Oxidation and Methylation in Human Brain: Implications for vaccines 1
Life can be viewed through the perspective of oxidation and reduction, which involves the loss and gain of electrons, respectively. 2
Life is thought to have first arisen in a sulfur-rich environment in the ocean depths, where sulfur compounds could serve as energy sources and antioxidants. 3
Oxidation is important throughout life and can be used to control development. 4
Outline of the presentation 5
Glutathione (GSH) is the primary intracellular antioxidant. It receives electrons that originate from glucose and other metabolites such as isocitrate. These electrons are passed via a series of reactions which include thioredoxin and the selenoprotein thioredoxin reductase. The balance between oxidation and reduction is known as the redox state of the cell. 6
The redox state exerts powerful and fundamental control over all cells. When cells are highly reduced they tend to divide, but when they are more oxidized they stop proliferating and undergo differentiation. Under extreme conditions of oxidative stress cells die by either apoptosis or necrosis. 7
The sulfur amino acid cysteine is rate limiting for synthesis of GSH, and changes in cysteine levels will lead to changes in the redox state of cells. Oxidation of GSH leads to formation of gluathionine disulfide (GSSG), and the ratio of GSH to GSSG is an indicator of the redox state of a cell. 8
The body obtains cysteine via uptake from the GI tract, which involves the cysteine transporter known as EAAT3. Once absorbed via GI epithelial cells, cysteine can be taken up from the blood by different tissues. In the liver, some cysteine is oxidized to its dimeric cystine form, which is the only form that can cross the blood-brain barrier. In the brain cystine is taken up by astrocytes which convert it to GSH. Excess GSH is released and hydrolyzed to cysteine, which is available for uptake by neurons via the same EAAT3 transporter found in the GI tract. This is the primary source of cysteine for neurons to make GSH. 9
The EAAT3 cysteine transporter has been demonstrated to be present in the small intestine (ileum), especially the later portion, which is where autistic children commonly display inflammation, reflecting too little antioxidant. 10
Brain-specific redox features 11
The available amount of cysteine is much lower (100-fold) in cerebral spinal fluid surrounding the brain than in plasma, and this relative scarcity makes cysteine uptake very important to neurons to maintain a normal redox state. 12
In addition to EAAT3-mediated uptake, neurons can also create cysteine by transsulfuration of homocysteine (HCY), which is a product of the four-step methylation cycle. 13
However, in neurons the transsulfuration pathway operates at a lower level than in other cells, limiting the supply of cysteine. 14
The lower rate of transsulfuration is evident as a higher level of cystathionine, and cystathionine levels are remarkably higher in the brain of humans compared to other species, although this is not observed in other tissues. This evolutionary trend indicates a progressive decrease in transsulfuration, which places increase importance on EAAT3-mediated cysteine uptake to meet cellular antioxidant needs. 15
Increased dependence upon cysteine uptake allows neurotrophic growth factors to strongly influence methylation reactions, including DNA methylation, by increasing EAAT3-mediated cysteine uptake. The folate and vitamin B12 (cobalamin)-dependent enzyme methionine synthase is highly sensitive to redox status and synthesis of the active form of vitamin B12 requires GSH (Green; upper right). 16
Methylation of DNA is the primary event in epigenetic regulation of gene expression, which is particularly important during development. 17
The D4 dopamine receptor carries out a four-step cycle of phospholipid methylation that is stimulated by dopamine and is dependent upon methionine synthase for an ongoing supply of methyl groups (Blue; lower left). This unique activity was first discovered in out laboratory and it appears to play an important role in attention, by promoting synchronization of neural networks. If oxidative stress causes a decrease in methionine synthase activity, it will compromise dopamine-stimulated phospholipid methylation. Notably, it is generally recognized that the D4 receptor gene is the most important genetic risk factor for ADHD (attention-deficit hyperactivity disorder). 18
A specific methionine residue in the D4 dopamine receptor (yellow) is activated by ATP and then transfers its methyl group (i.e. carbon atom) to adjacent phospholipid molecules. A new methyl group is then provided from methylfolate by the vitamin B12-dependent enzyme methionine synthase. 19
It has been previously reported that measles virus infection completely blocks the ability of the catcholamine isoproterenol to stimulate phospholipid methylation, raising the possibility that measles virus infection might also interfere with dopamine-stimulated phospholipid methylation. 20
Methionine synthase controls all methylation reactions, including DNA methylation. 21
The molecular structure of methionine synthase consists of five separate domains, including binding sites for homocysteine (HCY) (pink), methylfolate (green), cobalamin (red) and S-adenosylmethionine (SAM) (blue), along with a Cap domain (yellow) which partially protects cobalamin from oxidation. During enzyme activity the methyl group from methylfolate is transferred to cobalamin, creating methylcobalamin, after which the HCY domain brings HCY close enough to methylcobalamin to pick up the methyl group, converting HCY to methionine. Oxidation of the cobalamin can occur while it awaits the next methyl group. If the cobalamin is oxidized, SAM, along with an auxillary reductase, is required to reactivate the enzyme. 22
Our measurement of the level of methionine synthase mrna in postmortem human cortex revealed a progressive decrease across the lifespan, amounting to about 400-fold. This gradual decrease in methionine synthase promotes increased transsulfuration of HCY to cysteine and GSH, but at the expense of methylation. This is a useful adaptive response since the need for antioxidant increases with age. This finding also suggests that changes in methionine synthase will cause progressive changes in DNA methylation with epigenetic consequences across the lifespan. 23
PCR analysis of methionine synthase mrna from postmortem human cortex from a 24 yr old control subject shows the presence of all five domains. 24
However, PCR analysis of methionine synthase mrna from postmortem human cortex from an 80 yr old control subject shows only four domains, with the Cap domain missing. 25
Quantitative analysis confirms that the Cap domain mrna is absent from methionine synthase mrna in older subjects. 26
Deletion of the Cap domain portion of the mrna reflects age-dependent alternative splicing of methionine synthase pre-mrna. 27
When the Cap domain is absent, the enzyme is more easily oxidized, increasing the probability of HCY diversion to cysteine and GSH synthesis. This is another useful adaptive redox response during aging. 28
Rates of autism have steadily increased in the U.S. since about 1988, and it has been proposed that a portion of this increase may be due to vaccine-derived mercury. 29
Autistic children show significant abnormalities in thiol metabolites from the methylation and redox pathways. Specifically, GSH 30
We carried out a comparison of methionine synthase mrna levels in postmortem brain samples of autistic and non-autistic subjects. Levels were noticeably lower in autistic subjects, especially at younger ages. This was observed for both cobalamin-binding and Cap domains, indicating that transcription is decreased. The decrease is suggestive of increased oxidative stress in autism, and the decrease in methionine synthase transcription is likely to result in epigenetic consequences and dysregulated neurodevelopment. 31
Effects of neurotrophic growth factors and TNF-alpha on EAAT3 activity 32
Neurotrophic growth factors increase PI3 kinase activity which can augment EAAT3-mediated uptake of cysteine. 33
Brain-derived growth factor (BDNF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-1) increase cysteine uptake in cultured human neuroblastoma cells. Their effects are blocked by the PI3 kinase inhibitor wortmannin. 34
IGF-1 increases cysteine, GSH and SAM levels, and increases the ratio of GSH to GSSG, indicating a more reduced intracellular redox state. It also increases the ratio of SAM to SAH, indicating an increase in methylation potential. 35
IGF-1 stimulates methionine synthase activity and increases DNA methylation in human neuroblastoma cells. These changes reflect the more reduced intracellular redox environment caused by increased cysteine uptake, an example of redox signaling. 36
Thus activation of EAAT3 allows neurotrophic growth factors to exert powerful control over DNA methylation, thereby producing epigenetic effects on cells. 37
The pre-inflammatory cytokine TNF-alpha inhibits EAAT3-mediated cysteine uptake in neuronal cells 38
The pre-inflammatory cytokine TNF-alpha, whose levels increase following vaccination, reduces the level of methionine synthase mrna in neuronal cells. 39
Although TNF-alpha inhibits cysteine uptake, intracellular levels of cysteine are increased, which reflects activation of transsulfuration (i.e. conversion of homocyseine to cystathionine and cysteine). 40
TNF-alpha inhibits cysteine uptake and methionine synthase, but increases transsulfuration. 41
Redox effects of mercury and aluminum: Implications for immune response 42
EAAT3-mediated cysteine uptake in cultured human neuronal cells is inhibited by heavy metals and is potently inhibited by the vaccine preservative thimerosal. 43
The vaccine preservative thimerosal potently lowers GSH levels and inhibits methionine synthase activity. 44
Thus the EAAT3 cysteine transporter is stimulated by growth factors, but inhibited by TNF-alpha, mercury, aluminum and food-derived opiate peptides from casein and gluten. 45
Mercury has an extraordinarily high affinity for binding to selenocysteine (10 45 ), which approaches irreversible binding. Thus mercury will bind to and inhibit selenoproteins at low concentrations.
Thimerosal inhibits the selenoprotein thioredoxin reductase with high potency, whereas thioredoxin, which lacks a selenium, is less potently inhibited. 47
Mercury binds to selenoprotein P (SelP), which contains 11 selenocysteines and functions as a supplier of selenium to cells when it is taken up. SelP binds to beta amyloid and to neurofibrillary tangles, both of which are pathological features of Alzheimer s disease. By interfering with selenoprotein function, mercury may contribute to Alzheimer s disease. 48
Aluminum potently inhibits methionine synthase activity in human neuroblastoma cells. 49
Aluminum has been shown to inhibit the mitochondrial form of NADP + -dependent isocitrate dehydrogenase. This enzyme is normally activated by calcium, and is a major source of NADPH, which is essential for maintaining GSH levels in mitochondria. Aluminum inhibition of NADP + -dependent isocitrate dehydrogenase will increase ROS levels, and can thereby contribute to autoimmunity, as well as its role as a vaccine adjuvant (see below). 50
Isocitrate dehydrogenase is also inhibited by glutathionylation, which is carried out by the transfer of GSH from GSSG (oxidized glutathione) to a cysteine residue. Thus oxidative stress conditions will reinforce NADP + -dependent isocitrate dehydrogenase inhibition and further increase mitochondrial ROS levels. 51
Recent studies have clarified the role of aluminum as a vaccine adjuvant, showing that increased ROS levels promote activation of the NLRP3 inflammasome. Aluminum s inhibition of mitochondrial NAD P + -dependent isocitrate dehydrogenase increases ROS, and shifts thioredoxin to its oxidized form. The reduced form of thioredoxin (Txn) binds to TXNIP, an inhibitor of the NLRP3 inflammasome, but thioredoxin oxidation releases TXNIP and promotes the immune response. Notably, mercury potently inhibits thioredoxin reductase (TxnR) and contributes to immune activation by decreasing Txn reduction. 52
Redox signaling also plays a central role in the immune response at the level of T cell activation by antigen-presenting cells (APCs, also known as dendritic cells). APCs release GSH, which is converted to cysteine in the extracellular space, analogous to the role of astrocytes in the brain. Cysteine is taken up by T o cells which are bound to APCs via MHC/T-cell receptor interaction. Cysteine uptake shifts the redox state of T cells, activating them and promoting their ability to initiate clonal development. 53
Our lab compared the levels of GSH in autoimmune-prone SJL mice vs. normal mice and found a significantly lower level associated with autoimmunity. Methionine synthase activity was also significantly lower in SJL mice. Together these findings indicate that oxidative stress and impaired methylation, which are caused by aluminum and mercury, are associated with increased autoimmunity. 54
Gluten and casein-derived opiate peptides 55
Hydrolysis of casein and gluten yields proline-containing peptides that have the ability to stimulate opiate receptors, similar to morphine. Human and bovine casein-derived peptides (beta casomorphin-7 or BCM7) differ in two amino acids. Presence of a histidine (His) residue at the carboxyl terminus of the bovine peptide in A1 cows facilitates its release from the parent casein, whereas a proline (Pro) residue present in A2 cows inhibits peptide release. Thus milk from A1 cows yields higher levels of opiate peptides. 56
Bovine beta casein has a SNP at position 67. The histidine allele facilitates cleavage and release of BCM7, while the proline allele restricts cleavage and yields little BCM7. 57
A comparison of A1 milk consumption in different countries shows a strong association with the prevalence of type I diabetes, which may correlate with autoimmunity. 58
A recent study in Finland compared the development of type I diabetes and auto-antibody titers in children who were fed either standard formula (bovine casein) or a hydrolyzed casein version of the same formula. A decrease of about 50% was observed in both the incidence of diabetes and in the level of auto-antibodies. Thus it appears that casein makes a substantial contribution to auto-immune diseases such as type I diabetes, which may involve the opiate peptide effects of bovine BCM7. 59
Morphine causes a dose-dependent decrease in EAAT3-mediated cysteine uptake by human neuroblastoma cells. 60
Bovine and human forms of BCM7 inhibit cysteine uptake in neuroblastoma cells after 30 min of incubation, and the bovine form is more effective. 61
Food-derived opiate peptides and morphine inhibit EAAT3-mediated cysteine uptake in human GI epithelial cells. Bovine BCM7 is more inhibitory than human BCM7. 62
Bovine BCM7 causes significant changes in cellular thiol levels in human neuroblastoma cells. Cysteine and GSH levels are decreased, indicative of oxidative stress. The reduction in methionine and increase of homocysteine is indicative of methionine synthase inhibition. 63
The inhibitory effects of morphine and food-derived opiates on cysteine uptake are blocked by the mu and delta opiate receptor antagonist naltrexone. 64
Notably, naloxone, a mu and delta opiate receptor antagonist, is as effective a vaccine adjuvant as aluminum, which may reflect its effects on EAAT3-mediated cysteine uptake. 65
Opiate peptides can restrict whole-body availability of cysteine via their effects on EAAT3-mediated cysteine uptake in the distal ileum. The decrease in circulating cysteine will lead to decreased GSH and oxidative stress, as well as decreased cystine for uptake by the brain and ultimately leading to oxidative stress in neurons, which can adversely affect methylation with epigenetic consequences for development. 66
Summary of major points. 67
Summary of major points. 68
Thanks are due to the students from the Deth lab who carried out this research. 69
Acknowledgements 70