Redox-based epigenetic status in drug addiction: a potential contributor to gene priming and a mechanistic rationale for metabolic intervention

Alcohol and other drugs of abuse, including psychostimulants and opioids, can induce epigenetic changes: a contributing factor for drug addiction, tolerance and associated withdrawal symptoms. DNA methylation is the major epigenetic mechanism and it is one of more than 200 methylation reactions supported by methyl donor S-adenosylmethionine (SAM). The levels of SAM are controlled by cellular redox status via the folate and vitamin B12-dependent enzyme methionine synthase (MS), for example; under oxidative conditions MS is inhibited, diverting its substrate homocysteine (HCY) to the transsulfuration pathway. Alcohol, dopamine and morphine, can alter intracellular levels of glutathione (GSH)-based cellular redox status, subsequently affecting S-adenosylmethionine (SAM) levels and DNA methylation status. In this discussion, we compile this and other existing evidence in a coherent manner to present a novel hypothesis implicating the involvement of redox-based epigenetic changes in drug addiction. Next, we also discuss how gene priming phenomenon can contribute to maintenance of redox and methylation status homeostasis under various stimuli including drugs of abuse. Lastly, based on our hypothesis and some preliminary evidence, we discuss a mechanistic explanation for use of metabolic interventions / redox-replenishers as symptomatic treatment of alcohol addiction and associated withdrawal symptoms. Hence, the current review article strengthens the hypothesis that neuronal metabolism has a critical bidirectional coupling with epigenetic changes in drug addiction and we support this claim via exemplifying the link between redox-based metabolic changes and resultant epigenetic consequences under the effect of drugs of abuse

Role of a Redox-Based Methylation Switch in mRNA Life Cycle (Pre- and Post-Transcriptional Maturation) and Protein Turnover: Implications in Neurological Disorders

Homeostatic synaptic scaling in response to neuronal stimulus or activation, and due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions (Turrigiano and Nelson, 2004). Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia, etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic; Cajigas et al., 2010). This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation, and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition, and behavior (Cajigas et al., 2010). Thus a regulatory switch, which controls the lifespan, maturation, and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at (1) the pre-transcription level, by regulating precursor-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and (2) the post-transcription level by modulating the regulatory functions of ribonucleoproteins and RNA binding proteins in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione anti-oxidant levels (Lertratanangkoon et al., 1997), the redox status of neurons might be the central regulatory switch for methylation-based changes in mRNA processing, protein expression, and turnover. Lastly, we also describe experimental methods and techniques which might help researchers to evaluate the suggested hypothesis

Zero Entropy Interval Maps And MMLS-MMA Property

We prove that the flow generated by any interval map with zero topological entropy is minimally mean-attractable (MMA) and minimally mean-L-stable (MMLS). One of the consequences is that any oscillating sequence is linearly disjoint with all flows generated by interval maps with zero topological entropy. In particular, the M\"obius function is orthogonal to all flows generated by interval maps with zero topological entropy (Sarnak's conjecture for interval maps). Another consequence is a non-trivial example of a flow having the discrete spectrum.Comment: 12 page

Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences

Dietary interventions like gluten-free and casein-free diets have been reported to improve intestinal, autoimmune and neurological symptoms in patients with a variety of conditions; however, the underlying mechanism of benefit for such diets remains unclear. Epigenetic programming, including CpG methylation and histone modifications, occurring during early postnatal development can influence the risk of disease in later life, and such programming may be modulated by nutritional factors such as milk and wheat, especially during the transition from a solely milk-based diet to one that includes other forms of nutrition. The hydrolytic digestion of casein (a major milk protein) and gliadin (a wheat-derived protein) releases peptides with opioid activity, and in the present study, we demonstrate that these food-derived proline-rich opioid peptides modulate cysteine uptake in cultured human neuronal and gastrointestinal (GI) epithelial cells via activation of opioid receptors. Decreases in cysteine uptake were associated with changes in the intracellular antioxidant glutathione and the methyl donor S-adenosylmethionine. Bovine and human casein-derived opioid peptides increased genome-wide DNA methylation in the transcription start site region with a potency order similar to their inhibition of cysteine uptake. Altered expression of genes involved in redox and methylation homeostasis was also observed. These results illustrate the potential of milk- and wheat-derived peptides to exert antioxidant and epigenetic changes that may be particularly important during the postnatal transition from placental to GI nutrition. Differences between peptides derived from human and bovine milk may contribute to developmental differences between breastfed and formula-fed infants. Restricted antioxidant capacity, caused by wheat- and milk-derived opioid peptides, may predispose susceptible individuals to inflammation and systemic oxidation, partly explaining the benefits of gluten-free or casein-free diets

Regulation of Cellular Cobalamin Acquisition and Processing by Nrf2 and mTORC1 Downstream of Neurotrophic Factors

Objective. Define the roles of Nrf2 and mTORC1 in regulating cobalamin content downstream of neurotrophic factors in SH-SY5Y neuroblastoma cells. Background. Vitamin B12 (cobalamin) serves as a cofactor for methionine synthase, which catalyzes the regeneration of methionine from homocysteine. Cellular cobalamin processing requires proper lysosomal acidification and the availability of cytoplasmic glutathione. Mechanistic target of rapamycin complex 1 (mTORC1) abrogates lysosomal acidification and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) promotes glutathione production. Both mTORC1 and Nrf2 are activated downstream of neurotrophic factor-induced receptor tyrosine kinase signaling. We seek to understand the individual roles of Nrf2 and mTORC1 in regulating cobalamin content in this setting. Methods. SH-SY5Y cells were pre-treated with temsirolimus (TEMS) or treated with either neuregulin-1 (NRG-1) or brain-derived neurotrophic factor (BDNF). RT-qPCR was performed to measure gene expression. Cobalamin analysis was done via HPLC. Results. Both 1 nM and 100 nM NRG-1 decreases LMBRD1 (probable lysosomal cobalamin transporter) expression, but only 1 nM NRG-1 decreased ATP6V1H (V-type proton ATPase subunit H) mRNA. 10 nM BDNF decreased both LMBRD1 and ATP6V1H mRNA levels. 10 nM BDNF may selectively increase adenosylcobalamin. Furthermore, pre-treatment of cells with 100 nM TEMS and treatment with 100 nM NRG-1 increased cobalamin content over either agent alone. Conclusion. Our research suggests that different neurotrophic factors play unique roles in regulating cobalamin processing, NRG-1 may have opposing effects at various concentrations, and that mTORC1 activation can limit cobalamin content in SH-SY5Y cells. Grants. This research was partially funded by the A2 Milk Company

Neuregulin-1/Phosphatidylinositol 3-kinase Signaling Regulates Expression of Genes Involved in Cellular Cobalamin Uptake and Processing

Objective. To determine whether the Neuregulin-1/Phosphatidylinositol 3-kinase signaling transcriptionally regulates proteins involved with the uptake and processing of Vitamin B12 (cobalamin) in SH-SY5Y neuroblastoma cells. Background. Cobalamin is required as a cofactor for methionine synthase, which transfers folate-derived methyl groups to homocysteine to form methionine. Cobalamin must be imported and processed by cells into activated species. The antioxidant glutathione is needed to process intracellular cobalamin. Neurotrophic factors, such as neuregulin-1, increase glutathione in neurons. Neuregulin-1 and downstream phosphatidylinositol 3-kinase (PI3K) signaling promote glutathione-dependent cobalamin processing and methionine synthase activity in SH-SY5Y cells. In the work presented here, we sought to understand whether neuregulin-1/PI3K signaling might additionally regulate cobalamin status via parallel mechanisms complementary to stimulating glutathione formation. Methods. SH-SY5Y cells in 6-well plates were maintained under low-serum conditions. Cells were exposed to 1 nM neuregulin-1 for 1- or 4h. Cells were pre-treated with PI3K inhibitors pictilisib or wortmannin for 30 min and then some cells were co-treated with neuregulin-1 for 1h. Real-Time quantitative polymerase chain reaction (RT-qPCR) was used to assess gene expression of cobalamin-interacting proteins. Results. 1h neuregulin-1 generally increased expression of cobalamin processing genes, which was blocked by PI3K inhibitors. However, PI3K inhibitors increased expression when given alone. Transcripts were decreased or no longer increased after 4h exposure. Conclusion. Neuregulin-1 regulates expression of cobalamin-related genes in a temporal- and PI3K-dependent manner. Grants. This research was supported with a fellowship from the American Foundation for Pharmaceutical Education (AFPE)

Methylation and D4 Dopamine Receptors in Autism and Schizophrenia

Objective. To evaluate the role of methylation in regulating the D4 dopamine receptor in autism and schizophrenia Background. D4 dopamine receptors have the unique ability to catalyze methylation of membrane phospholipids, involving the vitamin B12 and folate-dependent enzyme methionine synthase. This process is proposed to be centrally involved in dopamine-mediated attention by synchronization of neural network activity. Impaired methylation has been reported in autism and schizophrenia. Methods. Levels of vitamin B12 (cobalamin) in postmortem brain samples were analyzed by HPLC. DNA methylation of CpG sites in intron 1 of the D4 receptor were measured by pyrosequencing and global DNA methylation was measured via an Elisa-based assay. Results. Vitamin B12 levels, especially methylcobalamin, were decreased in both autism and schizophrenia frontal cortex. Methylation of the D4 dopamine receptor was significantly higher in both autism and schizophrenia. Conclusion. Methylation status of the D4 dopamine receptor gene is abnormal in autism and schizophrenia, associated with lower levels of vitamin B12. Grants. This study was funded in part by the Autism Research Institute

Multifunctional Polymeric Micelles Co-loaded with Anti-Survivin siRNA and Paclitaxel Overcome Drug Resistance in an Animal Model of Ovarian Cancer

Ovarian cancer is a dreadful disease estimated to be the second most common gynecologic malignancy worldwide. Its current therapy, based on cytoreductive surgery followed by the combination of platinum and taxanes, is frequently complicated by the onset of multidrug resistance (MDR). The discovery that survivin, a small antiapoptotic protein, is involved in chemoresistance provided a new prospect to overcome MDR in cancer, because siRNA could be used to inhibit the expression of survivin in cancer cells. With this in mind, we have developed self-assembly polymeric micelles (PM) able to efficiently co-load an anti-survivin siRNA and a chemotherapeutic agent, such as paclitaxel (PXL; survivin siRNA/PXL PM). Previously, we have successfully demonstrated that the downregulation of survivin by using siRNA-containing PM strongly sensitizes different cancer cells to paclitaxel. Here, we have evaluated the applicability of the developed multifunctional PM in vivo. Changes in survivin expression, therapeutic efficacy, and biologic effects of the nanopreparation were investigated in an animal model of paclitaxel-resistant ovarian cancer. The results obtained in mice xenografed with SKOV3-tr revealed a significant downregulation of survivin expression in tumor tissues together with a potent anticancer activity of survivin siRNA/PXL PM, while the tumors remained unaffected with the same quantity of free paclitaxel. These promising results introduce a novel type of nontoxic and easy-to-obtain nanodevice for the combined therapy of siRNA and anticancer agents in the treatment of chemoresistant tumors