The Cholinergic System, the Adrenergic System and the Neuropathology of Alzheimer’s Disease

Neurodegenerative diseases are a major public health problem worldwide with a wide spectrum of symptoms and physiological effects. It has been long reported that the dysregulation of the cholinergic system and the adrenergic system are linked to the etiology of Alzheimer’s disease. Cholinergic neurons are widely distributed in brain regions that play a role in cognitive functions and normal cholinergic signaling related to learning and memory is dependent on acetylcholine. The Locus Coeruleus norepinephrine (LC-NE) is the main noradrenergic nucleus that projects and supplies norepinephrine to different brain regions. Norepinephrine has been shown to be neuroprotective against neurodegeneration and plays a role in behavior and cognition. Cholinergic and adrenergic signaling are dysregulated in Alzheimer’s disease. The degeneration of cholinergic neurons in nucleus basalis of Meynert in the basal forebrain and the degeneration of LC-NE neurons were reported in Alzheimer’s disease. The aim of this review is to describe current literature on the role of the cholinergic system and the adrenergic system (LC-NE) in the pathology of Alzheimer’s disease and potential therapeutic implications

Methyl Donors, Epigenetic Alterations, and Brain Health: Understanding the Connection

Methyl donors such as choline, betaine, folic acid, methionine, and vitamins B6 and B12 are critical players in the one-carbon metabolism and have neuroprotective functions. The one-carbon metabolism comprises a series of interconnected chemical pathways that are important for normal cellular functions. Among these pathways are those of the methionine and folate cycles, which contribute to the formation of S-adenosylmethionine (SAM). SAM is the universal methyl donor of methylation reactions such as histone and DNA methylation, two epigenetic mechanisms that regulate gene expression and play roles in human health and disease. Epigenetic mechanisms have been considered a bridge between the effects of environmental factors, such as nutrition, and phenotype. Studies in human and animal models have indicated the importance of the optimal levels of methyl donors on brain health and behavior across the lifespan. Imbalances in the levels of these micronutrients during critical periods of brain development have been linked to epigenetic alterations in the expression of genes that regulate normal brain function. We present studies that support the link between imbalances in the levels of methyl donors, epigenetic alterations, and stress-related disorders. Appropriate levels of these micronutrients should then be monitored at all stages of development for a healthier brain

Neuroprotective Effects of Choline and Other Methyl Donors

Recent evidence suggests that physical and mental health are influenced by an intricate interaction between genes and environment. Environmental factors have been shown to modulate neuronal gene expression and function by epigenetic mechanisms. Exposure to these factors including nutrients during sensitive periods of life could program brain development and have long-lasting effects on mental health. Studies have shown that early nutritional intervention that includes methyl-donors improves cognitive functions throughout life. Choline is a micronutrient and a methyl donor that is required for normal brain growth and development. It plays a pivotal role in maintaining structural and functional integrity of cellular membranes. It also regulates cholinergic signaling in the brain via the synthesis of acetylcholine. Via its metabolites, it participates in pathways that regulate methylation of genes related to memory and cognitive functions at different stages of development. Choline-related functions have been dysregulated in some neurodegenerative diseases suggesting choline role in influencing mental health across the lifespan

Epigenetic effects of fetal alcohol exposure on hypothalamic proopiomelanocortin gene

Hypothalamic POMC neurons, one of the major regulators of the HPA axis, immune functions, and energy homeostasis, are vulnerable to the adverse effects of fetal alcohol exposure (FAE) exhibiting a significant decrease in POMC gene expression and functions in the arcuate area of the hypothalamus of adult offspring. This permanent deficit in gene expression could be caused by epigenetic mechanisms such as histone modifications and DNA methylation induced by alcohol exposure during critical period of development. We found that FAE decreased significantly the protein and mRNA levels of histone-modifying enzymes that methylate H3K4me2,3 (Set7/9), acetylate H3K9 (CBP) or phosphorylate H3S10. These are activation marks that correlate with gene expression. FAE significantly increased the protein levels and gene expression of G9a and Setdb1 that methylate the repressive mark H3K9me2 in β-endorphin-producing POMC neurons of adult offspring. These changes were associated with increased levels of the DNA-methyltranferase Dnmt1 and the methyl-CpG-binding protein 2 MeCP2 but not Dnmt3a. Microarray analysis confirmed that alcohol exposure modulated the gene expression profile of the epigenetic machinery in LCM-captured POMC neurons. ChIP assay revealed a significant reduction in the activation mark H3K4me3 along Exon 3 of POMC gene in alcohol-exposed rats associated with no change in the repressive mark H3K9me2 in Exon 3 and promoter region of POMC gene. We then examined whether gestational choline supplementation, a major methyl donor, could mitigate alcohol adverse effects on POMC neurons. Gestational choline normalized in alcohol-exposed rats the methylation of H3K4 and H3K9 with no significant effect on other histone marks such as acetylated H3K9 or phosphorylated H3S10. Similarly, gestational choline normalized the protein levels and gene expression of histone-modifying and DNA-methylating enzymes in POMC neurons. This data correlated with normalization of POMC gene methylation, POMC gene expression and β-EP peptide production. In conclusion, these studies demonstrate that FAE induces long-lasting epigenetic modifications of POMC gene in the hypothalamus by altering histone marks and methylation state along POMC gene. The hypermethylation state of POMC gene might be a cause for induction of lower β-endorphin activity and its inhibitory regulation of stress axis function in the adult offspring.Ph. D.Includes bibliographical referencesby Rola Aldana Bekdas

Fetal alcohol exposure alters proopiomelanocortin gene expression and hypothalamic-pituitary-adrenal axis function via increasing MeCP2 expression in the hypothalamus.

Proopiomelanocortin (POMC) is a precursor gene of the neuropeptide β-endorphin in the hypothalamus and is known to regulate various physiological functions including stress response. Several recent reports showed that fetal alcohol exposure programs the hypothalamus to produce lower levels of POMC gene transcripts and to elevate the hypothalamic-pituitary-adrenal (HPA) axis response to stressful stimuli. We investigated the role of methyl CpG binding protein (MeCP2) in the effects of prenatal ethanol on POMC gene expression and hypothalamic-pituitary-adrenal (HPA) axis function. Pregnant Sprague Dawley rats were fed between GD 7 and 21 with a liquid diet containing 6.7% alcohol, pair-fed with isocaloric liquid diet, or fed ad libitum with rat chow, and their male offsprings were used at 60 days after birth in this study. Fetal alcohol exposure reduced the level of POMC mRNA, but increased the level of DNA methylation of this gene in the arcuate nucleus (ARC) of the hypothalamus where the POMC neuronal cell bodies are located. Fetal alcohol exposed rats showed a significant increase in MeCP2 protein levels in POMC cells, MeCP2 gene transcript levels as well as increased MeCP2 protein binding on the POMC promoter in the arcuate nucleus. Lentiviral delivery of MeCP2 shRNA into the third ventricle efficiently reduced MeCP2 expression and prevented the effect of prenatal ethanol on POMC gene expression in the arcuate nucleus. MeCP2-shRNA treatment also normalized the prenatal ethanol-induced increase in corticotropin releasing hormone (CRH) gene expression in the hypothalamus and elevated plasma adrenocorticotrophic hormone (ACTH) and corticosterone hormone responses to lipopolysaccharide (LPS) challenge. These results suggest that fetal alcohol programming of POMC gene may involve recruitment of MeCP2 on to the methylated promoter of the POMC gene to suppress POMC transcript levels and contribute to HPA axis dysregulation

Primer sequences.

<p>Forward primer (FP), Reverse primer (RP), Methyl forward primer (MFP), Methyl reverse primer (MRP), Un methyl forward primer (UFP), Un methyl reverse primer (URP), Promoter forward primer (PFP), Promoter reverse primer (PRP).</p><p>Primer sequences.</p

Changes in MeCP2 gene and protein levels in MBH of fetal alcohol exposed rat offspring.

<p><b>A</b>. MeCP2 mRNA levels in MBH of AD, PF and AF rat offspring. MeCP2 mRNA levels were measured by quantitative RT-PCR and the amounts were normalized with GAPDH values and expressed as relative mRNA levels. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, <i>P</i><0.05, AF vs. AD or PF. <b>B</b>. A representative western blot gel demonstrating changes in MeCP2 protein levels (top) and actin levels (loading control, bottom) in MBH of AD, PF and AF rat offspring along with quantification measurements were represented as relative protein level (MeCP2/actin). Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; **, <i>P</i><0.01, AF vs. AD or PF. <b>C</b>. MeCP2 protein levels in β-endorphin neurons in the ARC of AD, PF and AF rat offspring were measured by double immunofluorescence methods (MeCP2 proteins are shown in green and β-endorphin shown in red). Representative photographs show double-labeled cells in each group. Histograms show the mean ± SEM (n = 6) values of percent β-EP cells expressing MeCP2 and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; ***, <i>P</i><0.001, AF vs. AD or PF.</p

Changes in MeCP2 binding onto POMC promoter in ARC and PVN of fetal alcohol exposed rat offspring.

<p><b>A</b>. Schematic representation of POMC promoter with CpG sites and primers flanking MeCP2 binding site. <b>B</b>. The levels of MeCP2 binding on POMC promoter in ARC and PVN of AD, PF and AF rat offspring were measured by ChIP assay. MeCP2 bound DNA pulled by its specific antibody was amplified by real time PCR using primers specific for the POMC promoter. MeCP2 enriched DNA was normalized with GAPDH and expressed as fold enrichment (MeCP2/GAPDH). AD and AF are ARC samples of <i>ad libitum</i>-fed and alcohol-fed rats. ADP and AFP are PVN samples of <i>ad libitum</i>-fed and alcohol-fed rats. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, <i>P</i><0.05, AF vs. AD; ^a, P<0.001, ADP and AFP versus AD or AF.</p

Effects of lentiviral knockdown of MeCP2 on POMC gene expression levels in fetal alcohol exposed rat offspring.

<p><b>A</b>. A representative western blot gel showing MeCP2 protein levels (top) and actin levels (bottom) in MBH of scr sh (scr) or MeCP2 sh (MeCP) RNA-treated AD, PF and AF rat offspring. Histograms showing MeCP2 and actin ratio values in MBH of AD, PF and AF rats.treated with scr sh or MeCP2 sh RNA. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, MeCP2 sh vs. scr-sh; ^a, P<0.05, AF vs. AD or PF. <b>B</b>. POMC mRNA levels in MBH of scr-sh and MeCP2 sh RNA of AD, PF and AF rat offspring. POMC mRNA amounts were normalized with GAPDH and expressed as relative mRNA level. Data are mean ± SEM (n = 6) and were analyzed using one-way ANOVA with Newman-Keuls post-hoc test; *, P<0.05, AF- vs. AD- or PF-scr-sh-treated groups; ^a, P<0.05, MeCP2 sh vs. scr-sh (AF group).</p