Epigenome-wide association study of alcohol consumption in N = 8161 individuals and relevance to alcohol use disorder pathophysiology: identification of the cystine/glutamate transporter SLC7A11 as a top target

Alcohol misuse is common in many societies worldwide and is associated with extensive morbidity and mortality, often leading to alcohol use disorders (AUD) and alcohol-related end-organ damage. The underlying mechanisms contributing to the development of AUD are largely unknown; however, growing evidence suggests that alcohol consumption is strongly associated with alterations in DNA methylation. Identification of alcohol-associated methylomic variation might provide novel insights into pathophysiology and novel treatment targets for AUD. Here we performed the largest single-cohort epigenome-wide association study (EWAS) of alcohol consumption to date (N = 8161) and cross-validated findings in AUD populations with relevant endophenotypes, as well as alcohol-related animal models. Results showed 2504 CpGs significantly associated with alcohol consumption (Bonferroni p value < 6.8 × 10−8) with the five leading probes located in SLC7A11 (p = 7.75 × 10−108), JDP2 (p = 1.44 × 10−56), GAS5 (p = 2.71 × 10−47), TRA2B (p = 3.54 × 10−42), and SLC43A1 (p = 1.18 × 10−40). Genes annotated to associated CpG sites are implicated in liver and brain function, the cellular response to alcohol and alcohol-associated diseases, including hypertension and Alzheimer’s disease. Two-sample Mendelian randomization confirmed the causal relationship of consumption on AUD risk (inverse variance weighted (IVW) p = 5.37 × 10−09). A methylation-based predictor of alcohol consumption was able to discriminate AUD cases in two independent cohorts (p = 6.32 × 10−38 and p = 5.41 × 10−14). The top EWAS probe cg06690548, located in the cystine/glutamate transporter SLC7A11, was replicated in an independent cohort of AUD and control participants (N = 615) and showed strong hypomethylation in AUD (p < 10−17). Decreased CpG methylation at this probe was consistently associated with clinical measures including increased heavy drinking days (p < 10−4), increased liver function enzymes (GGT (p = 1.03 × 10−21), ALT (p = 1.29 × 10−6), and AST (p = 1.97 × 10−8)) in individuals with AUD. Postmortem brain analyses documented increased SLC7A11 expression in the frontal cortex of individuals with AUD and animal models showed marked increased expression in liver, suggesting a mechanism by which alcohol leads to hypomethylation-induced overexpression of SLC7A11. Taken together, our EWAS discovery sample and subsequent validation of the top probe in AUD suggest a strong role of abnormal glutamate signaling mediated by methylomic variation in SLC7A11. Our data are intriguing given the prominent role of glutamate signaling in brain and liver and might provide an important target for therapeutic intervention.

Opponent vesicular transporters regulate the strength of glutamatergic neurotransmission in a C. elegans sensory circuit

At chemical synapses, neurotransmitters are packaged into synaptic vesicles that release their contents in response to depolarization. Despite its central role in synaptic function, regulation of the machinery that loads vesicles with neurotransmitters remains poorly understood. We find that synaptic glutamate signaling in a C. elegans chemosensory circuit is regulated by antagonistic interactions between the canonical vesicular glutamate transporter EAT-4/VGLUT and another vesicular transporter, VST-1. Loss of VST-1 strongly potentiates glutamate release from chemosensory BAG neurons and disrupts chemotaxis behavior. Analysis of the circuitry downstream of BAG neurons shows that excess glutamate release disrupts behavior by inappropriately recruiting RIA interneurons to the BAG-associated chemotaxis circuit. Our data indicate that in vivo the strength of glutamatergic synapses is controlled by regulation of neurotransmitter packaging into synaptic vesicles via functional coupling of VGLUT and VST-1.

TAMI-63. INHIBITION OF LINEAGE STATES WITHIN GBM OVERCOMES CHEMO-RESISTANCE

OBJECTIVE Novel insights into the developmental trajectory exhibited by glioblastoma (GBM) have shown that it has the capability to respond to its microenvironment by clonal selection of specific transcriptional phenotypes. Using the same mechanisms, malignant GBM develop intrinsic mechanisms to resist chemotherapeutic treatments. In this study, we determine the role of the metabotropic glutamate receptor 3 (mGluR3) in chemoresistance and show that inhibition of mGluR3 leads to significant loss of tumor growth volume when concomitantly treated with Temozolomide. METHODS Transcriptomic analysis was carried out post mGlur3 receptor inhibition using LY341495 (100nM), to study loss of transcriptional programs. Morphological and cellular kinetic analysis was carried out post inhibition, to determine apoptosis/necrosis rate, when combined with Temozolomide. The efficacy of the treatment within a human like environment was assessed using our novel human cortical GBM model. RESULTS We present transcriptional and morphological evidence that mGluR3 inhibition using LY341495 leads to loss of both lineage (FDR&lt; 0.01) and reactive (FDR&lt; 0.01) transcription programs. This leads to a loss of ability to evade cytotoxicity when combined with Temozolomide treatment, validated using kinetic apoptosis (p&lt; 0.01). When our cortical GBM model was used to study the effect of treatment within the appropriate microenvironment, we see that the combination therapy leads to a significant reduction in tumor growth (p&lt; 0.01) over the course of 10 days. CONCLUSION Through the integration of diversified molecular-biological analyses, we illustrate a new picture of how glutamate signaling via mGluR3 interacts with phenotypical GBM transcriptional programs to evade effective therapy, the inhibition of which leads to loss of chemo-resistance.

Spider Neurotoxins as Modulators of NMDA Receptor Signaling

Molecules that selectively act on N-methyl-D-aspartate (NMDA) receptors may have a multidirectional effect by modulating the activity of NMDARs, affecting their active sites as well as by changing the composition of their subunits. The results of the clinical trials conducted so far in mood disorders and schizophrenia indicate that such agents may become new effective drugs for the treatment of these diseases. Number of spider neurotoxins e.g. ctenitoxins extracted from Phoneutria sp. venom act as potent and selective NMDAR blockers that do not disturb cortical and hippocampal glutamate signaling, LTP generation and synaptic neurochemistry. Possibly this intriguing kind of promising neuroregulatory peptides and polyamines can be clinically applicable in a wide spectrum of neuropsychiatric disorders, including epilepsy, neurotrauma and ischemic injuries. These novel medications can potentially be helpful in the future treatment of stroke and several neurodegenerative diseases.

Circuit contributions to sensory-driven glutamatergic drive of olfactory bulb mitral and tufted cells during odorant inhalation

In the mammalian olfactory bulb (OB), mitral/tufted (MT) cells respond to odorant inhalation with diverse temporal patterns that are thought to encode odor information. Much of this diversity is already apparent at the level of glutamatergic input to MT cells, which receive direct, monosynaptic excitatory input from olfactory sensory neurons (OSNs) as well as multisynaptic excitatory drive via glutamatergic interneurons. Both pathways are also subject to modulation by inhibitory circuits in the glomerular layer of the OB. To understand the role of direct OSN input versus postsynaptic OB circuit mechanisms in shaping diverse dynamics of glutamatergic drive to MT cells, we imaged glutamate signaling onto MT cell dendrites in anesthetized mice while blocking multisynaptic excitatory drive with ionotropic glutamate receptor antagonists and blocking presynaptic modulation of glutamate release from OSNs with GABAB receptor antagonists. GABAB receptor blockade increased the magnitude of inhalation-linked glutamate transients onto MT cell apical dendrites without altering their inhalation-linked dynamics, confirming that presynaptic inhibition impacts the gain of OSN inputs to the OB. Surprisingly, blockade of multisynaptic excitation only modestly impacted glutamatergic input to MT cells, causing a slight reduction in the amplitude of inhalation-linked glutamate transients in response to low odorant concentrations and no change in the dynamics of each transient. Postsynaptic blockade also modestly impacted glutamate dynamics over a slower timescale, mainly by reducing adaptation of the glutamate response across multiple inhalations of odorant. These results suggest that direct glutamatergic input from OSNs provides the bulk of excitatory drive to MT cells, and that diversity in the dynamics of this input may be a primary determinant of the temporal diversity in MT cell responses that underlies odor representations at this stage.

Inhibition of metabotropic glutamate receptor III facilitates sensitization to alkylating chemotherapeutics in glioblastoma

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.

Transcriptomic Profiling of Canine Atrial Fibrillation Models after One Weekof Sustained Arrhythmia

Background - Atrial fibrillation (AF), the most common sustained arrhythmia, is associated with increased morbidity, mortality, and health-care costs. AF develops over many years and is often related to substantial atrial structural and electrophysiological remodeling. AF may lack symptoms at onset and atrial biopsy samples are generally obtained in subjects with advanced disease, so it is difficult to study earlier-stage pathophysiology in humans. Methods - Here, we characterized comprehensively the transcriptomic (miRNAseq and mRNAseq) changes in the left atria of two robust canine AF-models after one week of electrically-maintained AF, without or with ventricular rate-control via atrioventricular node-ablation/ventricular pacing. Results - Our RNA-sequencing experiments identified thousands of genes that are differentially expressed, including a majority that have never before been implicated in AF. Gene-set enrichment analyses highlighted known (e.g. extracellular matrix structure organization) but also many novel pathways (e.g. muscle structure development, striated muscle cell differentiation) that may play a role in tissue remodeling and/or cellular trans-differentiation. Of interest, we found dysregulation of a cluster of non-coding RNAs, including many microRNAs but also the MEG3 long non-coding RNA orthologue, located in the syntenic region of the imprinted human DLK1-DIO3 locus. Interestingly (in the light of other recent observations), our analysis identified gene-targets of differentially expressed microRNAs at the DLK1-DIO3 locus implicating glutamate signaling in AF pathophysiology. Conclusions - Our results capture molecular events that occur at an early stage of disease development using well-characterized animal models, and may therefore inform future studies that aim to further dissect the causes of AF in humans.

Alpha-synuclein induces epigenomic dysregulation of glutamate signaling and locomotor pathways

Background: Mutations and multiplications in the gene encoding for alpha-synuclein are associated with Parkinson's disease (PD). However, not all individuals with alpha-synuclein variants develop PD, suggesting that additional factors are involved. We hypothesized that increased alpha-synuclein might alter epigenetic regulation of PD pathways. Objectives: To identify genome-wide DNA methylation and hydroxymethylation changes induced by overexpression of two alpha-synuclein variants in human dopaminergic neurons, and to relate these to the corresponding transcriptome. Methods: We assessed DNA methylation and hydroxymethylation at >850,000 CpGs using the EPIC BeadChip in LUHMES cells differentiated to dopaminergic neurons. Control LUHMES neurons, LUHMES neurons overexpressing wild type alpha-synuclein, and LUHMES neurons overexpressing A30P alpha-synuclein were compared. We used SMITE network analysis to identify functionally related genes with altered DNA methylation, DNA hydroxymethylation, and/or gene expression, incorporating LUHMES H3K4me1 ChIP-seq to delineate enhancers in addition to the default promoter and gene body regions. Results: Using stringent statistical thresholds, we found that increased expression of wild type or A30P mutant alpha-synuclein induced DNA methylation changes at thousands of CpGs and DNA hydroxymethylation changes at hundreds of CpGs. Differentially methylated sites in both genotypes were enriched for several processes including movement-associated pathways and glutamate signaling. For glutamate and other signaling pathways (i.e. PDGF, insulin), this differential DNA methylation was also associated with transcriptional changes. Conclusions: Our results indicated that alpha-synuclein altered the DNA methylome of dopaminergic neurons, influencing regulation of pathways involved in development, signaling, and metabolism. This supports a role for alpha-synuclein in the epigenetic etiology of PD.

Inhibition of Metabotropic Glutamate Receptor III facilitates sensitization to alkylating chemotherapeutics in Glioblastoma

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.