Early-Life Pb Exposure Might Exert Synapse-Toxic Effects Via Inhibiting Synapse-Associated Membrane Protein 2 (VAMP2) Mediated by Upregulation of miR-34b

Abstract

Background:

Early-life Pb exposure can cause behavioral and cognitive problems and induce symptoms of hyperactivity, impulsivity, and inattention in children. Studies showed that blood lead levels were highly correlated with neuropsychiatric disorders, and effects of neurotoxicity might persist and affect the incidence of neurodegenerative diseases, for example Alzheimer’s disease (AD).

Objective:

To explore possible mechanisms of developmental Pb-induced neuropsychiatric dysfunctions.

Methods:

Children were divided into low blood lead level (BLL) group (0–50.00μg/L) and high BLL group (> 50.00μg/L) and blood samples were collected. miRNA array was used to testify miRNA expression landscape between two groups. Correlation analysis and real-time PCR were applied to find miRNAs that altered in Pb and neuropsychiatric diseases. Animal models and cell experiments were used to confirm the effect of miRNAs in response to Pb, and siRNA and luciferase experiments were conducted to examine their effect on neural functions.

Results:

miRNA array data and correlation analysis showed that miR-34b was the most relevant miRNA among Pb neurotoxicity and neuropsychiatric disorders, and synapse-associated membrane protein 2 (VAMP2) was the target gene regulating synapse function. In vivo and in vitro studies showed Pb exposure injured rats’ cognitive abilities and induced upregulation of miR-34b and downregulation of VAMP2, resulting in decreases of hippocampal synaptic vesicles. Blockage of miR-34b mitigated Pb’s effects on VAMP2 in vitro.

Conclusion:

Early-life Pb exposure might exert synapse-toxic effects via inhibiting VAMP2 mediated by upregulation of miR-34b and shed a light on the underlying relationship between Pb neurotoxicity and developmental neuropsychiatric disorders.

Keywords

Lead miRNA neuropsychiatric disorders synapse function

INTRODUCTION

Lead (Pb), as a ubiquitous environmental pollutant, can accumulate in human bodies and give rise to irreversible damages to many systems, especially to the central nervous system (CNS) [1 –5]. Children and pregnant women are the most liable to suffer Pb poisoning. Pb exposure during early-life period causes more severe neural problems and may persist across the life course [1, 6], later leading to the process of neurodegenerative diseases. Studies have shown that developmental excessive Pb exposure is an important environmental risk factor in children’s neuropsychiatric disorders, including attention deficit/hyperactivity disorder (ADHD), autism spectrum disorders (ASDs), etc. [7 –9]. However, how Pb exposure affects neuropsychiatric disorders remains unclear. In the present studies, we tried to investigate whether Pb’s neurotoxic effects are mediated by alteration of miRNA landscape.

Human genes is controlled by several genetic and epigenetic mechanisms, and microRNA (miRNA) has been identified as a vital arbiter of gene expression [10]. miRNAs are single-stranded non-coding RNAs that act as regulators by combining with the 3^′ untranslated regions of mRNAs, and leading to the degradation or inhibition of target genes [10]. Previous studies established that exposure to heavy metals might effectively change miRNA expression [11 –13], suggesting that miRNAs played an important role in heavy metal-mediated neural damages. Our previous studies showed that some miRNAs were involved in Pb-induced neural damages [12 , 15]. Recent researches have uncovered the existence of a possible relationship between miRNAs and neuropsychiatric disorders pathophysiology [16], thus a hypothesis came up that miRNA alteration might be the key regulator in Pb-mediated neuropsychiatric disorders. In the present studies, experimental studies and epidemiological studies were combined to explore whether Pb exposure mediated miRNA expression alterations were involved in developmental neuropsychiatric disorders.

Multiple mechanisms are involved in Pb-induced neural damages. Synapse structures were one of the most important targets in Pb-neurotoxicity. Our previous studies indicated that Pb-induced neurotoxicity was mediated by decreasing density of synapse and affecting vesicles transmission [17], but whether miRNAs were involved in Pb-synapse dysfunctions needs to be further clarified. Synapse-associated membrane protein 2 (VAMP2) plays an important role in the maintenance of synaptic function. Together with SNAP25 and syntaxin, VAMP2 is a key component of SNARE (soluble N-ethylmaleimide sensitive factors attachment protein) [18]. VAMP2 concerns the release of neurotransmitters as well as the docking of synaptic vesicles and has been proved to be closely related to a variety of neuropsychiatric and neurodegenerative disorders [19]. In our experiments, we wondered whether VAMP2 were involved in Pb-induced synapse dysfunctions.

In this study, we found that early-life Pb exposure might exert synapse-toxic effects via inhibiting VAMP2 mediated by upregulation of miR-34b. Our data shed a light on the underlying relationship between Pb neurotoxicity and developmental neuropsychiatric disorders.

MATERIALS AND METHODS

Study subjects

Children aged from 2∼5 years selected from different kindergartens in Wei nan city, Shaanxi province. Children were eligible for inclusion if 1) they were living for at least one year at their current address and 2) their parents were able to complete a questionnaire. The exclusion criteria included a) children who self-reported disease histories including flu, fever, chronic inflammation, and heart or lung diseases; b) children who have taken medicines in the past month; c) children who failed to offer samples of their blood. After exclusion, 195 children (100 boys and 95 girls) were selected to participate in the experiment. Written informed consent was requested from their parents. Then, 4 mL of heparin-anticoagulated fasting venous blood was collected from each child. Additional information, such as addresses, ages, etc., were obtained the day before the examination via questionnaires filled out by parents. The study was approved by the Ethics Committee of the Medical Faculty of Fourth Military Medical University (IRB protocol number is KY20143247-1), and all procedures were conducted in accordance with the ethical principles for medical research.

Study design

Based on the national toxicology program suggestion, we firstly classified children as low BLL group (BLL≤50.00μg/L) and high BLL group (BLL > 50.00μg/L). We detected items of health examination, trace elements, and blood indexes in all the children population. For the miRNA array, 40 children were randomly selected from high BLL group (n = 20) and low BLL group (n = 20). General characteristics, including ages, body mass indexes (BMI) and genders between the two groups showed no significant difference. Given the shortage of blood samples and requirements for subsequent validation experiments, 250μL of blood samples from each subject were blended to form a 4 ml plasma pool of each group. Then, miRNA array analysis was carried out to compare differences in miRNA expression profiles between these two groups. The remaining samples were then used for quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) validation.

Agilent human miRNA microarray

The ligation of Solexa adaptors were conducted with purified RNA molecules as well as reverse transcription into cDNA. Agilent Technologies’ Agilent Human miRNA microarray (Agilent Technologies Inc., ShanghaiBio Corporation, China) was employed for cluster generation and sequencing analyses of the purified cDNA. The obtained images were further processed into data in digital forms. Following the removal of adaptors and the elimination of contaminated as well as low-quality reads, miRNAs were identified by comparing the clean reads with miR-Base 21.0. miRNAs were selected based on the following criteria: a) demonstrated at least a 1.5-fold lower or higher expression in the high BLL group compared with low BLL group and b) were intersected with our previous miRNAs array data in a Pb exposure rat model published in 2014 [14].

Blood lead level measurement

Blood lead levels were measured by using graphic furnace atomic absorption spectrometry via using standardized protocols including confirmation that storage materials were not contaminated with background lead. Whole blood specimens were processed, stored, and transported to the Department of Occupational & Environmental Health (FMMU) for analysis. No samples were below the analytical limit of detection (< 0.10μg/L).

RNA isolation and qRT-PCR

Total RNA was isolated from the EDTA-anticoagulated plasma pools. RNA was transcribed to cDNA with One Step Prime Script® miRNA cDNA Synthesis Kit (TAKARA). qRT-PCR was performed with SYBR Premix Ex Taq TM Kit (TAKARA) via the applied biosystem 7500 fast real-time PCR system (Applied Biosystem, Madrid, Spain). The relative gene expression was normalized to internal control U6. The miRNA expression levels were calculated by the 2^–Δ Ct method, where Ct is the cycle threshold: ΔCt = C_t miRNA- C_tU6.

Bioinformatics analysis

Through employing DAVID Bioinformatic Resources [20, 21] (version 6.8), we predicted the potential targets of miRNAs that were differentially regulated in these two groups, for the purpose of enrichment analysis of Gene Ontology (GO) terms as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [22].

For the identification of chosen miRNAs’ target genes, three databases for predicting miRNA targets, and TargetScan (https://www.targetscan.org/), PicTar (https://pictar.mdc-berlin.de/), and microRNA.org (https://www.microrna.org) were employed to seek candidate genes.

Venn diagram was used to filter possible miRNAs involved in children’s neuropsychiatric disorders and Pb exposure. The miRNA pools of ASDs [16 , 23–30] and ADHD [16 , 31–34] were selected from PubMed (https://pubmed.ncbi.nlm.nih.gov/).

Animals and treatments

Forty developmental male Sprague Dawley (SD) rats (3 w∼3.5 w) were obtained from the animal center of FMMU. Rats were divided randomly according to the experiment plan and were raised under controllable lighting conditions (12 h/12 h light/dark cycle), temperature (25±2°C), and humidity (40∼60%). After acclimation, we randomized rats into 3 groups (n = 10 in each group): Con group (control), 100 ppm Pb group (Pb exposure of 100 ppm), 300 ppm Pb group (Pb exposure of 300 ppm). 100 ppm and 300 ppm Pb solutions were prepared by dissolving Pb acetate in deionized water. Rats of each group were allowed to drink freely. Duration of Pb exposure was 8 weeks. Blood samples were collected from rats’ tails and testified 4 weeks after Pb treatment. Morris water maze was conducted for behavioral analysis. Brain tissues were dissected closely with the use of tiny forceps. Hippocampus tissues were separated and stored in 0.1 M PBS (pH = 7.4) at the temperature of –80°C. Hippocampus tissues were used for transmission electron microscopy examinations and immunoblotting analysis. All procedures above were carried out strictly according to the international standards of animal care guidelines (Permit Number: SCXK-2015-0371).

Transmission electron microscopy

0.1% sodium cacodylate buffer, with 2% glutaraldehyde contained, was used to fix animal tissues by keeping 12 h at 4°C. When this process was completed, tissues were washed for 5 min with 0.1% sodium cacodylate buffer. After that, fixation solution with 1% osmium tetroxide as well as 2% K4Fe contained, was used to post-fix tissues. Tissues were stained with 1% uranyl acetate and were pelleted in 2% agar. Graded ethanol solution was used for the dehydration of the pellets, which were then wrapped in spur resin. With a Reichert Ultra cut microtome, the wrapped pellets were sliced into 60 nm pieces. With Rhodanimu 400-mesh grids, these ultrathin slices were collected. The collected sliced samples were post-stained with lead citrate and uranyl acetate. In the end, sections were washed in water. Transmission electron microscope (Joel, JEM-2000EX, Tokyo, Japan) was employed to examine the sliced samples.

Immune electron microscopy

Brain tissues were immersed in the fixative (with 4% PFA + 0.0125% glutaraldehyde) for 4 h, and hippocampal tissue was sliced into 50 nm ultrathin sections with a frozen ultrathin microtome. Ultrathin sections were put into a wet box, then rinsed for 8∼10 times, letting sections sink to the bottom. Sections were then dehydrated in 30% sucrose solution, and frozen in liquid nitrogen. After rinsing with PBS and blocking with 5% BSA, sections were incubated with the primary anti-VAMP2 antibody (1:100) (at 4°C, overnight), then with antirabbit IgG conjugated with 1.4-nm gold particles (Nanoprobes, Stony Brook, NY) (1:100). Sections were postfixed in 2% glutaraldehyde for 1 h. Silver enhancement color development, sequential dehydration, embedding, and subsequent tissue removal on the resin block were completed by staff in the electron microscope room. Electron micrographs were captured by the Gatan digital camera and its application software (832 SC1000, Gatan, Warrendale, PA).

Cell culture

PC 12 cells (a cell line derived from a pheochromocytoma which shows neural features) and HT22 (an immortalized mouse hippocampal cell line) cells were purchased from American Type Culture Collection (ATCC, USA). Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) added with 10% fetal bovine serum (FBS, Gibco, USA) was used to culture HT22 cells and PC12 cells. Cells were both cultured at 37°C with 5% CO₂. For in vitro studies, HT22 cells and PC12 cells were divided into 3 groups: Control group (Con), cells without Pb exposure; 5μM Pb: Pb acetate exposure of 5μmol/L; 10μM Pb (Pb acetate exposure of 10μmol/L. The dosages of Pb acetate were used based on our previous studies [35]. Culture medium were changed every 48 h.

Transfection of miRNA inhibitor/mimics

DEPC-treated water was used for the dissolution of miRNA oligonucleotides. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used following its instructions for the transfection of sub-confluent proliferating cells. The ultimate concentration of inhibitors and mimics was 50 nM.

Recombinant plasmid construction and dual-luciferase reporter assay

The protocol of plasmid construction and dual-luciferase reporter assay was based on our previous study [36]. Rno-miR-34b has three binding locations with 3^′UTR of VAMP2 mRNA, which located on 1083–1089, 1215–1221, and 1267–1274 bps. We chose the binding site of 1267–1274 bps because this site has a complete match of 8 pairs of complementary bases (while the others are 7–8 pairs), and the binding site has a context score percentile is 98, suggesting the most likely binding site with VAMP2 mRNA. Briefly, the entire vamp2 3′ UTR of mRNA was inserted into the XbaI site utilizing the following primer: vamp2 forward: 5^′-CAGCTGGTGTGTAAGTGTCTTGGAG-3^′, reverse: 5^′-GCAGCAGATCAGGCAGATGG-3^′. PC12 cells were used to perform the dual- luciferase reporter assay. The primer of miR-34b mimics was shown as followed. Sense: 5^′-AGGCAGUGUAAUUAGCUGAUUGU-3^′, anti-sense: 5^′-AAUCAGCUAAUUACACUGCCUUU-3^′. Luciferase enzymatic activity was detected with the Dual-Luciferase Reporter Assay System according to the manufacturer’s protocol (Promega, USA).

Western blot

Lysis buffer that contains protease inhibitors was used to extract proteins from hippocampus tissues of rats, HT22 cells/PC12 cells, then western blotting was employed to detect the protein expression. To be detailed, the detection kit of BCA-200 (Thermo, USA) was used to quantify the proteins. 20 mg of the protein sample was then treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After this procedure, the protein was further transferred on a PVDF western blotting membrane, which was kept for two hours in TRIS-buffered saline (Sigma, USA) that contains 5% skimmed milk powder supplemented with 1% Tween 20 (Sigma, USA). Next, the membrane was incubated in the fridge for a night with the following primary antibodies (VAMP2, 1:500, Abcam, UK) at the temperature of 4°C. After that, horseradish peroxidase-conjugated antibodies (Goat anti-Mouse IgG, 1:2000, CST, USA; Goat anti-Rabbit IgG, 1:2000, CST, USA) were incubated for two hours at the normal temperature. Then the enhanced chemiluminescence system (BioRad, USA) was employed to visualize the protein bands. After being normalized by using β-actin, image J was adopted to measure the protein expression.

Statistical analysis

Data were presented in the form of mean± standard error of the mean (SEM). We evaluated the differences of general characteristics between different groups (low BLL group versus high BLL groups) by Student’s t-test (for continuous variables). Hierarchical clustering for the miRNAs was performed to display different expression of the two groups. In the validation stage, the values of real-time PCR were by student’s t-test. For animal and cell experiments, western blot and RT-PCR results were analyzed by ANOVA, followed by Turkey test for post-hoc analysis. All statistical analyses were conducted using SPSS 20.0 for Windows.

The equation in the form of y = a + bx was generated from the linear regression model, where x stands for BLL, and y represents the expression of miR-34b. In the equation, coefficient a = intercept and b = regression coefficient. To figure out the relationship, the calculation of a Pearson correlation coefficient (R) as well as p-value was also conducted.

RESULTS

No markable difference was found in development indexes between the low BLL group and high BLL group in the children population

Characteristics of subject were shown in Table 1. According to national toxicology program, the children population were divided into two groups, BLL levels were assayed and quite different between the two groups (31.47±9.74 versus 56.71±14.44, p < 0.001). Items of health examination, trace elements and blood indexes were examined between the low BLL group and high BLL group. Data showed that no significant difference was found in health indexes, which is height, weight, and body mass index (BMI). To further investigate the role of Pb on children’s development growth, head/chest/waist circumferences were examined, and no evident difference was found. Blood routine examination data showed that no difference, such as erythrocytes, leukocytes etc., was found between these two groups. Trace elements analysis showed that copper levels were unexpectedly higher in the high BLL group than those in low BLL group (15.88±3.19 versus 17.27±2.97, p = 0.023).

Table 1

General characteristics of Weinan population

Weinan city	Low BLL group (n = 165)		High BLL group (n = 30)		p
	Mean	SD	Mean	SD
Age (y)	3.54	0.92	3.73	0.91	0.286
Items of health examination
Hight (cm)	105.71	6.65	106	8.42	0.823
Weight (Kg)	16.96	2.8	17.67	3.96	0.214
BMI (Kg/m²)	15.1	1.26	15.53	1.76	0.091
Head circumference	49.3	3.32	49.33	1.87	0.956
Chest circumference	52.86	4.04	53.7	4.51	0.286
Waist circumference	51.61	4.1	52.78	5.41	0.153
Trace elements
Blood lead (μg/L)	31.47	9.74	66.71	14.44	< 0.001^*
Manganese (mg/L)	15.95	5.22	15.84	4.2	0.912
Calcium (mg/L)	1.68	0.13	1.67	0.09	0.578
Iron (mg/L)	7.75	0.52	7.85	0.65	0.354
Zinc (mg/L)	69.29	11.26	69.08	17.88	0.928
Magnesium (mg/L)	1.49	0.14	1.53	0.17	0.241
Copper (mg/L)	15.88	3.19	17.27	2.97	0.023^*
Blood indexes
Erythrocyte (10¹²/L)	4.73	0.33	4.85	0.32	0.051
Hemoglobin (g/L)	127.5	9.75	129.8	7.91	0.213
Thrombocyte (10⁹/L)	290.44	66.24	288	61.8	0.847
Leukocyte (10⁹/L)	6.61	1.73	7.21	1.89	0.073
Lymphocyte (10⁹/L)	3.47	1.62	3.42	0.75	0.857

Pb exposure induced alteration of neural-related miRNA expression profiles

miRNA array analysis was adopted to detect the differences between miRNA expression of these two groups. Based on the miRNA selection criteria and our previous miRNA assay in rats, these 27 miRNAs were highly differentially expressed miRNAs (Fig. 1A). KEGG pathway classification revealed that the major target genes of these 27 miRNAs participated in regulation of axon guidance, neurotrophic signaling pathways, and synaptic vesicular transportation (Fig. 1B). In addition, the top 30 of enriched pathways predicted by both GO terms and KEGG terms showed that target genes of these miRNAs are enriched in synaptic vesicular transportation (Fig. 1C, D). The findings above suggested that these differentially expressed miRNAs are closely related with neural synaptic functions.

Fig. 1

miRNA expression profiles and bioinformatic analysis. A) Heat map of the log₂ –transformed expression levels of 27 miRNAs significantly changed (|FC| ≥ 1.5) between Low BLL group and High BLL group. B) Number of differentially expressed miRNAs (out of 27) where its predicted targets are enriched for KEGG terms. C) Top 30 of enriched pathways predicted by GO terms based on the mRNA targets of all differentially regulated miRNAs. D) Top 30 of enriched pathways predicted by KEGG terms based on the mRNA targets of all differentially regulated miRNAs.

miR-34b was the most liable to connect early-life Pb neurotoxicity and developmental neuropsychiatric diseases

To further confirm the expression of 27 miRNAs in two groups, RT- PCR was used to validate data of miRNA array. The values of miR-18a between the two group (low BLL group versus high BLL group) is 0.627±0.044 versus 0.787±0.063 (p < 0.001), and miR-34b is 1.205±0.077 versus 1.431±0.080 (p < 0.001), miR-106b is 0.939±0.078 versus 1.159±0.051 (p < 0.001), and miR-494 is 1.361±0.098 versus 1.008±0.079 (p < 0.001) (Fig. 2A-D). miR-106b, 34b, and 18a were significantly upregulated, miR-494 was downregulated, which was in good consistency with miRNA array data. The expression of the other 23 miRNAs showed no significant differences (p > 0.05, Table 2). It is worth mentioning that although the sequencing results showed that changes of miR-93 were more significant than that of miR-106b, 34b, 18a, and miR-494, no statistically significant difference of miR-93 was detected after verification by qRT-PCR.

Fig. 2

miRNA Validation and correlation analysis of miRNA in children population. A–D) miRNA levels of miR-106b, miR-34b, miR-18a, and miR-494 measured by qRT-PCR in Low BLL group and High BLL group (n = 20, ^*p < 0.05, mean±SEM). E) Venn diagram reporting overlapping miRNA between our sequencing results and miRNAs presumed to be associated with ADHD and ADSs in human blood subjects. F) Linear regression analysis for relationship between the expression of miRNAs and BLL.

Table 2

Values of miRNA expression with no statistically significant difference after verification by qRT-PCR

	Low BLL group (n = 20)		High BLL group (n = 20)
	Mean	SD	Mean	SD	p
miR-15b-5p	1.149	0.619	1.213	0.921	0.265
miR-16-1-3p	0.826	0.060	0.937	0.051	0.131
miR-17-5p	1.311	0.049	1.314	0.128	0.196
miR-185-3p	1.576	0.105	1.649	0.055	0.137
miR-21-5p	2.382	0.387	2.274	0.306	0.384
miR-30b-5p	1.341	0.136	1.229	0.110	0.132
miR-20a-5p	12.560	0.356	12.784	0.338	0.633
miR-326	2.034	0.673	2.161	0.452	0.311
miR-20b-5p	12.033	0.691	13.547	0.810	0.098
miR-23a-3p	1.237	0.174	1.348	0.119	0.212
miR-23b-5p	12.599	0.565	12.445	0.602	0.287
miR-424-3p	10.047	0.256	11.221	2.087	0.179
miR-409-3p	1.257	0.180	1.193	0.174	0.199
miR-93-5p	1.208	0.115	1.309	0.135	0.412
miR-466-5p	1.295	0.213	1.107	0.196	0.333
miR-539-5p	10.225	0.598	9.646	0.580	0.231
miR-150-3p	5.797	1.068	5.761	1.433	0.480
miR-151a-3p	1.693	0.782	2.108	0.821	0.356
miR-24-3p	4.949	0.809	4.097	0.862	0.240
miR-145-5p	4.989	0.921	4.245	0.938	0.271
miR-199a-5p	3.108	0.664	2.466	0.548	0.324
miR-513c-5p	2.011	0.603	2.283	0.640	0.089
miR-30c-1-3p	1.667	0.585	2.173	0.837	0.071

To analysis the relationship between BLL and children’s neuropsychiatric disorders, Venn analysis was conducted to filter the most liable miRNA involved. As shown in Fig. 2E, we summarized related miRNAs in ASD or ADHD detected in peripheral blood samples, and Venn analysis showed that miR-34b, miR-145 and miR-21-5p were changed both in Pb and ASDs. miR-34b, 18a-5p, and 106b-5p were altered both in Pb and ADHD. Intriguingly, miR-34b seems the most liable miRNA in Pb-induced neuropsychiatric disorders. In addition, linear regression analysis showed that the correlation coefficient between blood lead level and miR-34b is 0.247, p < 0.001, indicating that miR-34b was correlated with BLL.

Pb increased miR-34b expression in vivo and in vitro

To investigate the role of miR-34b in Pb neurotoxicity, a Pb exposure rat model was established. Administration with Pb for 4 w increased BLL to ∼100μg/L in 100 ppm group and ∼200μg/L in 300 ppm group, which is statistically significant to Con group. There was no significant difference among 4 w, 6 w, and 8 w (Fig. 3A). Morris water maze results showed that 300 ppm Pb treatment significantly affected rats’ spatial memory abilities compared with Con group, while neurotoxic effect of 100 ppm Pb exposure did not show effects on memory abilities (Fig. 3C, D). Next, transmission electron microscopy results showed that 300 ppm Pb exposure decreased densities of synaptic vesicles in hippocampus tissues (p < 0.05, Fig. 3I, J). RT-PCR data showed that 100 ppm and 300 ppm Pb exposure markedly upregulated miR-34b expression, and the enhancing effect was more evident in 300 ppm group than in 100 ppm group (p < 0.05, Fig. 3B). To further confirm the expression of miRNA in response to Pb treatment, PC12 cells and HT22 cells were applied for in vitro studies. PC12 cells data showed that miR-34b was upregulated after treatment with 5μM Pb for 24 h and 48 h, and the upregulation effect was more significant after treatment with 10μM Pb (Fig. 3E, G). miR-34b was also upregulated in response to Pb in HT22 cells, and the effect of 10μM Pb was much more evident than that of 5μM Pb (Fig. 3F, H).

Fig. 3

Pb increased miR-34b expression in vivo and in vitro. A) Effects of 100 ppm and 300 ppm lead exposure on blood lead in rats (n = 12, mean±SEM, ^*p < 0.05, ^** p < 0.01 versus Con group). B) Effects of lead exposure on blood miR-34b expression in rats (n = 3, mean±SEM, ^*p < 0.05, ^**p < 0.01 versus Con group). C) Representative rats’ frequency of shuttles to the target quadrant where the platform located in water maze test (n = 6, mean±SEM, ^*p < 0.05 versus Con group). D) Representative latency periods of rats finding the hidden platform (n = 6, mean±SEM, ^*p < 0.05 versus Con group). E–H) miR-34b expression in PC12 cells and HT22 cells in response to lead exposure (n = 3, mean±SEM, ^*p < 0.05, ^**p < 0.01 versus Con group). I) Vesicles in synaptic structures of rats’ hippocampus observed by transmission electron microscopy. J) Statistical analysis vesicle densities in synaptic structures (16 synapses/4 mice for all groups, mean±SEM, ^*p < 0.05).

Vesicle associated membrane protein 2 (VAMP2) is the target gene of miR-34b

Using bioinformatics algorithms PicTar, TargetScan and microRNA.org, we found that miR-34b was predicted as a potential binding motif on the 3^′UTR of vamp2 (vesicle associated membrane protein 2), nav1 (neuron navigator 1), traf3 (TNF receptor associated factor 3), jag1 (jagged canonical notch ligand 1), and ergic-32 (endoplasmic reticulum-Golgi intermediate compartment 32) (Fig. 4A). As VAMP2 is a synaptic vesicle protein and has been demonstrated to be closely associated with multiple neuropsychiatric and neurodegenerative disorders [19], we chose VAMP2 as the target gene for further research (Fig. 4B). This binding site was highly conserved among different species. To verify the combination of miR-34b and vamp2, luciferase reporter gene plasmid was constructed. The plasmid construction was shown in Fig. 4C. After transfecting the plasmid into PC12 cells, luciferase activity was significantly suppressed by co-transfection of miRNA-34b mimics, indicating that miR-34b combined with vamp2 3^′UTR region and interfered stabilities of vamp2 mRNA. To confirm the effect of Pb on vamp2 expression, immune electron microscopy data showed that Pb reduced the number of VAMP2-positive spots in rats’ hippocampus tissues (Fig. 4E, F). These results indicated that lead exposure resulted in upregulation of miR-34b via directly regulating vamp2 mRNA.

Fig. 4

Vesicle associated membrane protein 2 (VAMP2) is the target gene of miR-34b. A) Prediction of miR-34b target genes by bioinformatics methods. B) The binding site of miR-34b and VAMP2 mRNA. C, D) Diagram of luciferase reporter gene plasmid structure and results (^**p < 0.01). E) The expression of VAMP2 protein in synapses of rats’ hippocampus observed by immunoelectron microscopy. F) Statistical analysis of VAMP2 positive vesicles (n = 10, mean±SEM, ^*p < 0.05).

Pb exposure suppresses VMAP2 by regulating miR-34b in vitro

To further validate the relationship between VAMP2 and miR-34b in response to Pb exposure, HT22 cells and PC12 cells were adopted for in vitro studies. RT-PCR data showed that 5μM and 10μM Pb exposure markedly downregulated expression of vamp2 mRNA in both HT22 cells and PC12 cells after 24 h and 48 h, the impact on vamp2 mRNA was more evident in 10μM group than in 5μM group (Fig. 5A-D). Western blot data results showed that VAMP2 was significantly downregulated after exposure to 10μM Pb, while 5μM Pb exposure showed no obvious effect on the protein levels of VAMP2 in PC12 cells (Fig. 5E, F). Similar results were found that 10μM Pb exposure, not 5μM Pb, decreased VAMP2 protein levels in HT22 cells (Fig. 5G, H). To determine the effect of miR-34b on regulating VAMP2, inhibition of miR-34b was performed in PC12 cells. RT-PCR data showed that miR-34b was notably downregulated after transfection of miR-34b inhibitor in PC12 cells, suggesting that the transfection was effective (Fig. 5I). Vamp2 mRNA showed significant upregulation after the inhibition of miR-34b in PC12 cells (Fig. 5J), indicating that miR-34b did regulate the protein expression of VAMP2. To further investigate the effects of miR-34b on regulating VAMP2, western blot data showed that inhibition of miR-34b significantly reversed the downregulation of VAMP2 induced by 5μM and 10μM Pb exposure in PC12 cells (Fig. 5I, J). Compared with NC + 5μM Pb group, VAMP2 protein level was increased in si-miR-34b + 5μM Pb group in PC12 cells (p < 0.05). For HT22 cells, inhibition of miR-34b significantly increased vamp2 mRNA level, even with Pb treatment (Fig. 5K, L). These data suggested that miR-34b was involved in the downregulation of VAMP2 caused by Pb exposure.

Fig. 5

Pb exposure suppresses VMAP2 by regulating miR-34b in vitro. A–D) mRNA levels of Vamp2 in leads exposed HT22 cells and PC12 cells measured by RT-PCR (n = 3, mean±SEM, ^*p < 0.05, ^**p < 0.01 versus Con group). E-H) Protein levels of VAMP2 in leads exposed HT22 cells and PC12 cells measured by western blotting (n = 3, mean±SEM, ^*p < 0.05 versus Con group). I) Representative immunoblotting of VAMP2 in PC12 cells after lead exposure with or without inhibition of miR-34b. J) Gray intensity analysis of VAMP2 (n = 3, mean±SEM). K) Levels of miR-34b in leads exposed HT22 cells after transfection of miR-34b inhibitor (n = 3, mean±SEM, ^*p < 0.05). L) mRNA levels of Vamp2 in leads exposed HT22 cells after transfection of miR-34b inhibitor (n = 3, mean±SEM, ^*p < 0.05).

DISCUSSION

A growing number of scientific evidence have confirmed that Pb can give rise to multiple systems disorders, and CNS is the top target in acute or chronic lead exposure. Studies revealed that Pb exposure in children stage may lead to long-lasting epigenetic, neurological, and behavioral changes, which may significantly elevate the risk of neurodegenerative diseases (such as AD and Parkinson’s disease) in old age [37, 38]. Reports of Lanphear et al. suggested an intelligence decline correlated with blood lead concentrations below 100μg/L [39]. Blood lead levels are also particularly correlated with symptoms of hyperactivity, impulsivity, and inattention in children [40 –42]. Recent studies have revealed the relationship between blood lead level and ADHD in children [43, 44]. ADHD is a common neurodevelopmental disorder with a global prevalence of up to 5% –10% in children, and the pathogenesis is yet to be clarified [45]. A meta-analysis recently suggested that even a blood lead level of lower than 30μg/L may still be related with symptoms of ADHD in children [46], indicating that Pb exposure plays an important role in the triggering of ADHD. Studies also revealed that Pb exposure may induce the onset of ASDs [8, 47]. Based on previous studies, we tried to investigate the underlying approach of Pb on neurodevelopmental disorders. To be honest, blood lead level was not the best parameter in response to Pb toxicity, compared to bone lead level, but its accessibility made it perfect under our present condition. In our present studies, we firstly analyzed health indexes, trace elements and blood examination between the low BLL group and high BLL group, and no significant difference was found in health indexes and blood examination, suggesting that the difference of BLL might not affect physical development. Copper levels were significantly different between the two groups, and this might be due to geographical factors. For some uncontrollable reason, we failed to perform intelligence or behavioral tests in children population. As a result, we used in vivo and in vitro experiments to validate the possible mechanism between Pb and developmental neuropsychiatric disorders. Developmental rats’ Pb exposure model has confirmed Pb’s damaging effects on behavioral and synaptic functions, but the effective dosage in rats was higher than in children, which may be due to species diversity. Results of two neural cell lines were in consistence with rats’ results, which further confirmed Pb’s neurotoxicity.

The regulation of miRNAs in neurodegenerative diseases has been widely investigated. Alternations of miRNAs were detected in plasma and cerebrospinal fluid of AD patients, involving processes of amyloid-β plaques [48], cognitive impairments [49], inflammation [50], and especially synaptic functions [51, 52]. Synaptic dysfunction is a common and critical factor among many neurological disorders. Synaptic failure induced by loss of dendritic spines has been associated with the cognitive impairment in multiple pathologies such as AD [53]. In modulation of synaptic functions, miRNAs can regulate aspects such as dendritic outgrowth, spine morphology, and density of dendritic spines, contributing to the structural and functional organization of synapses as well as synaptic strength and excitability [54, 55]. In this context, it has already demonstrated that miRNAs play essential roles in the synaptic regulation and neural survival, thus influencing cognitive function in both physiological and pathological conditions [53]. Besides, researchers have also proofed variations in miRNAs of ADHD subjects [56]. The expression of miRNAs, as potential signatures of environmental exposure, was also a potential biomarker of lead exposure [36 , 58]. Based on these previous studies, we tried to investigate whether there existed some miRNAs changed both in Pb exposure and neuropsychiatric disorders. In our data, miRNA chip array and subsequence validation revealed that 4 miRNAs, miR-18a, 34b, 106b, and 494, were highly differentially altered. Previous studies have shown effects of miR-18a, 106b, and 494 in the neural-related field. miR-18a was previously identified as apoptotic inducer [59]. Plasma levels of miR-18a were related to spinocerebellar ataxia type 7 (SCA7), an inherited neurodegenerative disorder [60]. The miR-106b expression change is associated with stress-induced process [61]. Hebert et al. found that the expression level of miR-106b was decreased in patients with sporadic AD [62], and upregulation of miR-106b has been shown to have protective effects on in vitro models of AD [63, 64]. Raised level of miR-106b also alleviated neuronal apoptosis and enhance neuronal autophagy in Parkinson’s disease [65]. Our previous studies also confirmed the role of miR-106b in Pb-induced neural damages [36]. Additionally, miR-494 reduces DJ-1 expression and exacerbates neurodegeneration [66], and our previous study found that miR-494 was involved in Pb-induced hippocampus injuries [14]. In the present study, miR-34b was determined to be our research target based on the intersection of existing research on ADHD and ASDs. Correlation analysis has confirmed the relationship between miR-34b and BLL, and the upregulation of miR-34b was confirmed in animal and cell experiments. Bioinformatic analysis showed that some target genes of miR-34b are highly related to neural functions. Among these target genes, Nav 1 participates in regulation of axonal growth, Jag1 participates in maintaining peripheral nerve integrity through Notch pathway, and Vamp2 participates in maintaining synaptic homeostasis [67 –69]. It is worth mentioning that although miR-449-5p, 34a, 34b, and 34c have the same seed sequence, and belong to a functionally related miRNA family, only miR-34b were altered between high BLL group and low BLL group according to the present study. These microRNAs share similar functions and alter expressions of all these microRNAs may lead to severe disturbances in nervous system development [70]. Our subsequent studies will focus on why only miR-34b was affected by high blood lead, while other microRNAs belonging to the same gene cluster were not significantly changed.

Pb exerted neurotoxicity by multiple mechanisms, including regulation of Ca^2 +-mediated homeostasis [71], altering releasing and uptake of neurotransmitters [72, 73], and inhibiting synaptic plasticity [17]. VAMP2 is a small molecule SNARE protein, which is mainly composed of a short N-terminal sequence, a SNARE sequence, and a C-terminal transmembrane domain. Studies showed that knocking out of VAMP2 affected the release of neurotransmitters in mice brain [69, 74]. VAMP2 is closely related to learning and memory functions, including the extension of axons and the plasticity of synapses in response to damages of the CNS [75]. It is reported that learning and memory functions of rats were impaired after exposure to formaldehyde, and these impairments might be related to alternations in protein expression of SNARE in hippocampal synaptosome [76]. Our data revealed that expression of VAMP2 in the presynaptic membrane of hippocampal tissues decreased significantly after 300 ppm of Pb exposure, indicating that Pb exposure might exert injuring effects on synapses by affecting VAMP2. VAMP2 was the target gene of miR-34b, and luciferase reporter assay and miR-34b inhibitor experiments confirmed the regulating effect of miR-34b on VAMP2. Combined with electron microscopy results which revealed decreases in the number of neuronal synaptic vesicles after lead exposure, we speculated that lead exposure affected the stability of vamp2 mRNA and its protein expression through miR-34b, which affected the formation and release of synaptic vesicles. Further studies are required to confirm the role of miR-34b and VAMP2 in lead-induced neurotoxicity.

Conclusions

Our studies indicated that early-life Pb exposure might exert synapse-toxic effects via inhibiting VAMP2 mediated by upregulation of miR-34b and shed a light on the underlying relationship between Pb neurotoxicity and developmental neuropsychiatric disorders.

Footnotes

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China (81872576, 81903274, 82020108026).

Authors’ disclosures available online ().

References

Dórea

(2019) Environmental exposure to low-level lead (Pb) co-occurring with other neurotoxicants in early life and neurodevelopment of children. Environ Res 177, 108641.

Santa Maria

, Hill

, Kline

(2019) Lead (Pb) neurotoxicology and cognition. Appl Neuropsychol Child 8, 272–293.

Wang

, Zhang

, Xu

(2020) Epigenetic basis of lead-induced neurological disorders. Int J Environ Res Public Health 17, 4878.

Fang

, Lu

, Liang

, Peng

, Aschner

, Jiang

(2021) Signal transduction associated with lead-induced neurological disorders: A review. Food Chem Toxicol 150, 112063.

Mason

, Harp

, Han

(2014) Pb neurotoxicity: Neuropsychological effects of lead toxicity. Biomed Res Int 2014, 840547.

Téllez-Rojo

, Hernández-Avila

, Lamadrid-Figueroa

, Smith

, Hernández-Cadena

, Mercado

, Aro

, Schwartz

, Hu

(2004) Impact of bone lead and bone resorption on plasma and whole blood lead levels during pregnancy. Am J Epidemiol 160, 668–678.

Muñoz

, Rubilar

, Valdés

, Muñoz-Quezada

, Gómez

, Saavedra

, Iglesias

(2020) Attention deficit hyperactivity disorder and its association with heavy metals in children from northern Chile. Int J Hyg Environ Health 226, 113483.

Skogheim

, Weyde

, Engel

, Aase

, Surén

, Øie

, Biele

, Reichborn-Kjennerud

, Caspersen

, Hornig

, Haug

, Villanger

(2021) Metal and essential element concentrations during pregnancy and associations with autism disorder and attention-deficit/hyperactivity spectrum disorder in children. Environ Int 152, 106468.

Donzelli

, Carducci

, Llopis-Gonzalez

, Verani

, Llopis-Morales

, Cioni

, Morales-Suárez-Varela

(2019) The association between lead and attention-deficit/hyperactivity disorder: A systematic review. Int J Environ Res Public Health 16, 382.

10.

Cao

, Yeo

, Muotri

, Kuwabara

, Gage

(2006) Noncoding RNAs in the mammalian central nervous system. Annu Rev Neurosci 29, 77–103.

11.

Hou

, Wang

, Baccarelli

(2011) Environmental chemicals and microRNAs. Mutat Res 714, 105–112.

12.

Guo

, Yang

, Zhang

, Chen

, Ren

, Gao

(2019) Associations of blood levels of trace elements and heavy metals with metabolic syndrome in Chinese male adults with microRNA as mediators involved. Environ Pollut 248, 66–73.

13.

Wallace

, Taalab

, Heinze

, Tariba Lovaković

, Pizent

, Renieri

, Tsatsakis

, Farooqi

, Javorac

, Andjelkovic

, Bulat

, Antonijević

, Buha Djordjevic

(2020) Toxic-metal-induced alteration in miRNA expression profile as a proposed mechanism for disease development. Cells 9, 901.

14.

, Cai

, Che

, Yu

, Cao

, Liu

, Zhao

, Jing

, Shen

, Liu

, Du

, Chen

, Luo

(2014) The changes of miRNA expression in rat hippocampus following chronic lead exposure. Toxicol Lett 229, 158–166.

15.

, Zhao

, Cao

, Zhang

, Aschner

, Luo

(2015) Mir-203-mediated tricellulin mediates lead-induced in vitro loss of blood-cerebrospinal fluid barrier (BCB) function. Toxicol In Vitro 29, 1185–1194.

16.

Paul

, Reyes

, Garza

, Sharma

(2020) MicroRNAs and child neuropsychiatric disorders: A brief review. Neurochem Res 45, 232–240.

17.

Zhao

, Du

, Wang

, Cao

, Chen

, Song

, Zheng

, Shen

(2021) Maternal lead exposure impairs offspring learning and memory via decreased GLUT4 membrane translocation. Front Cell Dev Biol 9, 648261.

18.

Zylbersztejn

, Galli

(2011) Vesicular traffic in cell navigation. FEBS J 278, 4497–4505.

19.

Margiotta

(2021) Role of SNAREs in neurodegenerative diseases. Cells 10, 991.

20.

Huang

, Sherman

, Lempicki

(2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44–57.

21.

Huang

, Sherman

, Lempicki

(2009) Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37, 1–13.

22.

Ashburner

, Ball

, Blake

, Botstein

, Butler

, Cherry

, Davis

, Dolinski

, Dwight

, Eppig

, Harris

, Hill

, Issel-Tarver

, Kasarskis

, Lewis

, Matese

, Richardson

, Ringwald

, Rubin

, Sherlock

(2000) Gene ontology: Tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25, 25–29.

23.

Xiong

, Sun

, Jiang

, Ma

, Zhang

(2020) ASDmiR: A stepwise method to uncover miRNA regulation related to autism spectrum disorder. Front Genet 11, 562971.

24.

, Li

, Zheng

(2020) Recent progress on relevant microRNAs in autism spectrum disorders. Int J Mol Sci 21, 5904.

25.

Jyonouchi

, Geng

, Toruner

, Rose

, Bennuri

, Frye

(2019) Serum microRNAs in ASD: Association with monocyte cytokine profiles and mitochondrial respiration. Front Psychiatry 10, 614.

26.

Popov

, Minchev

, Naydenov

, Minkov

, Vachev

(2018) Investigation of circulating serum microRNA-328-3p and microRNA-3135a expression as promising novel biomarkers for autism spectrum disorder. Balkan J Med Genet 21, 5–12.

27.

Vaccaro

, Sorrentino

, Salvador

, Veit

, Souza

, de Almeida

(2018) Alterations in the microRNA of the blood of autism spectrum disorder patients: Effects on epigenetic regulation and potential biomarkers. Behav Sci (Basel) 8, 75.

28.

Kichukova

, Popov

, Ivanov

, Vachev

(2017) Profiling of circulating serum MicroRNAs in children with autism spectrum disorder using Stem-loop qRT-PCR assay. Folia Med (Plovdiv) 59, 43–52.

29.

Mundalil Vasu

, Anitha

, Thanseem

, Suzuki

, Yamada

, Takahashi

, Wakuda

, Iwata

, Tsujii

, Sugiyama

, Mori

(2014) Serum microRNA profiles in children with autism. Mol Autism 5, 40.

30.

Nakata

, Kimura

, Funabiki

, Awaya

, Murai

, Hagiwara

(2019) MicroRNA profiling in adults with high-functioning autism spectrum disorder. Mol Brain 12, 82.

31.

Aydin

, Kabukcu Basay

, Cetin

, Gungor Aydin

, Tepeli

(2019) Altered microRNA 5692b and microRNA let-7d expression levels in children and adolescents with attention deficit hyperactivity disorder. J Psychiatr Res 115, 158–164.

32.

Nuzziello

, Craig

, Simone

, Consiglio

, Licciulli

, Margari

, Grillo

, Liuni

, Liguori

(2019) Integrated analysis of microRNA and mRNA expression profiles: An attempt to disentangle the complex interaction network in attention deficit hyperactivity disorder. Brain Sci 9, 288.

33.

Kandemir

, Erdal

, Selek

, Ay

, Karababa

, Kandemir

, Ay

, Yılmaz

, Bayazıt

, Taşdelen

(2014) Evaluation of several micro RNA (miRNA) levels in children and adolescents with attention deficit hyperactivity disorder. Neurosci Lett 580, 158–162.

34.

Coskun

, Karadag

, Gokcen

, Oztuzcu

(2021) miR-132 and miR-942 Expression levels in children with attention deficit and hyperactivity disorder: A Controlled Study. Clin Psychopharmacol Neurosci 19, 262–268.

35.

, Zhang

, Wang

, Aschner

, Cao

, Zhao

, Wang

, Chen

, Luo

(2016) Genistein alleviates lead-induced neurotoxicity in vitro and in vivo: Involvement of multiple signaling pathways. Neurotoxicology 53, 153–164.

36.

Xue

, Kang

, Su

, Wang

, Zhao

, Zhang

, Wang

, Lang

, Cao

(2020) MicroRNA-106b-5p participates in lead (Pb)-induced cell viability inhibition by targeting XIAP in HT-22 and PC12 cells. Toxicol In Vitro 66, 104876.

37.

Reuben

(2018) Childhood lead exposure and adult neurodegenerative disease. J Alzheimers Dis 64, 17–42.

38.

Mazumdar

, Xia

, Hofmann

, Gregas

, Ho Sui

, Hide

, Yang

, Needleman

, Bellinger

(2012) Prenatal lead levels, plasma amyloid β levels, and gene expression in young adulthood. Environ Health Perspect 120, 702–707.

39.

Lanphear

, Hornung

, Khoury

, Yolton

, Baghurst

, Bellinger

, Canfield

, Dietrich

, Bornschein

, Greene

, Rothenberg

, Needleman

, Schnaas

, Wasserman

, Graziano

, Roberts

(2005) Low-level environmental lead exposure and children’s intellectual function: An international pooled analysis. Environ Health Perspect 113, 894–899.

40.

Hong

, Im

, Kim

, Park

, Shin

, Kim

, Yoo

, Cho

, Bhang

, Hong

, Cho

(2015) Environmental lead exposure and attention deficit/hyperactivity disorder symptom domains in a community sample of South Korean school-age children. Environ Health Perspect 123, 271–276.

41.

Huang

, Hu

, Sánchez

, Peterson

, Ettinger

, Lamadrid-Figueroa

, Schnaas

, Mercado-García

, Wright

, Basu

, Cantonwine

, Hernández-Avila

, Téllez-Rojo

(2016) Childhood blood lead levels and symptoms of attention deficit hyperactivity disorder (ADHD): A cross-sectional study of mexican children. Environ Health Perspect 124, 868–874.

42.

Joo

, Lim

, Ha

, Kwon

, Yoo

, Choi

, Paik

(2017) Secondhand smoke exposure and low blood lead levels in association with attention-deficit hyperactivity disorder and its symptom domain in children: A community-based case-control study. Nicotine Tob Res 19, 94–101.

43.

Braun

, Kahn

, Froehlich

, Auinger

, Lanphear

(2006) Exposures to environmental toxicants and attention deficit hyperactivity disorder in U.S. children. Environ Health Perspect 114, 1904–1909.

44.

Liu

, Liu

, Wang

, McCauley

, Pinto-Martin

, Wang

, Li

, Yan

, Rogan

(2014) Blood lead concentrations and children’s behavioral and emotional problems: A cohort study. JAMA Pediatr 168, 737–745.

45.

Polanczyk

, de Lima

, Horta

, Biederman

, Rohde

(2007) The worldwide prevalence of ADHD: A systematic review and metaregression analysis. Am J Psychiatry 164, 942–948.

46.

, Ning

, Huang

(2019) Low blood lead levels and attention-deficit hyperactivity disorder in children: A systematic review and meta-analysis. Environ Sci Pollut Res Int 26, 17875–17884.

47.

Goel

, Aschner

(2021) The effect of lead exposure on autism development. Int J Mol Sci 22, 1637.

48.

Cosín-Tomás

, Antonell

, Lladó

, Alcolea

, Fortea

, Ezquerra

, Lleó

, Martí

, Pallás

, Sanchez-Valle

, Molinuevo

, Sanfeliu

, Kaliman

(2017) Plasma miR-34a-5p and miR-545-3p as early biomarkers of Alzheimer’s disease: Potential and limitations. Mol Neurobiol 54, 5550–5562.

49.

Giuliani

, Gaetani

, Sorgentoni

, Agarbati

, Laggetta

, Matacchione

, Gobbi

, Rossi

, Galeazzi

, Piccinini

, Pelliccioni

, Bonfigli

, Procopio

, Albertini

, Sabbatinelli

, Olivieri

, Fazioli

(2021) Circulating inflamma-miRs as potential biomarkers of cognitive impairment in patients affected by Alzheimer’s disease. Front Aging Neurosci 13, 647015.

50.

, Aloi

, Garden

(2016) MicroRNAs mediating CNS inflammation: Small regulators with powerful potential. Brain Behav Immun 52, 1–8.

51.

Salpietro

, Malintan

, Llano-Rivas

, Spaeth

, Efthymiou

, Striano

, Vandrovcova

, Cutrupi

, Chimenz

, David

, Di Rosa

, Marce-Grau

, Raspall-Chaure

, Martin-Hernandez

, Zara

, Minetti

, Bello

, De Zorzi

, Fortuna

, Dauber

, Alkhawaja

, Sultan

, Mankad

, Vitobello

, Thomas

, Mau-Them

, Faivre

, Martinez-Azorin

, Prada

, Macaya

, Kullmann

, Rothman

, Krishnakumar

, Houlden

(2019) Mutations in the neuronal vesicular SNARE VAMP2 affect synaptic membrane fusion and impair human neurodevelopment. Am J Hum Genet 104, 721–730.

52.

Wang

, Qin

, Tang

(2019) microRNAs in Alzheimer’s disease. Front Genet 10, 153.

53.

Reza-Zaldivar

, Hernández-Sápiens

, Minjarez

, Gómez-Pinedo

, Sánchez-González

, Márquez-Aguirre

, Canales-Aguirre

(2020) Dendritic spine and synaptic plasticity in Alzheimer’s disease: a focus on microRNA. Front Cell Dev Biol 8, 255.

54.

Olde Loohuis

, Kos

, Martens

, Van Bokhoven

, Nadif Kasri

, Aschrafi

(2012) microRNA networks direct neuronal development and plasticity. Cell Mol Life Sci 69, 89–102.

55.

, Xu

, Su

, He

(2016) Role of microRNA in governing synaptic plasticity. Neural Plast 2016, 4959523.

56.

Zadehbagheri

, Hosseini

, Bagheri-Hosseinabadi

, Rekabdarkolaee

, Sadeghi

(2019) Profiling of miRNAs in serum of children with attention-deficit hyperactivity disorder shows significant alterations. J Psychiatr Res 109, 185–192.

57.

Masoud

, Bihaqi

, Alansi

, Dash

, Subaiea

, Renehan

, Zawia

(2018) Altered microRNA, mRNA, and protein expression of neurodegeneration-related biomarkers and their transcriptional and epigenetic modifiers in a human tau Transgenic mouse model in response to developmental lead exposure. J Alzheimers Dis 63, 273–282.

58.

Ochoa-Martínez

, Varela-Silva

, Orta-García

, Carrizales-Yáñez

, Pérez-Maldonado

(2021) Lead (Pb) exposure is associated with changes in the expression levels of circulating miRNAS (miR-155, miR-126) in Mexican women. Environ Toxicol Pharmacol 83, 103598.

59.

Ferreira

, Santos

, Amar

, Gong

, Chen

, Tahara

, Giannotta

, Hofman

(2014) Argonaute-2 promotes miR-18a entry in human brain endothelial cells. J Am Heart Assoc 3, e000968.

60.

Borgonio-Cuadra

, Valdez-Vargas

, Romero-Córdoba

, Hidalgo-Miranda

, Tapia-Guerrero

, Cerecedo-Zapata

, Hernández-Hernández

, Cisneros

, Magaña

(2019) Wide profiling of circulating microRNAs in spinocerebellar ataxia type 7. Mol Neurobiol 56, 6106–6120.

61.

, Luna

, Qiu

, Epstein

, Gonzalez

(2009) Alterations in microRNA expression in stress-induced cellular senescence. Mech Ageing Dev 130, 731–741.

62.

Hébert

, Horré

, Nicolaï

, Bergmans

, Papadopoulou

, Delacourte

, De Strooper

(2009) microRNA regulation of Alzheimer’s amyloid precursor protein expression. Neurobiol Dis 33, 422–428.

63.

Pan

, Guo

, Xue

, Tu

(2021) Estradiol exerts a neuroprotective effect on SH-SY5Y cells through the miR-106b-5p/TXNIP axis. J Biochem Mol Toxicol 35, e22861.

64.

Madadi

, Saidijam

, Yavari

, Soleimani

(2020) Downregulation of serum miR-106b: A potential biomarker for Alzheimer disease. Arch Physiol Biochem, doi: 10.1080/13813455.2020.1734842.

65.

Bai

, Dong

, Zhao

, Yao

, Wang

(2021) microRNA-106b-containing extracellular vesicles affect autophagy of neurons by regulating CDKN2B in Parkinson’s disease. Neurosci Lett 760, 136094.

66.

Xiong

, Wang

, Zhao

, Li

, Chen

, Zhang

, Wang

, Wu

, Li

, Ding

, Chen

(2014) microRNA-494 reduces DJ-1 expression and exacerbates neurodegeneration. Neurobiol Aging 35, 705–714.

67.

Sullivan

, Motley

, Johnson

, Aisenberg

, Marshall

, Barwick

, Kong

, Huh

, Saavedra-Rivera

, McEntagart

, Marion

, Hicklin

, Modarres

, Baple

, Farah

, Zuberi

, Lutz

, Gaudet

, Traynor

, Crosby

, Sumner

(2020) Dominant mutations of the Notch ligand Jagged1 cause peripheral neuropathy. J Clin Invest 130, 1506–1512.

68.

Muley

, McNeill

, Marzinke

, Knobel

, Barr

, Clagett-Dame

(2008) The atRA-responsive gene neuron navigator 2 functions in neurite outgrowth and axonal elongation. Dev Neurobiol 68, 1441–1453.

69.

Schoch

, Deák

, Königstorfer

, Mozhayeva

, Sara

, Südhof

, Kavalali

(2001) SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294, 1117–1122.

70.

, Bao

, Kim

, Yuan

, Tang

, Zheng

, Mastick

, Xu

, Yan

(2014) Two miRNA clusters, miR-34b/c and miR-449, are essential for normal brain development, motile ciliogenesis, and spermatogenesis. Proc Natl Acad Sci U S A 111, E2851–2857.

71.

Loikkanen

, Naarala

, Savolainen

(1998) Modification of glutamate-induced oxidative stress by lead: The role of extracellular calcium. Free Radic Biol Med 24, 377–384.

72.

Bressler

, Belloni-Olivi

, Forman

, Goldstein

(1996) Distinct mechanisms of neurotransmitter release from PC 12 cells exposed to lead. J Neurosci Res 46, 678–685.

73.

Suszkiw

(2004) Presynaptic disruption of transmitter release by lead. Neurotoxicology 25, 599–604.

74.

Deák

, Shin

, Kavalali

, Südhof

(2006) Structural determinants of synaptobrevin 2 function in synaptic vesicle fusion. J Neurosci 26, 6668–6676.

75.

Montecucco

, Schiavo

, Pantano

(2005) SNARE complexes and neuroexocytosis: How many, how close? Trends Biochem Sci 30, 367–372.

76.

Liu

, Ye

, Yang

, Zhou

, Fan

, He

, Chui

(2010) Disturbances of soluble N-ethylmaleimide-sensitive factor attachment proteins in hippocampal synaptosomes contribute to cognitive impairment after repetitive formaldehyde inhalation in male rats. Neuroscience 169, 1248–1254.