Differential expression of SLC30A10 and RAGE in mouse pups by early life lead exposure

It is well known that SLC30A10 and RAGE play a crucial role in regulating the transport and accumulation of A β plaques. Our previous studies have shown that early exposure to lead can cause cerebral damage to pups due to the accumulation of A β and the deposition of amyloid plaques. However, the effect of lead on the protein expression levels of SLC30A10 and RAGE remains unclear. This study aimed to verify that maternal exposure to lead-containing drinking water during pregnancy would affect the expression of SLC30A10 and RAGE proteins in mice offspring, further verifying the lead-induced neurotoxicity. Four groups of mice were exposed to 0 mM, 0.25 mM, 0.5 mM, and 1 mM of lead for 42 consecutive days from pregnancy to weaning, and the offspring mice were tested on postnatal day 21. The levels of lead in the blood, hippocampus, and cerebral cortex were examined; the learning and memory abilities of the mice were investigated using the Morris water maze; the expression levels of SLC30A10 and RAGE in the hippocampus and cerebral cortex were examined using Western blotting and immuno�uorescence. The results showed that the lead concentration in the brain and blood of the mice increased along with the lead content of the mothers during the lead exposure period (P < 0.05). In the Morris water maze test, the spatial memory of the lead exposure group was lower than that of the control group (P < 0.05). Both Immuno�uorescence and Western blot analysis showed that the hippocampal and cerebral cortex of the offspring were proportionally affected by differential levels of lead exposure. The expression levels of SLC30A10 were negatively correlated with lead doses (P < 0.05). Surprisingly, under the same conditions, the expression of RAGE in the hippocampus and cortex of offspring was positively correlated with lead doses (P < 0.05). SLC30A10 may play a differential role in aggravated A β accumulation and transportation compared with RAGE. A difference in RAGE and SLC30A10 expression in the brain could contribute to lead-induced neurotoxicity.

downregulated in the hippocampus of AD patients. It has also been shown that SLC30A10 expression is signi cantly reduced in the frontal cortex of APP/PS1 mice [9].
Receptors for advanced glycation end products (RAGE) belong to the immunoglobulin superfamily [14]. Abnormal expression of RAGE can lead to certain chronic diseases, such as diabetes, in ammation, and AD [15]. In the pathological development of AD, RAGE acts as a cell surface receptor to bind Aβ to the neurons, blood-brain barrier (BBB), and microglia [16]. In cerebral endothelial cells, RAGE guides Aβ to penetrate the BBB of the cerebrum [17]. RAGE also mediates the in ux of circulating Aβ through the BBB [17]. In neurons, RAGE mediates Aβ intraneuronal transport, Aβ-induced oxidative stress and causes mitochondrial dysfunction [14,18]. The up-regulated expression of RAGE in neurons leads to decreased learning and memory and disturbances in neuronal circuits related to Aβ assembly in APP transgenic mice [19]. In microglia, RAGE enhances Aβ-mediated in ammation [20]. Interestingly, in an Aβ-rich environment, the expression of RAGE is signi cantly increased in the endothelium and neurons. It can also amplify Aβ-induced adverse effects such as cerebrum and BBB in the cerebrum.
Since both SLC30A10 and RAGE regulate the transport and accumulation of Aβ, it is valuable to study the effect of maternal lead exposure on the expression of SLC30A10 and RAGE in the offspring. This study aimed to characterize the expression of SLC30A10 and RAGE in the brains of offspring when pregnant rats were drink lead-containing water and to understand the potential mechanisms of lead-induced neurotoxicity.

Animal grouping and handling
Forty pregnant mice (C57BL/6) were purchased from Henan Experimental Animal Center. Upon arrival, the mice were kept in a room with a temperature control of 21 ± 2℃ and natural light/dark cycle. Before the experiment, the mice were adapted to the animal center of Zhengzhou University for one week. This study was approved by the Animal Use and Care Ethics Committee of Zhengzhou University, and the relevant standards of animal use laws and regulations were strictly throughout the experiment.
The pregnant mice were randomly divided into four groups of ten each. Three groups of Mice were exposed to lead for 42 consecutive days from pregnancy until weaning and were exposed to either 0.25 mM, 0.5 mM, and 1 mM (low, medium, and high doses) lead acetate, respectively. The remaining group was given lead-free distilled water as a control group. After birth, 12 mice from each group were randomly selected for subsequent experiments.

Determination of lead levels in blood and brain samples
This study aimed to investigate the mechanism of cerebral damage in the offspring of mice exposed to lead during pregnancy, blood samples were collected from the tail on the 21st day after birth in clean grade PND21 rat pups. The mouse pups were anesthetized with sodium pentobarbital (35mg/kg, i.p.).
Hippocampal and cerebral cortical tissues were collected accordingly [21]. Whole blood (100 µl) with 0.5 N ultrapure nitric acid (3.9 ml) (Luoyang Chemical Agent Factory, China) was vortexed for 10 seconds and placed in a centrifuge (7500 r/min) for 10 min at 37℃. The supernatant was used to analyze the lead concentration. Brain tissues, such as the hippocampus and cerebral cortex, were digested in a digestion solution consisting of 0.5 N perchloric acid, 0.5 N nitric acid, and 0.01% Triton X-100 (Tianjin Chemical Agent Factory, China) with a 1:10 (w/v) tissue/digestion solution ratio.
The method for determining the lead concentration in blood and cerebrum tissue can be found in the literature [22,23]. When necessary, the samples were further diluted with 1.0% (v/v) HNO3 to a concentration range of 0-20 µg/L for reading Hitachi Flameless Graphite Furnace AAS with a wavelength setting of 283.3nm (HITACHI, Japan) was used for scanning the lead concentrations of the samples.

Water maze detection
The Morris water maze test evaluated the learning and memory of the offspring of 12 PND21 mice exposed to lead during the perinatal period [24,25]. The water maze was a white berglass pool with a 1.5 m. The water temperature was kept at around 22°C. The water was made opaque using a non-toxic opaque Funstuff © liquid tempera black paint. Designate four points on the pool's edge and divide them evenly into four quadrants. The escape platform was a 15 cm 2 Plexiglas square. First, place it 1 cm below the horizontal and in the center of the rst quadrant. The platform was kept in the same position during the learning phase. It was removed during the probe phase.
After the experiment began, mice were tested four times a day for ve days. The mice were placed in the water facing the wall, and if they could nd the platform within 60 seconds, they were placed on it for 5 seconds. If they could not nd the platform, they were guided to the platform and left there for 20 seconds.
Moreever, record the time each group of mice spent to reach the platform for these 5 days. The probe test was started on day six by placing the mice in the water facing the wall and recording the number of times the mice crossed the platform position and the time spent in the target quadrant within 60 seconds.

Immuno uorescence
Immuno uorescence is often used to detect the expression of target proteins, which has been reported in several studies [26][27][28]. The study was performed using immuno uorescence of SLC30A10 and RAGE in mice induced on PMD21 with or without maternal exposure to lead [29,30]. Speci cally, three mice samples from each group were randomly selected for IHC. Mice were anesthetized and brain tissue was carefully removed and kept overnight in 4% paraformaldehyde in phosphate buffer (PBS; pH 7.4) at 4°C, followed by immersion in increasing concentrations of sucrose (15% and 30%). Different regions of the brain were subsequently sectioned and collected for storage at -20°C.
Next, brain sections were blocked with PBS containing 5% goat serum for 1 hour at 37°C. Subsequently, brain sections were incubated with SLC30A10 antibody (1:300, Abcam) and RAGE antibody (1:400, Abcam) separately overnight at 4°C. The next day the sections were washed with PBS and incubated with Alexa Flour 488/594 secondary antibody (1:200, Abcam) for 1 hour, followed by staining with DAPI for 5 minutes, nally placed under an inverted uorescence microscope for visualization.
After heating at 95˚C for about 10 minutes, all samples were separated with 10% Tris-acetate/SDS/Glycine acrylamide gel. Proteins were transferred to PVDF membranes and blocked with 5% skimmed milk for about two hours at room temperature. After washing 3X with 0.5% Tween − 20 in TBS and incubated with rabbit anti-SLC30A10 (1:6, Abcam) and rabbit anti-RAGE (1:700, Abcam) for approximately two hours, the PVDF membranes were placed in the second antibody for incubation and immunoglobulin at 37˚C for about one hour. The protein signal was captured using Super Signal West Pico Chemiluminescent Substrate (Pierce Chemical Company) and imaging Detection System (Syngene Gene Company). β-actin was visualized using the same method.

Statistical analysis
All data were expressed as mean ± SEM using SPSS 12.0 (IBM). One-way ANOVA was used to analyze the data using the LSD test, and if the variance was not homogeneous, the data were analyzed using Tamhane T2 test. Differences between treatment means were considered statistically signi cant at P < 0.05.

Lead levels in the hippocampus, cerebral cortex and blood of the offspring
The blood lead level of the offspring in the three lead exposure groups was signi cantly higher than that of the control group by 9.3, 18.4 and 41.6 times (P < 0.05) ( Table 1). The lead concentration in the hippocampus of the low, medium, and high dose lead exposure group was 16.0, 29.7, and 33.3 times that of the control, respectively (P < 0.05). In the cerebral cortex, the lead content of the three different dose groups was 8.8, 14.6, and 18.2 times that of the control, respectively (P < 0.05). Mice exposed to lead in all groups did not have morbidity or death.

Maternal drinking lead-containing water impairs learning and memory of offspring
The results of the learning and memory abilities of the offspring mice in different groups are shown in Fig. 1.
In the Morris water maze test, learning and memory abilities are evaluated by the number of platform site crossings, escape delay, and the time spent to reach the target area.
In training tests, mice exposed to lead during the perinatal period took longer to nd the hidden platform than the control group. The period of escape to uncover the platform in lead exposure groups was longer than that of the control (P < 0.05) on days 2, 3, 4, and 5. On the second and third days, the lead exposure group showed that the concealment period of the platform was more extended than that of the control group (P < 0.05). There was no statistical difference between the low concentration group and the control group on the second day of training (P > 0.05) (Fig. 1a). Probe testing showed that mice in the lead-exposed group had fewer platform-site crossings and spent less time in the reach area compared to the control (P < 0.05) (Fig. 1b, c).
Among all experimental groups, the mice in the high-dose group had the worst spatial memory ability. The above results indicate that lead impairs the spatial learning ability of mice and causes memory impairment.

Effects of lead exposure on SLC30A10 in the hippocampus and cerebral cortex
The immuno uorescence result of SLC30A10 in the hippocampal CA1 area of the offspring is shown in Fig.   2a-b. The SLC30A10 protein is highly expressed in the brain of the control group. However, the uorescence intensity in the hippocampal CA1 region of the offspring mice exposed to lead (1 mM) was signi cantly reduced (P < 0.05) (Fig. 2c). As shown in Fig. 2d-e, the qualitative and quantitative analysis of SLC30A10 by Western blot further con rmed that at 0.25 mM, 0.5 mM and 1 mM lead doses, the protein expression of SLC30A10 in the hippocampus decreased by 26.7%, 46.7%, and 73.3%, respectively (P < 0.05).
This study used the same experimental method to detect the expression level of SLC30A10 in the cerebral cortex. The results of immuno uorescence showed that the expression of SLC30A10 was signi cantly reduced in the cerebral cortex of the offspring mice in the lead-exposed group compared to the control group (Fig. 3a-c) (P < 0.05).The results of western blotting showed that the expression of SLC30A10 in the cerebral cortex of the offspring mice from different lead exposure groups decreased by 30.8%, 68.3%, and 85.0%, respectively, compared with the control group (P < 0.05) (Fig. 3d-e).

Effects of lead exposure on RAGE in the hippocampus and cerebral cortex
This study further con rmed the expression of RAGE in the brains of offspring with perinatal lead exposure. Immuno uorescence results are shown in Fig. 4a-b and 5a-b. The results showed that the RAGE protein was expressed in the CA1 region of the hippocampus and cerebral cortex of the offspring. The results of immuno uorescence showed that RAGE expression was signi cantly elevated in the hippocampus and cerebral cortex of the offspring mice in the lead-exposed group compared to the control group(P < 0.05) (Fig.   4c,5c). It was noted that the RAGE positive signals overlapped slightly with the nuclear staining in the control group (Fig. 4a, 5a). This overlap was more evident in groups exposed to lead (Fig. 4b, 5b).
The analysis of Western blot analysis illustrates the same results (Fig. 4d, 5d). The expression of RAGE was signi cantly higher in the hippocampus and cerebral cortex of the offspring mice in the lead exposure group compared to the control group (P < 0.05). The expression of RAGE in the hippocampus of the offspring of different lead exposure groups increased 7.8, 1.8, and 2.6-fold, respectively, compared to the control group (P < 0.05) (Fig. 4e). The expression of RAGE in the cerebral cortex of the offspring of different lead exposure groups increased 3.5, 4.5, and 10.5-fold, respectively, compared with the control group (P < 0.05) (Fig. 5e).

Correlation analysis
As shown in Table 2, correlation analysis shows that the expression of SLC30A10 in blood, the hippocampus, and the cerebral cortex is negatively related to lead content and spatial memory, and the difference was statistically signi cant (P < 0.05). However, the level of RAGE was positively correlated with it (P < 0.05).

Discussion
In this study, the effects of lead exposure in pregnant mice on the expression of SLC30A10 and RGEA in the cerebral cortex and hippocampus of offspring mice were examined by protein blotting and immuno uorescence. The expression of SLC30A10 in the hippocampus and cerebral cortex was negatively correlated with lead concentration. The expression of RAGE in the hippocampus and cerebral cortex was positively correlated with lead concentration. In addition, it was found in the Morris water maze experiment that the higher the concentration of lead exposure, the poorer the learning memory ability of the offspring mice of pregnant lead-exposed mothers.
During pregnancy, childhood exposure or exposure to lead can cause severe brain damage, including coma, cerebral oedema, lead encephalopathy, and convulsions. Many studies have reported early lead exposure is a potential risk factor for AD progression in older adults [33,34]. Environmental lead exposure has been shown to increase AD progression in mice by targeting the blood-brain barrier (BBB) [35]. Ashok et al. found that exposure to a mixture of As, Cd, and lead induces the amyloid processing pathway of APP, leading to Aβ aggregation and cognitive impairment in young mice through oxidative stress-dependent neuroin ammation [36]. Other studies have found that juvenile lead exposure causes cognitive dysfunction in adult rats [37]. Lead is a widespread environmental neurotoxin, signi cantly affecting the developing nervous system, but the detailed mechanism of neurotoxicity is poorly understood. All these ndings are at the adult or later life phase stage.
Our previous studies have found that lead exposure in early life exacerbates Aβ accumulation and amyloid plaque deposition, inducing AD damage in mice [38]. Juvenile lead exposure causes cognitive dysfunction in adult rats [39]. Some studies have also found that prenatal lead exposure in mothers can cause neurological damage to the offspring [40]. In the current study, we found interestingly that in the Morris water maze test, female mice exposed to lead during the perinatal period had a decrease in the spatial memory of their offspring. Lead exposure interferes with Aβ transport by decreasing SLC30A10 expression and increasing RAGE expression. The toxic effects of lead worsened with increasing lead levels. In addition, the experimental results showed that the differential expression of SLC30A10 and RAGE was closely associated with learning memory ability. The above scenario suggests that the impaired learning memory ability of the offspring mice may be due to neurotoxicity caused by lead exposure.
SLC30A10 exists in the cerebrum, retina, and liver at the tissue level [11]. SLC30A10 has been signi cantly reduced in patients with Alzheimer's disease, and damage to SLC30A10 contributes to the development of diseases [9]. The publication has shown that SLC30A10 mRNA expression levels are low in the frontal cortex of female APP/PS1 transgenic mice [9]. Reports have also shown that Aβ can be accumulated in mice exposed to the early lead, resulting in impaired learning and memory [38,41]. In the neuroblastoma cell line model, SLC30A10 was found in the Golgi body, and its mRNA expression was downregulated in the AD cerebrum. In addition, the location of SLC30A10 suggests two possible mechanisms that may induce AD progression by increasing its expression [9]. First, localization to the Golgi body promotes the formation of Aβ by binding zinc to APP and inhibiting the β-secretase. Secondly, since high levels of Zn-induced transport of SLC30A10 to the plasma membrane may be proposed like SLC30A1, the ow of Zn into the extracellular space may provide Zn ions to initiate Aβ deposition and the formation of senile plaques [42]. In this experiment, the expression of SLC30A10 in the hippocampus and cerebral cortex of the offspring exposed to maternal lead was decreased, and its expression was negatively correlated with lead content and spatial memory capacity.
RAGE plays a crucial role in Aβ transport by regulating the circulation of Aβ; inhibition of RAGE activity protects against the accumulation of Aβ in the cerebral vasculature [43]. Several methods of clearing Aβ from the cerebrum have been reported, including uid drainage, mesenchymal microglial phagocytosis, and transport of Aβ across the BBB into the bloodstream, which is regulated by RAGE and the lipoprotein receptorassociated protein 1 (LRP1) in the endothelium [44]. As mentioned in the literature, in the brains of AD mice, RAGE increased Aβ expression by increasing β-secretase and γ-secretase [45]. In ammation has also been reported to play an essential role in AD's physiological and pathological processes by promoting the aggregation of Aβ [46]. RAGE is a critical player in the in ammatory response cycle, excising a critical role in various processes, including amyloid change, in ammation, cellular stress, and neuronal damage [47]. It has been reported that the binding of RAGE to ligands leads to the death of dopaminergic neurons and that blocking its progression may help to slow the progression of Parkinson's disease [48,49]. It has also been illustrated that short peptides bound to RAGE protect primary neuronal cells from beta-amyloid toxicity [50]. In this experiment, the expression level of RAGE in the hippocampus and cerebral cortex of the offspring exposed to lead increased, and its expression was positively correlated with lead content and spatial memory capacity. Thus, RAGE may impair the learning and memory abilities of mice by increasing the aggregation of Aβ.
This study provides strong evidence that perinatal exposure to lead in drinking water markedly the expression of SLC30A10 and RAGE in offspring hippocampus and cerebral cortex. In addition, we found that lead exposure resulted in a downregulation of SLC30A10 expression. However, RAGE expression was upregulated.
Our results suggest new potential mechanisms for the neuropathologic process of maternal lead exposure.

Availability of data and materials
All data generated or analyzed in the course of this study are included in this article or can be obtained from the corresponding authors. Tables   Table.1. Lead levels in blood, the hippocampus and the cerebral cortex of different lead-exposed animals at PND21.

Group
Blood (   Immuno uorescence result of SLC30A10 in the hippocampal CA1 region of the offspring following perinatal exposure to lead in drinking water from gestation to PND21 (a-c). Tissues were stained with a mouse anti-SLC30A10 antibody (red) and nuclei were stained with DAPI (blue).  Immuno uorescence result of SLC30A10 in offspring cerebral cortex following perinatal exposure to lead in drinking water from gestation to PND21 (a-c). Tissues were stained with a mouse anti-SLC30A10 antibody (red) and nuclei were stained with DAPI (blue).  Immuno uorescence result of RAGE in the hippocampal CA1 region of the offspring following perinatal exposure to lead in drinking water from gestation to PND21 (a-c). Tissues were stained with a mouse anti-RAGE antibody (green) and nuclei were stained with DAPI (blue).  Immuno uorescence result of RAGE in offspring cerebral cortex following perinatal exposure to lead in drinking water from gestation to PND21 (a-c). Tissues were stained with a mouse anti-RAGE antibody (green) and nuclei were stained with DAPI (blue).

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