Profile of N6-methyladenosine of Pb-exposed neurons presents epitranscriptomic alterations in PI3K-AKT pathway-associated genes.

Lead (Pb) is a pervasive heavy metal with multi-organ toxicity. However, the molecular mechanisms of Pb-induced neurotoxicity are not fully understood. The dynamics of N6-methylademine (m6A) is an emerging regulatory mechanism for gene expression, which is closely related to nervous system diseases. To elucidate the association between m6A modification and Pb-mediated neurotoxicity, primary hippocampal neurons exposed to 5 µM Pb for 48 h were used as the paradigm neurotoxic model in this study. According to the results, Pb exposure reprogrammed the transcription spectrum. Simultaneously, Pb exposure remodeled the transcriptome-wide distribution of m6A while disrupting the overall level of m6A in cellular transcripts. United analysis of MeRIP-Seq and RNA-Seq was applied to further identify the core genes whose expression levels are regulated by m6A in the process of lead-induced nerve injury. GO and KEGG analysis unveiled that the modified transcripts were overrepresented by the PI3K-AKT pathway. Mechanically, we elucidated the regulatory role of the methyltransferase like3 (METTL3) in the process of lead-induced neurotoxicity and the downregulation of the PI3K-AKT pathway. In conclusion, our novel findings shed new light on the functional roles of m6A modification in the expressional alternations of downstream transcripts caused by lead, providing an innovative molecular basis to explain Pb neurotoxicity.

Bisphenol-A exposure leads to neurotoxicity through upregulating the expression of histone deacetylase 2 in vivo and in vitro.

Bisphenol-A (BPA), an environmental endocrine disruptor, is toxic to the central nervous system. Although recent studies have shown BPA-induced neurotoxicity, it is far from clear what precisely epigenetic mechanisms are involved in BPA-induced cognitive deficits. In this study, pheochromocytoma (PC12) cells were treated with BPA at 1 µM for 36 h in vitro. In vivo, C57BL/6 mice were administered to BPA at a dose of 1 mg/kg/day for 10 weeks. The results showed that 1 µM BPA exposure for 36 h impaired neurite outgrowth of PC12 cells through decreasing the primary and secondary branches. Besides, BPA exposure decreased the level of Ac-H3K9 (histone H3 Lys9 acetylation) by upregulating the expression of HDAC2 (histone deacetylases 2) in PC12 cells. Furthermore, treatment of both TSA (Trichostatin A, inhibitor of the histone deacetylase) and shHDAC2 plasmid (HDAC2 knockdown construct) resulted in amelioration neurite outgrowth deficits induced by BPA. In addition, it was shown that repression of HDAC2 could markedly rescue the spine density impairment in the hippocampus and prevent the cognitive impairment caused by BPA exposure in mice. Collectively, HDAC2 plays an essential role in BPA-induced neurotoxicity, which provides a potential molecular target for medical intervention.

Nuclear accumulation of histone deacetylase 4 (HDAC4) by PP1-mediated dephosphorylation exerts neurotoxicity in Pb-exposed neural cells.

Lead (Pb) is an environmental contaminant that primarily affects the central nervous system, particularly the developing brain. Recently, increasing evidence indicates the important roles of histone deacetylases (HDACs) in Pb-induced neurotoxicity. However, the precise molecular mechanisms involving HDAC4 remains unknown. The purpose of this study was to investigate the role of HDAC4 in Pb-induced neurotoxicity both in vivo and in vitro. In vitro study, PC12 cells were exposed to Pb (10 µM) for 24 h, then the mRNA and protein levels of HDAC4 were analyzed. In vivo study, pregnant rats and their female offspring were treated with lead (50 ppm) until postnatal day 30. Then the pups were sacrificed and the mRNA and protein levels of HDAC4 in the hippocampus were analyzed. The results showed that HDAC4 was significantly increased in both PC12 cells and rat hippocampus upon Pb exposure. Blockade of HDAC4 with either LMK-235 (an inhibitor of HDAC4) or shHDAC4 (HDAC4-knocking down plasmid) ameliorated the Pb-induced neurite outgrowth deficits. Interestingly, HDAC4 was aberrantly accumulated in the nucleus upon Pb exposure. By contrast, blocking the HDAC4 shuffling from the cytosol to the nucleus with ΔNLS2-HDAC4 (the cytosol-localized HDAC4 mutant) was able to rescue the neuronal impairment. In addition, Pb increased PP1 (protein phosphatase 1) expression which in turn influenced the subcellular localization of HDAC4 by dephosphorylation of specific serine/threonine residues. What's more, blockade of PP1 with PP1-knocking down construct (shPP1) ameliorated Pb-induced neurite outgrowth deficits. Taken together, nuclear accumulation of HDAC4 by PP1-mediated dephosphorylation involved in Pb-induced neurotoxicity. This study might provide a promising molecular target for medical intervention with environmental cues.

Interplay of miR-137 and EZH2 contributes to the genome-wide redistribution of H3K27me3 underlying the Pb-induced memory impairment.

Compromised learning and memory is a common feature of multiple neurodegenerative disorders. A paradigm spatial memory impairment could be caused by developmental lead (Pb) exposure. Growing evidence implicates epigenetic modifications in the Pb-mediated memory deficits; however, how histone modifications exemplified by H3K27me3 (H3 Lys27 trimethylation) contribute to this pathogenesis remains poorly understood. Here we found that Pb exposure diminished H3K27me3 levels in vivo by suppressing EZH2 (enhancer of zeste homolog 2) expression at an early stage. EZH2 overexpression in Pb-treated rats rescued the H3K27me3 abundance and partially restored the normal spatial memory, as manifested by the rat performance in a Morris water maze test, and structural analysis of hippocampal spine densities. Furthermore, miR-137 and EZH2 constitute mutually inhibitory loop to regulate the H3K27me3 level, and this feedback regulation could be specifically activated by Pb treatment. Considering genes targeted by H3K27me3, ChIP-chip (chromatin immunoprecipitation on chip) studies revealed that Pb could remodel the genome-wide distribution of H3K27me3, represented by pathways like transcriptional regulation, developmental regulation, cell motion, and apoptosis, as well as a novel Wnt9b locus. As a Wnt isoform associated with canonical and noncanonical signaling, Wnt9b was regulated by the opposite modifications of H3K4me3 (H3 Lys4 trimethylation) and H3K27me3 in Pb-exposed neurons. Rescue trials further validated the contribution of Wnt9b to Pb-induced neuronal impairments, wherein canonical or noncanonical Wnt signaling potentially exhibited destructive or protective roles, respectively. In summary, the study reveals an epigenetic-based molecular change underlying Pb-triggered spatial memory deficits, and provides new potential avenues for our understanding of neurodegenerative diseases with environmental etiology.

Identification and Functional Characterization of a New Splicing Variant of EZH2 in the Central Nervous System.

EZH2 plays vital roles in epigenetic regulation, neuronal development and cancer progression. Here a novel EZH2 variant, namely EZH2-X9 (X9 for short) resulting from alternative splicing, was isolated, identified and functionally characterized. X9 was highly expressed in the brains of SD rats, indicating a potentially distinguished role in the central nervous system (CNS). Owing to a transcript profiling, X9 was enriched in multiple brain regions at very early stage of life. Immunostaining validated the presence of the protein form of X9, which was localized similarly with the wild-type form, EZH2-WT. To investigate the functional consequence of X9, genetic intervention was performed in PC-12 cell line, a classic cellular model for neuronal development. It revealed that the depletion of either variant was sufficient to impair neuronal proliferation and differentiation significantly, an evidence that roles of X9 could not be complemented by EZH2-WT. Considering epigenetic regulation, X9 lost the capability to recruit the histone mark H3K27me3, but retained the cooperation with EED, as well as the repressive aspects in governing gene expression. Nonetheless, through profiling the genes affected, it's discovered that EZH2-WT and X9 markedly differed in their regulatory targets, as X9 intended to repress cell cycle- and autophagy-related genes, like GSK and MapILC3. Overall, a novel Ezh2 variant was characterized in the mammal CNS, providing insight with the structural and functional delineation of this key developmental switch, Ezh2.

Pb disrupts autophagic flux through inhibiting the formation and activity of lysosomes in neural cells.

Lead (Pb) has long been known as a metallic toxin to exert detrimental effects on human health, particularly on the central nervous system (CNS). Misregulated autophagy was regularly associated with multiple cellular dysfunctions and human diseases. However, the role of autophagy underlying Pb-induced neurotoxicity remains to be elucidated. In this study, we demonstrated that Pb promoted the accumulation of autophagosomes in PC12 cells, and subsequent findings revealed that this autophagosome accumulation was primarily caused by the inhibition of autophagic flux. Moreover, the results showed that Pb affected autophagy course through increasing Beclin 1 and ATG5 expression levels. Specifically, by double labeling with LC3-II (a marker of autophagosome) and LAMP-1 (a marker of lysosome), Pb impaired fusion between autophagosomes and lysosomes. Additionally, Pb exposure significantly reduced the number or size of lysosomes via decreasing the level of LAMP1, which is confirmed by the LysoTracker Red staining. Furthermore, the impairment of lysosomal activity was also signaled by the altered pH value of this acidic organelle. Overall, Pb exposure led to injuries of autophagy of neural cells through inhibiting the genesis and activity of lysosomes. The data provides insight with the neurotoxicity of Pb in a novel perspective, autophagy.