Superfund Research Program

Genetic Mechanisms of Metal Neurotoxicity

Project Leader: Quan Lu
Co-Investigator: Tomas R. Guilarte (Columbia University)
Grant Number: P42ES016454
Funding Period: 2010-2015

Project-Specific Links

Final Progress Reports

Year:   2013 

Studies and Results

Project researchers have performed deep sequencing of transcripts in human neural stem cells (NSCs)-derived neurons that were exposed to lead (Pb) and identified both known and novel RNA transcripts that are responsive to Pb exposure. This complete transcriptomic analysis allows the researchers to propose and test novel hypotheses regarding Pb neurotoxicity. They also have performed deep sequencing of small RNAs in human neural stem cells (NSCs) as well as NSC-derived neurons that were exposed to Pb at a physiologically relevant level (1 µM for 24 hours). Bioinformatics analysis of the RNA-seq data identified at least eight differentially expressed transcripts in neurons, which show a greater transcriptional response to Pb than neural stem cells. Some of the differentially expressed genes are involved in oxidative stress pathways, suggesting that Pb exposure at even low levels can elicit oxidative stress responses. The researchers have focused on the characterization of two genes (SPP1 and F2RL2). They showed that these genes are novel, direct targets of the transcriptional factor Nrf2. These results point to new pathways that Pb may affect neuronal function trough inducing Nrf2-meidated oxidative stress response.

The researchers also identified several microRNAs whose expression was altered by Pb treatment. Among the novel Pb-induced transcripts is the host gene of mir-22 (MIR22HG). Using qRT-PCR, they confirmed that Pb increased the expression of mir-22 host gene. MicroRNAs are small (~22 nucleotides) RNAs processed from larger primary transcripts and direct posttranscriptional repression of target genes by complimentary pairing with mRNAs through seed sequences (nucleotide position 2-7 of a microRNA). Such miRNA/mRNA pairing usually leads to mRNA degradation, translational repression, or both. Mir-22 is predicted to target multiple genes encoding proteins with neuronal functions, including TrkB--the canonical receptor for BDNF. There are two putative mir-22 sites within the 3'-UTR (un-translated region) of TrkB transcript. BDNF-TrkB signaling plays a critical role in synaptic development. Several studies from Project co-leader Dr. Guilarte's laboratory has implicated the impaired TrkB signaling in Pb neurotoxicity. Highly relevant to this study, Pb reduces TrkB expression at both mRNA and protein level, however, the underlying mechanism remain unclear. The researchers hypothesize that mir-22 directly targets TrkB and that Pb induces mir-22 to suppress TrkB expression.

In addition to inducing gene expression, Pb also decreases the expression of some transcripts, including the one encoding mir-219-1. qRT-PCR validated the down-regulation of the mature form of mir-219-1 in Pb-treated neurons. Importantly and consistent with the fact that Pb is a potent NMDA receptor antagonist, a recent study has shown that pharmacological or genetic manipulations that inhibit NMDA receptor function decreases mir-219-1 levels. Mir-219-1 is a brain-specific microRNA that directly targets calcium/calmodulin-dependent protein kinase II γ subunit (CaMKIIγ), which is a critical component of the NMDA receptor signaling cascade. Studies including those from Project Co-leader Dr. Tomas Guilarte's laboratory have provided overwhelming evidence that Pb is a potent noncompetitive antagonist of NMDA receptor. The researchers identification of mir-219-1 down-regulation by Pb further supports a critical role of mir-219-1 in NMDA receptor signaling; more importantly, it offers a potential mechanistic explanation for the inhibitory effect by Pb on NMDA receptor signaling and the decrease in CaMKII protein levels and activity that the researchers have previously described in Pb-exposed animals.

In establishing comprehensive genetic pathways and networks affecting metal neurotoxicity, the researchers have used the Ingenuity Systems Pathway Analysis tool to analyze the RNAi hits from their arsenic-ER stress screen. Their initial analysis identified an enrichment of pathways related to intracellular stress signaling and oxidation-reduction that are known to be associated with arsenic and more generally metal exposure. The researchers also detected enrichment in pathways related to G-protein signaling, DNA repair and cell cycle, suggesting novel modes of action by arsenic and potentially other toxic metal exposures. They also work with the Environmental Statistics Core to analyze the deep sequencing data (both microRNA deep sequencing and RNA-seq). The researchers are currently doing pathway analysis on the genes identified as responsive to Pb exposure.

For investigating roles of isolated genes in metal-affected neuronal function, the researchers have collaborated with Dr. Guilarte's laboratory at Columbia University. Dr. Guilarte's laboratory recently proposed a working model for Pb effect on BDNF-TrkB signaling and synaptic development. BDNF vesicle trafficking and release is controlled by huntingtin (HTT) protein and its interaction partner HAP1. The researchers have obtained several lines of additional evidence that further support the effect of Pb on HTT/HAP1-mediated BDNF trafficking: 1) deep sequencing identified HAP1 as a novel Pb-induced gene, 2) Pb increases HTT expression but suppresses its phosphorylation, and 3) genetic variations in HAP1 interact with Pb to affect phenotypes that correlate with Pb-impaired neural functions. Interestingly, preliminary studies indicate that Mn exposure also increases HTT expression in hippocampal neurons, suggesting that perturbation of HTT/HAP1 BDNF pathway may be a unifying mechanism by which Pb and Mn affect neuronal and cognitive function. This notion is supported by the human epidemiological data (from the Epidemiology of Developmental Windows, Metal Mixtures and Neurodevelopment project) that show an interaction between Mn and Pb in neurodevelopment.

Significance

The overall goal of this project is to elucidate metal neurotoxic mechanisms by identifying and characterizing critical genes that determine the susceptibility of neuronal cells to toxic metal exposures. The researchers have utilized powerful Next-Gen deep sequencing technology to identify both coding and non-coding transcripts altered by Pb. The complete mapping of the transcriptome under different metal exposures will form the basis of hypothesis-driven mechanistic studies. These studies promise to advance the field of metal neurotoxicity by revealing novel insights into metal neurotoxicity and its impact on learning and memory. Relevant to the Harvard SRP program, this project integrates with epidemiological studies (the Epidemiology of Developmental Windows, Metal Mixtures and Neurodevelopment and the Genetic Epidemiology of Neurodevelopmental Metal Toxicity projects) that examine the gene-metal interactions in the susceptibility of human populations. Ultimately, knowledge gained from this study will be translated into better strategies for the prevention, diagnosis and treatment of neurological diseases that are caused by exposures to metal contaminants, including those found at Superfund sites.