidal epithelia was confirmed by immunohistochemistry (Fig. 9a), the observation that was in a good agreement with existing literature (Zhu et al. 2018; Dani et al. 2021). Subchronic Pb exposure significantly reduced the expression of Klotho by 35.6% in choroidal epithelial cells (Fig. 9a and c). Correspondingly, a decrease in Klotho expression was also observed in SVZ OPCs (Fig. 9b and c). OTX2 expression, although abundant in control choroidal epithelia, showed a marked decrease following Pb exposure by 46.7% (Fig. 9d and e). A similar pattern existed in the SVZ, where Pb exposure led to a 59.2% reduction in OTX2 expression (Fig. 9d and e). Collectively, these findings suggest that a decline in key regulatory proteins in the CP and SVZ may underpin the previously observed reduction in oligodendrogenesis.

Fig. 9.

Pb exposure decreases OTX2 and Klotho in the choroid plexus. (a) Altered Klotho expression in choroidal epithelia following subchronic Pb exposure. The dose regimen for subchronic Pb exposure can be found in Fig. 1c. (b) Modulation of Klotho presence in SVZ OPCs by Pb. (c) Relative Klotho expression in choroidal epithelia and SVZ OPCs post-Pb exposure. Data represent mean±SD (n = 4/group), *P < 0.05, **P < 0.01 as compared with controls. (d) OXT2 protein expression in choroidal epithelia and SVZ. (e) Relative OTX2 expression in choroidal epithelia and SVZ cells following Pb exposure. Data represent mean±SD (n = 4/group), *P < 0.05, ***P < 0.001.

CP restores Pb-inhibited oligodendrogenesis

To further elucidate the CP’s role in oligodendrogenesis amidst Pb toxicity, we employed an in vitro two-chamber co-culture system (illustrated in Fig. 1d). Neurospheres derived from SVZ tissues of acutely Pb-exposed mice (as described in Fig. 3f) showed limited growth and differentiation when cultured alone. However, when co-cultured with control CP tissues, a significant restoration in differentiation parameters was observed (Fig. 10a). This included enhanced migration (by 61%, Fig. 10b), increased neuronal differentiation (by 154.3%, Fig. 10c), and a rise in newborn oligodendrocytes (by 168.1%, Fig. 10d), coupled with an evident myelination on neurons (Fig. 10a). Additionally, Fig. 10a showed that the presence of CP apparently maintained OPCs in their characteristic bipolar morphology (Kuhn et al. 2019); this was a stark contrast to the negligible presence of OPC without CP for the co-culture (Fig. 10a). Thus, our data underscore the CP’s crucial role in sustaining SVZ OPCs and facilitating oligodendrogenesis, especially in the context of Pb-induced changes.

Fig. 10.

Choroid plexus restores the Pb-inhibited oligodendrogenesis. (a) Neurosphere differentiation and migration influenced by the presence of choroid plexus tissue, with labeling for Tuj1, MBP, and NG2. Magnified views highlight cellular morphology and oligodendrocyte–neuron interactions. (b–d) Quantitative assessments of migration extent, Tuj1⁺ neuron count, and MBP⁺ oligodendrocyte count in neurospheres, influenced by choroid plexus intervention. Data represent mean±SD (n = 6 per group), ***P < 0.001.

Discussion

Our study presents compelling evidence to support a detrimental effect of Pb exposure on demyelination in the corpus callosum, likely through inhibiting the oligodendrogenesis by SVZ OPCs. Multiple lines of observations support this conclusion. First, acute Pb exposure in vivo impaired oligodendrogenesis in SVZ-derived neurospheres by reducing newborn oligodendrocytes and weakening neuron–oligodendrocyte interactions. Second, subchronic Pb exposure induced demyelinating changes in the corpus callosum, correlating with a significantly reduced oligodendrogenesis in this region. Third, chronic Pb exposure in APP/PS1 mice resulted in evident demyelination of the corpus callosum, with diminished myelin signals observed using dual-echo ultrashort echo time (UTE) MRI. The study also sheds light on possible mechanisms underlying Pb toxicity on myelination: (i) Pb-induced reduction of signaling molecules intrinsic to SVZ OPCs, such as Rictor, may underlie defective oligodendrogenesis in SVZ; (ii) Pb-caused disruption of self-defense mechanisms in SVZ OPCs, the CP and the meninges, as mediated by enzymes like SOD4 and MT3, could increase the susceptibility of OPCs to Pb toxicity; (iii) Pb-stimulated reactive astrogliosis and microgliosis within the corpus callosum may further reduce oligodendrogenesis and contribute to demyelination; and finally (iv) Pb-inhibited secretion of oligodendrogenesis-promoting proteins such as OTX2 and Klotho by the CP may also contribute to the decreased oligodendrogenesis in the SVZ.

Pb-induced neurotoxicity has been extensively documented for its disruption of neural cell types, including neuronal cells via calcium mimicry (Virgolini and Aschner 2021), neural stem/progenitor cells via proliferation inhibition (Mousa et al. 2018), and endothelial cells affecting blood–brain barrier integrity (Bradbury and Deane 1993; Struzyńska et al. 1997; Wang et al. 2007; Gu et al. 2020). Pb exposure has also been associated with increased reactivity in astrocytes and microglia (Chen et al. 2015), diminished choroidal epithelial cells’ transthyretin secretion (Zheng et al. 1996, 2001), and myelin sheath disintegration in hippocampal oligodendrocytes (Dabrowska-Bouta et al. 1999). The findings presented in this report support a novel concept that Pb exposure can cause demyelination in the corpus callosum through its interaction with oligodendroglial lineage cells, specifically OPCs in the SVZ and oligodendrocytes in the corpus callosum. A primary function of the corpus callosum is to conduct information between the brain’s hemispheres. Recent studies also suggest that the corpus callosum plays roles in movement control, cognitive function, and vision performance (Clarke et al. 2021; Degraeve et al. 2023; Wright and Booth 2023). Importantly, demyelination-related axon damage in the corpus callosum is known to impair the performance on complex tasks during the disease progression in multiple sclerosis (Ozturk et al. 2010; Degraeve et al. 2023). The current study provides novel evidence suggesting that demyelination in the corpus callosum following Pb exposure could underlie the symptom overlapping between multiple sclerosis and Pb encephalopathy such as limb numbness, vision blurring, and cognitive decline (Fenga et al. 2016; Mesri et al. 2018; Aghdam et al. 2019; McGinley et al. 2021). Further ultrastructural assessment with electron microscopy on the corpus callosum would likely reveal subcellular mechanisms for Pb-induced corpus callosum alterations, particularly the neuron–oligodendrocyte interactions.

Evidence in the literature suggests that SVZ OPCs are particularly responsive to demyelination in the corpus callosum (Xing et al. 2014), in the manner by generating new oligodendrocytes that migrate to the corpus callosum to repair the neuronal injury (Menn et al. 2006). An increased Ki67 immunoreactivity in SVZ OPCs by immunohistochemistry suggests a highly dynamic cell cycle in this population, rendering them susceptible to Pb, which is known to cause cell cycle arrest (Wierzbicka 1999). The vulnerability of SVZ OPCs to Pb was further supported by changes in critical proteins including Klotho, Rictor, and MT3; Klotho and Rictor regulate oligodendrogenesis and OPC differentiation into oligodendrocytes for myelination, respectively, whereas MT3 functions to defense against cytotoxicity (Zawia et al. 1998; Deng et al. 2001; Wei et al. 2003; Grier et al. 2017; Dahl et al. 2022). Future studies should employ single-cell technologies to unravel the intrinsic programs in oligodendrocyte lineage cells, which could help explain the decreased oligodendrogenesis and compromised oligodendrocyte function.

The SVZ OPCs are also sensitive to external cues (Delgado et al. 2021). For OPCs in the SVZ, the CP likely serves as a critical source of external signaling molecules; Pb accumulation in this CSF-generating structure, by interfering the production and secretion of these signaling molecules, may indirectly affect oligodendrogenesis in the SVZ. The choroid polexus’s proximity to the SVZ via the CSF pathway suggests that substances released into the CSF by the CP are readily incorporated by SVZ cells. Given that SVZ cells prominently feature primary cilia that interface with the CSF to detect its constituents (Tong et al. 2014), it is plausible that Pb-induced alterations in the CP secretome may contribute to SVZ OPC dysfunction. Critical extrinsic molecules that influence oligodendrogenesis and myelination include Klotho and OTX2 (Chen et al. 2013; Hoch et al. 2015), both of which are predominantly present in the CP. Our data showed that Pb exposure significantly reduced Klotho and OTX2 in the CP, which may in turn disrupt the oligodendrogenesis of SVZ OPCs.

Although the presence of MT3 in SVZ has been recently recognized by our previous report (Liu et al. 2022), little was known about the existence of SOD4, another “self-defense” protein (Coyle et al. 2002; Johnson and Giulivi 2005), in the SVZ or CP. The current study clearly shows the evidence of abundant expression of SOD4 in both SVZ OPCs and choroidal endothelial cells. Noticeably, the structures of SVZ and CP allow the tissues constantly in contact with extracellular fluid compartment. In case of SVZ, type B1 neural stem cells, including a subpopulation primed as OPCs (Delgado et al. 2021), project apical processes into the CSF-filled ventricle where they form ciliated terminals to sense signals in the CSF (Falcão et al. 2012), whereas the choroidal endothelial cells directly touch substances present in the blood stream (Zheng and Chodobski 2005). Conceivably, a high expression of SOD4 in these two unique cell types may form the first line of defense against toxicants in extracellular fluids. Pb exposure-induced expression of SOD4 in both SVZ and CP offers yet another telling example to support SOD4’s defense function. However, the exact function, the mechanism of its induction, and epigenic regulation upon exposure to environmental toxicants remain largely unknown. Thus, a thorough understanding of SOD4 in these tissues deserves future investigation.

This study, for the first time in literature, reveals an extraordinarily high level of Pb accumulated in meninges. The meninges play a role in CSF drainage, blood supply, and immune cell infiltration (Alves de Lima et al. 2020). Our finding suggests several critical implications of the meninges in neurotoxicology. First, the meninges, like the CP, may act as yet another Pb storage site in the brain. Considering the tissue mass of the meninges versus the CP (3.52 ± 0.55 mg meninges versus 0.68 ± 0.14 mg CP, measured in adult mice in this lab by total wet weight), the meninges are about five times larger than the CP and accumulate even larger amounts of Pb than does the CP. Thus, it is highly likely that the meninges may act another “reservoir” of Pb in the brain, enabling a chronical release of Pb into the brain’s extracellular fluid. The mimicry of Pb²⁺ to calcium (Ca²⁺) is known to underlie the long storage of this toxic metal in bones, the most calcified tissues throughout the human body (Bridges and Zalups 2005). Given both the CP and meninges are the leading tissues in the brain susceptible to calcification (Halstead 1923; Whitehead et al. 2015), this Pb-Ca mimicry mechanism may underlie the observations of high Pb accumulation in both tissues. Second, Pb exposure may also affect the meninges-secreted signaling molecules. For example, TGF-β superfamily proteins released by the meninges to the CSF have been shown to directly regulate the oligodendrogenesis in the corpus callosum (Choe et al. 2012). Given the known negative effects on TGF-β superfamily (Tung et al. 2022), Pb exposure seems likely to alter meningeal TGF-β secretion thus affecting the myelination in the corpus callosum. Third, our findings indicate a significant presence of detoxifying proteins, specifically SOD4 and MT3, within the meninges. However, the exact function of these self-protective proteins in the meninges, either as an innate capacity or inducible response to counteract chemical insults in two fluid compartments, remains elusive. Thus, the results in this report call for more in-depth neurotoxicological studies focused on this critical brain tissue.

Finally, this study employed two new technical approaches. The ex vivo neurosphere approach used in this study is a valuable tool for examining adult brain’s regenerative capacity when exposed to toxicants. By using this model, which allows for cell’s unrestricted differentiation, our data revealed that Pb exposure not only suppressed oligodendrogenesis but also compromised myelination by newborn oligodendrocytes through oligodendrocyte–neuron interactions. Thus, the ex vivo neurosphere assay developed in this study could serve as a useful tool for screening other toxicants implicated in multiple sclerosis, such as mercury and aluminum (Exley and Clarkson 2020; Pamphlett et al. 2023). We also successfully used multiparametric mapping MRI method to quantify brain myelination status. The data from MRI analyses support the demyelination in the corpus callosum after subchronic Pb exposure in mice. Thus, this technique can be applied to other research projects aiming at myelination or demyelination quantification.

In summary, the data presented in this report suggest that Pb exposure impairs oligodendrogenesis in brain’s regenerative niche, i.e. the SVZ. In addition, Pb exposure results in demyelination in the corpus callosum detected by both immunohistochemistry and MRI approaches. Moreover, our initial mechanistic studies provide the evidence of Pb exposure-induced alterations in signaling, defensive, and regulatory molecules important to SVZ oligodendrogenesis and corpus callosum myelination. Hence, considering the critical role of oligodendrocytes in maintaining neuronal functions, Pb-induced demyelination and aberrant oligodendrogenesis could contribute to or potentiate the previously reported Pb neurotoxicities through neuronal damage.

Author contributions

Wei Zheng and Luke L. Liu (Conceptualization); Luke L. Liu, Uzay Emir, Huiying Gu, Stephen J. Sawiak, Jason R. Cannon, and Wei Zheng (Methodology); Luke L. Liu, Uzay Emir, Huiying Gu, Lara T. Sang, and Wei Zheng (Investigation); Luke L. Liu (Writing Original Draft); Wei Zheng, Luke L. Liu (Writing—Review & Editing); Wei Zheng, Yansheng Du (Funding Acquisition); Wei Zheng (Supervision).

Supplementary material

Supplementary material is available at Toxicological Sciences online.

Funding

The work was supported by the National Institute of Health/National Institute of Environmental Health Sciences (NIEHS R01 ES027078 to WZ and YD).

Conflicts of interest: None declared.