Underlying Factors Leading To Autism - Dr Amy Yasko

A Molecular Approach to Health Care

Underlying Factors Leading to Autism Dr. Amy Yasko

“What do schizophrenia, diabetes and autism have in common? The answer is that many different factors act together to influence their development. As well as fundamentals like age and gender, other genetic and environmental factors…may play a role in the onset of these so-called multifactorial diseases.”

Multifactorial Diseases:Choose your targets BIOPEOPLE, Summer 2004

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection Chronic Viral Infection Heavy Metal Burden HLA Type Relationship to Asthma DPT Vaccine

Predisposing Factors That Increase Susceptibility to Autism • Methylation Cycle Mutations • Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection Chronic Viral Infection Heavy Metal Burden HLA Type Relationship to Asthma DPT Vaccine

What is Methylation?

Methylation Methyl groups – CH3

H

H

C H

Bound to DNA, enzymes, vitamins

In many cancer cells, inappropriate methylation – the attachment of methyl (CH3) groups to DNA’s cytosine bases – can silence genes that suppress tumor growth.

The Importance of Methylation

“Methylation happens over a billion times a second. It is like one big dance, with biochemicals passing methyl groups from one partner to another.”

The H Factor Solution, Dr. James Braly and P. Holford

DNA Methylation “DNA methylation may maintain the large amount of non-coding DNA in an inert state.” “This process would help prevent the transcription of large parts of the genome… INSERTED VIRAL SEQUENCES.” “…consequences of loss of methylation… could cause the potentially harmful expression of inserted viral genes.” New England Journal of Medicine, November 20, 2003

Particular relevance of this function of methylation to autism

Methylation

•

•

DNA synthesis •

Megaloblastic anemia

•

T cells

•

Intestinal mucosa

Involved in DNA regulation •

Host

•

Viral

•

Myelination and pruning

•

Proper immune response to i.e. TB

•

Membrane fluidity, phospholipid methylation

•

Methylation and polyamine synthesis

•

Methylation and detoxification

•

•

Arsenic

•

Selenium

Enzymatic reactions requiring methylation •

Melatonin

•

Neurotransmitter levels : dopamine, norepinephrine

•

Tryptophan methylation: serotonin

Importance of the Methylation Cycle • • • • • • • • • • • • • •

Methylation and Nucleic Acid Synthesis DNA Methylation Methylation, Pregnancy and Neural Tube Defects Methylation and Heart Disease Methylation and Energy Production Methylation and ADD Protein Methylation Methylation and Myelin Methylation and the Immune System Methylation and Cancer Methylation and Allergic Reactions Methylation and Anesthesia Methylation and Environmental Toxins Methylation and Aging

• Methylation Affects Both Nature and Nurture

• Decreased methylation • Decreased BH4 • Increased ammonia • Elevated homocysteine

CONSEQUENCES OF MUTATIONS

• •

Psoriasis Memory issues

Consequences of Reduced SAMe

Phosphatidyl serine Mg++

Phosphatidyl ethanolamine

SAMe Phosphatidyl methyl ethanolamine

SAMe Phosphatidyl dimethyl ethanolamine SAMe Phosphatidyl choline

SAMe is involved in the synthesis of: • • • • • •

Creatinine Creatine Methylcobalamin Phosphatidylcholine Coenzyme Q10 Carnitine Methylation by SAMe is a critical step in the stabilization of many proteins including myelin.

Serotonin 5-HT; 5-hydroxytryptamine

Melatonin N-acetyl-5-methoxytryptamine

Vitamin K2 made by intestinal bacteria

Superfamily: S-adenosyl-L-methionine-dependent methyltransferases Lineage: Catechol O-methyltransferase, COMT (1) Plant O-methyltransferase, C-terminal domain (3) RNA methyltransferase FtsJ (1) Fibrillarin homologue (1) Hypothetical protein MJ0882 (1) Hypothetical protein HI0319 (YecO) (1) Glycine N-methyltransferase (1) Phenylethanolamine N-methyltransferase, PNMTase (1) Histamine methyltransferase (1) Guanidinoacetate methyltransferase (1) Arginine methyltransferase, HMT1 (2) lacks the last two strands of the common fold replaced with a beta-sandwich oligomerisation subdomain Protein-L-isoaspartyl O-methyltransferase (3) Glucose-inhibited division protein B (GidB) (1) Probable methyltransferase Rv2118c (1) contains additional N-terminal beta-sandwich domain, res. 1-67 Chemotaxis receptor methyltransferase CheR, C-terminal domain (1) contains additional N-terminal all-alpha domain, res. 11-91 RNA methylases (4) DNA methylases (5) Type II DNA methylase (3) circularly permuted version of the common fold Spermidine synthase (1) contains additional N-terminal tetramerisation all-beta domain, res. 1-71 Mycolic acid cyclopropane synthase (2)

•

SAMe is involved in polyamine synthesis

•

Haven’t discussed role of polyamines before

•

Few slides on these important compounds

The polyamines, e.g. putrescine, spermidine, and spermine, constitute a group of cell components that are important in the regulation of cell proliferation and cell differentiation. There is also evidence suggesting a role for polyamines in programmed cell death.

Aminopropylation converts polyamines (putrescine to spermidine and spermine), which are necessary for cell growth. The by-product: methylthioadenosine (MTA) functions in cellular regulation and growth.

Functions of Polyamines • • • • • •

Stimulation of Casein Kinase II activity Cell growth Gene expression and differentiation Inhibition of protein kinase C Inhibition of platelet aggregation Low concentrations lead to increased opening of NMDA channels

Polyamine synthesis is tied to the urea cycle via ornithine.

Polyamines help to regulate the level of methionine synthase All the polyamines tested in these experiments were found to stimulate methionine synthase. Spermine was the most active of the polyamines tested (EC50 8 mM), whereas putrescine caused stimulation only in the millimolar range. The number of positive charges associated with the polyamines was correlated with their ability to stimulate enzyme activity, because spermine has four positively charged groups, spermidine three and putrescine two. To test this hypothesis further, the effect of cadaverine, also with two charged groups, was examined and it was found to be similar to that of putrescine. Thus at least part of the effect of polyamines on methionine synthase was probably due to electrostatic binding. In most of their functions polyamines are documented to interact by electrostatic binding, although polyamines have been shown to bind covalently to amino acids and proteins Polyamines have also been reported to exhibit radical scavenging properties. Because the cobalt of the vitamin B12 prosthetic group is known to be susceptible to oxidation, polyamines acting as scavengers could offer protection against oxidizing radicals and hence stimulate enzyme activity.

Polyamine synthesis requires SAMe and the methylation pathway

Influence of Polyamines on Phosphatidyl Choline Levels Eur J Biochem. 1981 May;116(1):1-6 Induction of choline transport and its role in the stimulation of the incorporation of choline into phosphatidylcholine by polyamines in a polyamine auxotroph of Saccharomyces cerevisiae. Hosaka K, Yamashita S. A mutant of Saccharomyces cerevisiae, defective in ornithine decarboxylase, was isolated. A prolonged culture of the mutant in a polyamine-free medium resulted in a great decrease in the polyamine content and in cessation of growth. The addition of polyamines to the culture induced the growth after a lag period of 5--6.5 h. The growth rate in the presence of polyamine was comparable to that of the wild-type strain. The effectiveness of polyamines was as follows: spermidine greater than putrescine approximately equal to spermine. 2. Phosphatidylcholinesynthesizing activity during the lag phase of growth was determined by measuring the rate of incorporation of [14C]choline into phosphatidylcholine. The incorporation rate was markedly increased with time by polyamine prior to the initiation of cell division. Polyamines were effective in the following order: spermidine greater than putrescine approximately equal to spermine. Experiments with methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, showed that putrescine stimulates cell growth and choline incorporation into phosphatidylcholine after it has been converted into spermidine in the cell. 3. The induction of the choline transport system was shown to be responsible for the increase in the rate of incorporation of [14C]choline into phosphatidylcholine effected by polyamines. A low concentration of cycloheximide completely prevented the induction of choline transport by polyamines. The levels of the CDP-choline pathway enzymes such as choline kinase, cholinephosphate cytidyltransferase and cholinephosphotransferase were not significantly changed.

FEBS Lett 1987 Apr 20;214(2):375. Cyclic AMP-like effects of polyamines on phosphatidylcholine synthesis and protein phosphorylation in human promyelocytic leukemia HL60 cells. Comparison with the effects of phorbol ester. Kiss Z, Deli E, Kuo JF. Spermine or putrescine increased cAMP levels through a catalase-sensitive mechanism, resulting in, most notably, a dephosphorylation of protein A (Mr 45,000, pI 5.15) and protein B (Mr 45,000, pI 4.9) and slightly increased phosphatidylcholine (PC) synthesis in HL60 cells. Exogenous dibutyryl cAMP mimicked the polyamine effects. 12-O-Tetradecanoyl phorbol-13acetate (TPA) also promoted the protein dephosphorylation and PC synthesis, the effects augmented by R59022 and mimicked by exogenous 1-oleoyl-2-acetylglycerol. The effects of spermine (or dibutyryl cAMP) and TPA on PC synthesis were synergistic. It was suggested that cAMP-dependent protein kinase and protein kinase C might mediate, in an independent but inter-related manner, the effects of polyamines and TPA. PMID: 3030816 [PubMed - indexed for MEDLINE]

Casein kinase II is Stimulated by Polyamines • Casein kinase II plays a role in circadian rhythm • Casein kinase II plays a role in cell cycle regulation • Casein kinase is inhibited by sulfates (relationship to CBS up regulations) • Data suggests that the anti-proliferative activity of p53 is activated by phosphorylation at serine 386 by Casein kinase II and establishes a direct link between the covalent modification of a growth suppressor protein and regulation of its activity in mammalian cells. (Mutation of the casein kinase II phosphorylation site abolishes the anti-proliferative activity of p53. DM Milne, RH Palmer and DW Meek Nucleic Acids Research, Vol 20, Issue 21 5565-5570 1992)

Modifications for Regulation

Mutations

• Ubiquitination • Acetylation • Methylation

• UBE3A • NAT • Methylation cycle, MeCP2…

• Phosphorylation

• Casein Kinase II, milk

CLOSED to expression

OPEN to expression

How Does Methylation Control Synthesis of Proteins? One of the ways the cells control which genetic information they will use is to chemically modify the DNA. The illustration shows an enzyme (diagrammed in ribbons) adding methyl groups to some of the DNA (balls in the form of a double helix). This inactivates that part of the chromosome. It's as if we were to put glue on the edges of some of the books in the library; those pages would become unavailable to readers.

CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J. 1998 Sep 1;17(17):4905-8. Razin A.

• There is a three-way connection between DNA methylation, gene activity and chromatin structure . • Methylation plays a pivotal role in establishing and maintaining an inactive state of a gene by rendering the chromatin structure inaccessible to the transcription machinery.

Targeted Methylation Introducing methylated DNA at specific genomic loci affects local histone acetylation. By Jonathan Weitzman

Methylation of DNA at CpG dinucleotides represses gene transcription. Methylation plays an important role in development, imprinting, X-chromosome inactivation and tissue-specific gene expression, but the mechanisms of methylation-induced repression are still unclear. In the December Molecular and Cellular Biology, Schubeler et al. show that localized histone deacetylation can explain methylation-induced repression (Mol Cell Biol 2000, 20:9103-9112). The authors used an elegant technique called recombinase-mediated cassette exchange (RMCE) to introduce in vitro-methylated DNA at defined chromosomal positions. They used the Cre recombinase to insert methylated or unmethylated forms of the human β-globin gene promoter driving a green fluorescent protein (GFP) reporter gene. Methylation repressed GFP expression, and was stable in cells over at least 12 weeks in culture. Methylation did not affect DNA replication or global chromatin remodeling. However, methylation caused a hypoacetylation of histones H3 and H4 within the transgene. These observations support a model in which methylated DNA represses

DNA methylation in genomic imprinting, development, and disease The Journal of Pathology, September 2001, vol. 195, no. 1, pp. 97-110(14) Paulsen M.[1]; Ferguson-Smith A.C.[1]*, [1]University of Cambridge, Department of Anatomy, Cambridge CB2 3DY, UK [*]University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK

Changes in DNA methylation profiles are common features of development and in a number of human diseases, such as cancer and imprinting disorders like Beckwith– Wiedemann and Prader–Willi/Angelman syndromes. This suggests that DNA methylation is required for proper gene regulation during development and in differentiated tissues and has clinical relevance. DNA methylation is also involved in X-chromosome inactivation and the allelespecific silencing of imprinted genes. This review describes possible mechanisms by which DNA methylation can regulate gene expression, using imprinted genes as examples. The molecular basis of methylation-mediated gene regulation is related to changes in chromatin structure and appears to be similar for both imprinted and biallelically expressed genes.

The expression of many cellular genes is modulated by DNA methylation and histone acetylation. These processes can influence malignant cell transformation and are also responsible for the silencing of DNA constructs introduced into mammalian cells for therapeutic or research purposes.

Targeted Methylation Introducing methylated DNA at specific genomic loci affects local histone acetylation. By Jonathan Weitzman

• Methylation of DNA at CpG dinucleotides represses gene transcription. • Localized histone deacetylation can explain methylation-induced repression. (Mol Cell Biol 2000, 20:9103-9112).

• Methylation caused a hypoacetylation of histones. • Methylated DNA represses local transcription by recruiting histone deacetylase activity.

Universal lack of methylation and inability to produce nucleic acids necessary for RNA synthesis result in a situation where the body is lacking the required regulatory elements for genetic silencing. Silencing is a multistep process that involves RNA as well as methylation and deacetylation of histones.

Histone deacylation

Turn off Genes

DNA methylation

SIR2 Histone deacylation

Turn off Genes

DNA methylation

IGF

CH3

The Methylation Cycle

Methylation Cycle • Specifics to be discussed later today • Use of Nutrition to Bypass Genetic Mutations • RNA and Methylation

MTHFr zmethylation zpurine metab. zpyrimidine metab. zT cells yHistamine zBH4 ztyrosine zN methyl nicotin (stress uv) zmyelination yprecursors glut/glycine z5 methyl C z1 methyl tryp. yIDO ztryptophan yyIL6 zmethylation yyTNF zmethylation zmyelination yTNF zmethylation yTNF zmethylation zVit. K yIL6 yIL6 zmethylation yIDO ztryptophan

Glutamate R yglutamate y

Strep

Metals

Virus/Vaccine zzzT cells yIDO

Blood/HLA yIDO z1 methyl tryp

DPT

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations

Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection Chronic Viral Infection Heavy Metal Burden HLA Type Relationship to Asthma DPT Vaccine

Excitotoxins • Glutamate, Glutamic Acid, Glutamine, MSG • Aspartate, Aspartame, Aspartic Acid • Nutra-sweet • Cysteine (NOT N-acetyl-cysteine)

Glutamine can be converted to glutamate plus ammonia

Other “Names” For Excitotoxins monosodium glutamate glutamate natural flavor(s) natural flavoring(s) maltodextrin carrageenan gelatin spice(s) seasoning(s) seasoned salt dough conditioner(s) yeast extract autolyzed yeast autolyzed yeast extract autolyzed anything broth stock soup base

nutrasweet / aspartame hydrolyzed protein hydrolyzed vegetable protein (HVP) hydrolyzed plant protein hydrolyzed oat flour hydrolyze anything sodium caseinate calcium caseinate caseinate disodium guanylate disodium inosinate disodium caseinate chicken/pork/beef “flavoring” chicken/pork/beef “base” bouillon vegetable gum plant protein extract smoke flavoring(s)

malted barely flour malt extract malt flavoring(s) malted barley / barley malt malted anything textured protein guar gum soy extract soy protein soy protein concentrate soy protein isolate soy sauce whey protein whey protein isolate whey protein concentrate kombu extract l-cysteine ajinomoto

Protein Breakdown Proteins breakdown

Amino Acids

Glutamate Aspartate Cysteine

Natural Foods High in Glutamate • Peas • Tomatoes • Mushrooms • Parmesan Cheese Glutamate Receptors Glutamate Glutamate is an excititory neurotransmittor

• Sulfur containing amino acids lead to increased norepinephrine release. • This effect appears to be mediated via stimulation of glutamate receptors implying an excitotoxin mechanism. Note: relationship between excess sulfur via CBS upregulations and glutamate

The issue is both the amount of glutamate as well as the number of glutamate receptors

Glutamate Receptors Increased Number of Glutamate Receptors In conjunction with…

Excessive Glutamate Neurological Inflammation

Fragile X

FMRP

mRNA

glutamate receptors

Trends Neurosci 2004 Jul;27(7);370-7. The mGluR theory of fragile X… Mark F Bear Kimberly M Huber Stephen T Warren The Picower Center for Learning and Memory, Howard Hughes Medical Institute and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.Many of the diverse functional consequences of activating group 1 metabotropic glutamate receptors require translation of preexisting mRNA near synapses. One of these consequences is long-term depression (LTD) of transmission at hippocampal synapses. Loss of fragile X mental retardation protein (FMRP), the defect responsible for fragile X syndrome in humans, increases LTD in mouse hippocampus. This finding is consistent with the growing evidence that FMRP normally functions as a repressor of translation of specific mRNAs. Here we present a theory that can account for diverse neurological and psychiatric aspects of fragile X syndrome, based on the assumption that many of the protein-synthesisdependent functions of metabotropic receptors are exaggerated in fragile X syndrome. The theory suggests new directions for basic research as well as novel therapeutic approaches for the treatment of humans with fragile X, the most frequent inherited cause of mental retardation and an identified cause of autism.

Glutamate & Schizophrenia “Fine-tuning drugs to act on particular glutamate receptors could lead to treatments for anxiety, depression, addiction, and even schizophrenia.” “Bita Moghaddam of Yale University calls ‘a state of glutamatergic chaos’ that results in the kind of disorganized cortical activity found in schizophrenia.” Excited by Glutamate Science, 6/20/2003, Vol. 300, Issue 5627

Glutamate & Seizures

“Glutamate-stimulating drugs can induce seizures, for instance” Excited by Glutamate Science, 6/20/2003, Vol. 300, Issue 5627

Glutamate & ALS

“…abnormal post-transcriptional modification of mRNA encoding a glutamate receptor may promote sporadic ALS.” Sporadic ALS: Blame it on the Editor Nature Medicine, April 2004, Vol. 10, No. 4

Glutamate & Intelligence • Doogie mice have been genetically engineered to make more than the usual amount of a key subunit of a protein called the N-methyl-D-aspartate (NMDA) receptor. • Scientists theorize that such strengthening is the basis for learning and memory. • Doogie mice are better than normal mice at distinguishing between objects they have seen before and at recalling how to find a platform in a tank of murky water, for instance. Building a Branier Mouse Tsien, Scientific American, April 2000

Receptor Recycling Receptor recycling may give inaccurate measurements of the number of glutamate receptors on autistic brain cells.

Glutamate Receptors Actual amount of mRNA for glutamate receptors is higher in autistic children. Levels of mRNA in 10 autism (A = autism) and 10 control (C = control) postmortem cerebellum samples were compared using reverse transcriptase PCR (RT-PCR). The mean autism versus mean control band intensity (± SEM) is summarized to the right of each RT-PCR. The Y axis of each graph represents average normalized intensity of cDNA product signals. (A) RT-PCR confirmation of altered mRNA levels in autism. All gene transcripts are increased in autism postmortem cerebellum (*p < 0.05). (B) Investigation of mRNA levels of additional glutamate-related genes. Excitatory amino acid transporter 2 (EAAT 2) and the AMPA-type glutamate receptors 2 and 3 have increased mRNA levels in autism (*p < 0.05). Glyceraldehyde-3-phosphate dehydrogenase band intensity (right, lower panel) was used for normalization of cDNA product in each sample. Reference: Purcell, Neurology 2001; 57:1618-1628

Glutamate Receptors and Folate Do methylation cycle mutations combined with folate supplementation lead to excess glutamate. Does this increase the number of glutamate receptors in the growing child?

Entry

Definition

Tetrahydrofolyl-[Glu](n) Poly-L-glutamate + Tetrahydrofolate R04242 Equation

RPair Pathway Enzyme Ortholog

Reaction C03541 C05930 + C00101

RP: A03877 C00101_C03541 main PATH: rn00790 Folate biosynthesis 3.4.19.9 KO: K01307 gamma-glutamyl hydrolase

IMPORTANCE OF GLUTAMATE TO METAL TOXICITY

“In the absence of glutamate, neurons are unaffected by acute exposure to mercury, suggesting that neuronal dysfunction is secondary to disturbances in astrocytes.” “Coapplication of nontoxic concentrations of MeHG and glutamate leads to the typical appearance of neuronal lesions associated with excitotxin stimulation”

Brookes, 1992/Matyja and Albrecht, 1993

IMPORTANCE OF METAL TOXICITY TO GLUTAMATE

Aluminum interferes with glutamate dehydrogenase

Mercury inhibits glutamine synthase

X

mercury

X X

aluminum

Glutamate

excitotoxin damage

opioids

• A major function of hippocampal encocannabinoids is to regulate GABA release. • Endocannabinoid synthesis can be triggered by activation of glutamate receptors. • Calcium influx increases depolarizations that lead to cannabinoid increase. • mGluR and depolarization appear to be two independent pathways to endocannabinoid synthesis. • Activation of cannabinoid receptors suppresses CCK release in addition to suppressing GABA release.

“Neurons need to protect themselves against the risk of excessive activity that can lead to neurotoxicity. Protective mechanisms likely exist to provide on-demand defense in the case of unusually high neuronal-spiking activity. Marsicano et al. created conditional mouse mutants missing the cannabinoid receptor type 1 in pyramidal cells but not in interneurons of the forebrain. Protection against seizures induced by the excitotoxin kainic acid was exerted via CB1 receptors in glutamatergic but not GABAergic neurons. The seizures enhanced production of anandamide-endogenous cannabinoid-in wild type but not in mutant mice.

Thus, the activation of the endogenous cannabinoid system is an on-demand, early, and necessary step for physiological protection against excitotoxicity.” Protective Endocannabinoids Science, 2003, Vol. 302

CB1 Cannabinoid Receptors and On-Demand Defense Against Excitotoxicity. Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A, Azad SC, Cascio MG, Gutierrez SO, van der Stelt M, Lopez-Rodriguez ML, Casanova E, Schutz G, Zieglgansberger W, Di Marzo V, Behl C, Lutz B. 3 OCTOBER 2003

VOL 302 SCIENCE

www.sciencemag.org

October 15, 2005

This suggests that cannabinoids can boost neurogenesis

Opioids and non-opioid enantiomers selectively attenuate N-methyl-D-aspartate neurotoxicity on cortical neurons. Addition of 1 microM-1 mM methadone to the bathing medium produced a concentration-dependent reduction in the neurotoxicity of exogenously applied N-methyl-D-aspartate (NMDA) in murine cortical cell culture (EC50 about 100 microM); the reduction persisted at intense NMDA exposure, consistent with non-competitive inhibition. Methadone also protected against exposure to quinolinate but not quisqualate or kainate. Concentrations (100 microM-3 mM) of several other opioids - morphine, fentanyl, codeine, meperidine, dextropropoxyphene, and naltrexone - were additionally found to produce concentration-dependent reductions in NMDA neurotoxicity. This novel neuron-protective effect of opioids was not mediated by conventional opioid receptors: the non-opioid enantiomer of methadone and morphine exhibited a potency equal to or greater than that of the opioid enantiomer, and 1 mM naloxone did not act as an antagonist. The possibility that opioids, or especially non-opioid enantiomers of opioids, might provide a useful therapeutic approach in diseases states involving NMDA receptor-mediated neurotoxicity, warrants further study. Choi DW, Viseskul V. Eur J Pharmacol. 1988 Oct 11;155(1-2):27-35.

Fig. 3. Endocannabinoids and the inhibitory hippocampal network. Endocannabinoids selectively inhibit GABA release from regularspiking basket cells. These cells also release the neuromodulatory peptide CCK and are depolarized by acetylcholine and substance P. CB11 interneurons use only N-type Ca21 channels at the presynaptic bouton; the function of this presynaptic specialization is unknown, but may confer supersensitivity to presynaptic inhibition because N-type channels are strongly inhibited by Gbg. Another class of basket cells is negative for CB1 and CCK, fast-spiking, and uses only P/Q-type Ca21 channels for GABA release. Finally, regular-spiking cells forming synapses with slow kinetics on distal dendrites use both N- and P/Q-type channels for GABA release and are endocannabinoid insensitive.

Muscarinic receptors may facilitate GABA release while nicotinic receptors do not appear to play a role. Ming, Hu et al Chronic Ethanol Exposure Increases H-GABA Release in Rat Hippocampus by Presynaptic Muscarinic Receptor Modulation. Alcoholism Clinical & Experimental Research, 23: 1587, 1999.

Glutamate

glutathione

TNF alpha

leaky gut

“… HIV-infected persons and SIV-infected rhesus macaques have also, on the average, substantially increased plasma glutamate levels. Increased glutamate levels aggravate the cysteine deficiency by inhibiting the membrane transport of cystine. Even moderately elevated extracellular glutamate levels as they occur in HIV-infected persons cause a substantial decrease of intracellular cysteine levels. The cellular cysteine supply affects amongst others the intracellular glutathione level and IL-2-dependent proliferation of T cells and (inversely) also the activation of the transcription factor NF-kappaB. The cysteine deficiency of HIVinfected persons is, therefore, possibly responsible not only for the cellular dysfunction but also for the overexpression of tumor necrosis factor-alpha (TNFalpha), interleukin-2 receptor alpha-chain, and beta2-microglobulin. All the corresponding genes are associated with kappaB-like enhancer sequences. W. Cysteine and glutathione deficiency in AIDS patients: a rationale for the treatment with N-acetyl-cysteine. Pharmacology 46:61-65; 1993.

Microbes also play a role in leaky gut • Specifics to be discussed tomorrow • Microbes and Metals

Glutamate

sleep

Glutamate

eye contact

Glutamate

acetycholine

bladder contraction, strabismus

Glutamate

stims

Glutamate in relation to GABA levels Glutamate

GABA

Glutamate and Gaba Normally: GAD Glutamate

Gaba (calming neurotransmitter) Enzyme

Pancreas

Glutamate

GAD enzyme

Pancreas

Gaba

Pancreas

Glutamate

GAD enzyme

Gaba

Autoantibodies to GAD/rubella

Pancreas

Reelin mutation

GAD enzyme

Reelin, GAD67 and GAD65 mRNA levels (attomol/μg total RNA) in FPC of WTM, HRM and HG67M

Type of mice

Reelin

GAD67

WTM

190 ± 9.0

7.0 ± 0.80

HRM

99 ± 16*

4.2 ± 0.59*

Proc Natl Acad Sci U S A. 2001 March 13; 98 (6): 3477–3482 Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

Reelin • Methylation of the reelin gene is used to regulate its activity. • There are 100 potential sites for methylation on the reelin gene. • Lack of methylation may lead to the extra long versions of the reelin protein found by Kellers group. • Reelin is secreted by GABAergic interneurons. • Reelin is secreted by glutamergic granule cells in adults. • A reduced level of GABAergic interneurons would result in lower levels of reelin. Nucleic Acids Research July 2002, page 2930, PNAS March 29, 2000, page 3556

Dennis Grayson, Ph.D. Associate Professor of Molecular Biology and Neuroscience

Neuropsychiatric Institute University of Illinois at Chicago •

Down-regulation of reelin and GAD67 expression in post-mortem brains of patients diagnosed with schizophrenia and not in post-mortem material obtained from other psychiatric patients (Guidotti et al., 2000).

•

To date, reelin and GAD67 mRNA and protein levels are reduced by approximately 50% in every cortical area, in hippocampi and in cerebella of postmortem brains obtained from patients diagnosed with schizophrenia. In these same samples, there were no changes in the levels of GAD65 immunoreactivity and mRNA that are expressed in the same neurons that express reelin and GAD67. This suggests the possibility that the genes encoding reelin and GAD67 may be coordinately regulated and that there may be a defect in the regulation of the promoters of each gene that compromises expression.

•

Based on data obtained thus far, we suggest that methylation represents a switch that can be used to turn off reelin expression under appropriate conditions. We are addressing whether this may also be operative in the regulation of GAD67 and GAD65. The role that methylation plays in regulating gene expression in the nervous system is still under explored. We know that alterations in methylation can result in mental retardation. Mutations that occur in methyl CpG binding proteins have drastic consequences that occur postnatally.

Glutamate and Myelination

: Nature. 2005 Dec 21

NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia. Micu I, Jiang Q, Coderre E, Ridsdale A, Zhang L, Woulfe J, Yin X, Trapp BD, McRory JE, Rehak R, Zamponi GW, Wang W, Stys PK.

Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca(2+) accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca(2+) indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca(2+) increase in oligodendroglial cell bodies, but only modestly reduced the Ca(2+) increase in myelin. In contrast, the Ca(2+) increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca(2+) increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.

X

mercury

X X

aluminum

Glutamate and Gaba

Glutamate

Gaba

Gaba

language/speech

Gaba and Speech • Gaba is necessary for speech. • Gaba creates the “gap” between words GABA

word GAP word GAP word GAP word

Gaba

anxiety

Gaba

aggressive behavior

Gaba

social behavior

Gaba

eye contact

Gaba

bowel function

Gaba

nystagmus

It’s not just about glutamate CALCIUM

Glutamate is the gun. Calcium is the bullet. Calcium

Glutamate

Calcium

Calcium

Glutamate and Calcium Glutamate

calcium flow into neurons

calcium flow

nerve damage

nerve damage

inflammation

Interaction between glutamate and glycine

Nutrition Industry Executive, Nov/Dec 2002 Andrius Baskys, MD, PhD

polyamines

Calcium Transport • Calcium requires vitamin D and vitamin K for transport • Vitamin D and vitamin K are fat soluble vitamins • Lower vitamin D and lower vitamin K associated with gut absorption issues Remember vitamin K is made in the gut by bacteria and requires SAMe

Inflammation and Calcium • Limiting calcium will help limit inflammation • Magnesium blocks the door for calcium reversibly • Zinc blocks the door permanently! Too much zinc will stimulate glutamate at non NMDA glutamate receptors.

Consequences of Decreased Gaba Gaba

language/speech

Gaba

anxiety

Gaba

aggressive behavior

Gaba

social behavior

Gaba

eye contact

Gaba

bowel function

Gaba

nystagmus

Consequences of Elevated Glutamate Glutamate

excitotoxin damage

opioids

Glutamate

glutathione

TNF alpha

Glutamate

sleep

Glutamate

eye contact

Glutamate

acetycholine

Glutamate

stims

leaky gut

bladder contraction, strabismus

MTHFr zmethylation zpurine metab. zpyrimidine metab. zT cells yHistamine zBH4 ztyrosine zN methyl nicotin (stress uv) zmyelination yprecursors glut/glycine z5 methyl C z1 methyl tryp. yIDO ztryptophan

Glutamate R

Strep

yglutamate yglutamate intelligence brain size zglutathione (yTNF) leaky gut yexcitotoxin dam yopiods zsleep zeye contact yreckless behavior yacetylcholine bladder strabismus pupils contract zgaba zlanguage yanxiety zbowel function yaggression

zglutathione (yTNF) yglutamate

Metals

Virus/Vaccine

Blood/HLA

zzzT cells yIDO z1 methyl tryp

DPT

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Viral Infection

Chronic Bacterial Infection Heavy Metal Burden HLA Type Relationship to Asthma DPT Vaccine

Role of Microbes in Autism • Specifics to be discussed tomorrow • Relationship between Microbes and Metals

Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin. Temmerman S, Pethe K, Parra M, Alonso S, Rouanet C, Pickett T, Drowart A, Debrie AS, Delogu G, Menozzi FD, Sergheraert C, Brennan MJ, Mascart F, Locht C. Nat Med. 2004 Sep;10(9):935-41. Epub 2004 Aug 08.

• Methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. • Post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Receptor: • Strep • Measles • Herpes

Binding of aluminium ions by Staphylococcus aureus 893. Bradley TJ, Parker MS. Experientia. 1968 Nov 15;24(11):1175-6.

IOM Report May 17, 2004 Immunization Safety Review: Vaccines and Autism

IOM Report June 15, 2004 In recent years, a number of chronic diseases have been linked, in some cases definitively, to an infectious etiology: peptic ulcer disease with Helicobacter pylori, cervical cancer with several human papillomaviruses, Whipple's disease with Tropheryma whipplei, Lyme arthritis and neuroborreliosis with Borrelia burgdorferi, AIDS with the human immunodeficiency virus, liver cancer and cirrhosis with hepatitis B and C viruses, to name a few. The proven and suspected

roles of microbes does not stop with physical ailments; infections are increasingly being examined as associated causes of or possible contributors to a variety of serious, chronic neuropsychiatric disorders and to developmental problems, especially in children.

Infectious Agents and Schizophrenia •

“Most cases of schizophrenia are caused by infections and other environmental events occurring in genetically susceptible individuals.”

•

“Infections relating to schizophrenia occurring in this context would not…lead to disease in individuals who do not have the appropriate genetic susceptibility.”

•

There was an association between infection with HSV2 and higher rates of schizophrenia.

•

The authors found increased levels of antibodies to Toxoplasma gondii in individuals with recent onset schizophrenia.

On a related note: •

Parasitic infection impairs the immune response to the tetanus vaccine. (Infection and Immunity May 2004 )

NADase

H2O2 (neutrophils, monocytes, strep) glutatione peroxidase glutathione

NAD

dopamine T3T4

serotonin

antibiotics

glutathione glutamate

calcium dysregulation

tryptophan

tyrosine

Strep

normal flora

antibodies brain stem

Vit K

myelination

neuraminidase viral spread

streptokinase CD26

TNF

OCD, Tourettes, stims

GAGs glutathione leaky gut

IL6

TNF

Streptococcal infection

H2O2

Macrophages

H2O2

Neutrophils

H2O2

IUBMB Enzyme Nomenclature EC 1.11.1.8 Common name: iodide peroxidase

Reaction: iodide + H2O2 = iodine + 2 H2O Other name(s): iodotyrosine deiodase; iodinase; iodoperoxidase (heme type); thyroid peroxidase; iodide peroxidase-tyrosine iodinase; iodotyrosine deiodinase; monoiodotyrosine deiodinase; thyroperoxidase; tyrosine iodinase Systematic name: iodide:hydrogen-peroxide oxidoreductase

Decreased tyrosine leads to decreased dopamine

Chronic Streptococcal Infection Streptococcal infection

TNF alpha

TNF alpha is associated with… • • • • • • •

Tourettes syndrome OCD behavior “stims” PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections) perserverative speech rheumatoid arthritis leaky gut

Connectivity map of TNF

Nature Cell Biology, Feb. 2004, Vol. 6, No. 2

Tryptophan breakdown pathway is triggered by bacterial infection as well as interferon gamma

Tryptophan is an essential amino acid and the least abundant constituent of proteins. In parallel it represents a source for two important biochemical pathways: the generation of neurotransmitter 5hydroxytryptamine (serotonin) by the tetrahydrobiopterin-dependent tryptophan 5-hydroxylase, and the formation of kynurenine derivatives and nicotinamide adenine dinucleotides initiated by the enzymes tryptophan pyrrolase (tryptophan 2,3-dioxygenase, TDO) and indoleamine 2,3-dioxygenase (IDO). Whereas TDO is located in the liver cells, IDO is expressed in a large variety of cells and is inducible by the cytokine interferon-gamma. Therefore, accelerated tryptophan degradation is observed

in diseases and disorders concomitant with cellular immune activation, e. g. infectious, autoimmune, and malignant diseases, as well as during pregnancy. According to the cytostatic and antiproliferative properties of tryptophan-depletion on T lymphocytes, activated T-helper type 1 (Th-1) cells may down-regulate immune response via degradation of tryptophan. Especially in states of persistent immune activation availability of free serum tryptophan is diminished and as a consequence of reduced serotonin production, serotonergic functions may as well be affected. Accumulation of neuroactive kynurenine metabolites such as quinolinic acid may contribute to the development of neurologic/psychiatric disorders. Thus, IDO seems to represent a link between the immunological network and neuroendocrine functions with far reaching consequences in regard to the psychological status of patients. These observations provide a basis for the better understanding of mood disorder and related symptoms in chronic diseases. Curr Med Chem. 2003 Aug;10(16):1581-91 Wirleitner B, Neurauter G, Schrocksnadel K, Frick B, Fuchs D.

Journal of Neurochemistry Volume 93 Page 611 - May 2005 doi:10.1111/j.1471-4159.2005.03070.xVolume 93 Issue 3 Tryptophan metabolism and oxidative stress in patients with Huntington's diseaseN. Stoy*, G. M. Mackay†, C. M. Forrest†, J. Christofides‡, M. Egerton‡, T. W. Stone† and L. G. Darlington§

Abnormalities in the kynurenine pathway may play a role in Huntington's disease (HD). In this study, tryptophan depletion and loading were used to investigate changes in blood kynurenine pathway metabolites, as well as markers of inflammation and oxidative stress in HD patients and healthy controls. Results showed that the kynurenine : tryptophan ratio was greater in HD than controls in the baseline state and after tryptophan depletion, indicating increased indoleamine dioxygenase activity in HD. Evidence for persistent inflammation in HD was provided by elevated baseline levels of C-reactive protein, neopterin and lipid peroxidation products compared with controls. The kynurenate : kynurenine ratio suggested lower kynurenine aminotransferase activity in patients and the higher levels of kynurenine in patients at baseline, after depletion and loading, do not result in any differences in kynurenic acid levels, providing no supportive evidence for a compensatory neuroprotective role for kynurenic acid. Quinolinic acid showed wide variations in blood levels. The lipid peroxidation data indicate a high level of oxidative stress in HD patients many years after disease onset. Levels of the free radical generators 3-hydroxykynurenine and 3-hydroxyanthranilic acid were decreased in HD patients, and hence did not appear to contribute to the oxidative stress. It is concluded that patients with HD exhibit abnormal handling of tryptophan metabolism and increased oxidative stress, and that these factors could contribute to ongoing brain dysfunction.

Implication of microbial infection in Huntington’s Disease as well as glutamate

Tryptophan

serotonin melatonin

IDO

N methyl tryptophan

increased w/ hyperbaric O2

Kynurenic Acid

N methyl tryptophan

IDO breakdown products

(INH glutamate)

Increased IDO leads to decreased T cells

B6 Quinolinic Acid (

NMDA) Krebs

B3 Niacinamide

antibiotic activity helps kill virus helps kill lyme

protects β islet cells in pancreas

NADH

regenerate glutathione BH4

T cells serotonin

dopamine

Niacinamide • Antibiotic effects • Deficiency of niacinamide will drive the breakdown of tryptophan • Bacteria can breakdown tryptophan • May inhibit proviral integration • Helpful in reversing the arrest of T cell proliferation that is linked to tryptophan depletion

Vitamin B3 • • • • • • • •

Niacin Nicotinic acid Nicotinamide Niacinamide NAD+ Glutathione recycling NADH NADP+ BH2 and BH4 NADPH Dopamine

Serotonin

Breakdown Niacinamide

Breakdown Tryptophan

1/3

2/3 NAD+

serotonin

NADH

A deficiency in niacinamide will drive breakdown of tryptophan. Reduced levels of tryptophan will result in decreased levels of serotonin.

Cognitive Inflexibility After Prefrontal Serotonin Depletion H.F. Clarke, et al. Science, May 7, 2004

Selective depletion of serotonin produced perseverative behavior. serotonin

perseverative behavior

CO2

serotonin

acid

CO2

serotonin

Methylation cycle mutations

methylation vitamin B3

BH4

serotonin dopamine IGF

strep infection

Consequences of Streptococcal Infection Glutamate Glutathione

TNF detoxification

TNF alpha Streptokinase

TNF & IL6 CD26

NADase NAD

glutathione recycling

tryptophan

serotonin & melatonin

Consequences of Streptococcal Infection inflammatory mediators leaky gut T cells and B cells due to chronic infection Glutathione peroxidase (2GSH +H O 2

H2O2

2

Æ GSSG + 2H2O)

tyrosine T3/T4

Neuraminidase (aids in viral infection)

dopamine

MTHFr zmethylation zpurine metab. zpyrimidine metab. zT cells yHistamine zBH4 ztyrosine zN methyl nicotin (stress uv) zmyelination yprecursors glut/glycine z5 methyl C z1 methyl tryp. yIDO ztryptophan yyIL6 zmethylation yyTNF zmethylation zmyelination yTNF zmethylation yTNF zmethylation zVit. K yIL6 yIL6 zmethylation yIDO ztryptophan

Metals

Glutamate R

Strep

yglutamate yglutamate intelligence brain size zglutathione (yTNF) leaky gut yexcitotoxin dam yopiods zsleep zeye contact yreckless behavior yacetylcholine bladder strabismus pupils contract zgaba zlanguage yanxiety zbowel function yaggression zVit. K (calcium) yglutamate yglutamate yglutamate

zglutathione (yTNF) yglutamate yglutamate zglutathione (yTNF) yTNF ystreptokinase yTNF yIL6 binds CD26 zCD26 (DPPIV) gluten casein T cell memory yneuraminidase yNADase zNAD ztryp. yinflamm. mediator leaky gut deplete T & B cells chronic infection yH202 ztyrosine yT3/T4 zdopa. z?Vit. K (antibiotics) y?niacinamide ztryptophan zserotonin zmelotonin yquinolinic yglutamate yIDO depletion niacin. zT cells

y glutamzglutath z glutathy TNF

Virus/Vaccine zzzT cells yIDO yneuraminidase yIDO yMT

Blood/HLA ABO/strep auto antibodies NAC gluc zgags gut basal ganglia yIDO yIDO z1 methyl tryp

DPT

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection

Chronic Viral Infection Heavy Metal Burden HLA Type Relationship to Asthma DPT Vaccine

“Viruses use a “Trojan horse” strategy in which the victim assists the intruder. To extract assistance from the host cell, viruses use the detailed “inside information” that they have acquired during million of years of coevolution with their hosts.” Science Vol 304 April 9,2004

Viruses as Parasites • Induction of Metallothionein proteins by viral infection • Trapping of heavy metals by virus

Viral infection leads to the increased absorption or retention of metals in the body.

Role of Virus and Metals • Specifics to be discussed tomorrow • Relationship between Microbes and Metals

Impact of the Methylation Cycle on Viral Load

Decreased methylation creates a compromised host for the virus. “DNA methylation may maintain the large amount of non-coding DNA in an inert state.” “This process would help prevent the transcription of large parts of the genome… INSERTED VIRAL SEQUENCES.” “…consequences of loss of methylation… could cause the potentially harmful expression of inserted viral genes New England Journal of Medicine, November 20, 2003

Factors that may lead to Chronic Viral Infection • • •

Decreased T cell response due to methylation cycle mutations that impairs DNA synthesis needed for T cell clonal expansion Impaired B cell response due to lack of T helper cells, and T regulatory cells. Further induction of IDO by interferon gamma • Interferon gamma has been reported to increase intestinal permeability and blood-brain permeability

• • •

Increased IDO, impact on self versus non self Decreased vaccine efficiency, increased viral load Nature of retroviruses themselves

Measles and Mumps • Measles and mumps are retroviruses • Retroviruses have the ability to insert into host DNA • While Rubella is not a retrovirus, it can behave like a retrovirus in the presence of retroviruses like Measles and Mumps

Specific Viruses • Measles • Gut

• Mumps • Hormones

• Rubella • Pancreas

Measles Virus • Affects GI tract (work by Wakefield) Inflammatory mediators • Retrovirus

insert into host DNA

Mumps Virus • Affects sensitivity to pain • May affect hormone levels • May affect neurotransmitters • Retrovirus

insert into host DNA

Rubella Virus May affect pancreas gaba

CCK

opioids

secretin

pain

NYP1

bile

vitamin K

IL6

gastrin

histamine acid

anxiety

CCK • CCK coexists and interacts with dopamine, serotonin, norepinephrine, GABA, acetylcholine and vasopressin(Harro 1993, Copper 1991, Schafmayer 1988) • Norepinephrine and acetylcholine may regulate the synthesis of CCK in the brain. ( Monstein 1990) • CCK containing dopaminergic neurons may be implicated in schizophrenia and Parkinson’s disease. ( Crawley 1990) • GABA appears to modulate the mechanism that controls CCK release in the forebrain. ( Harro 1991).

CCK • CCK coexists with dopamine in neurons (Hokfelt, 1980) • CCK levels are altered by estrogen, caffeine, NMDA, opiates. (Micevych 1996) • Sulfation of CCK as well as gastrin is important for their activity. (Huang 1989). Three out of four tyrosine residues of CCK contain sulfate groups. Sulfation is needed for correct sorting and processing of CCK • In animal models, CCK when injected alone had no effect. CCK modulated dopamine mediated behavior ( Crawley 1994)

• CCK potentates dopamine release via CCK 1 receptor • CCK inhibited dopamine release via CCK 2 receptor

CCK • Opioids may help to decrease excitotoxin damage, however they attenuate the estrogen induced expression of CCK. • T4 is able to antagonize certain actions of estrogen, however T4 appears not to affect the action of estrogen on CCK. • Thyroxine may potentiate the effect of estrogen on CCK. Interaction of Thyroxine and Estrogen on the Expression of Estrogen Receptor, Cholecystokinin and Preproenkepahalin Messenger RNA in the Limbic Hypothalmic Circuit. Holland et al.

Pancreas

Glutamate

GAD enzyme

Pancreas

Gaba

Pancreas

Glutamate

GAD enzyme

Pancreas

Gaba

Pancreas GAD enzyme Gaba 2nd highest source of vitamin K vitamin K

Secretin Gastrin Histamine Acid

CCK Opioids Pain Anxiety Bile

inflammatory mediator IL6

Calcium Transport • Calcium requires vitamin D and vitamin K for transport • Vitamin D and vitamin K are fat soluble vitamins • Lower vitamin D and lower vitamin K associated with gut absorption issues

Herpes • Implicated in MS • Implicated in seizure activity

Chronic Viral Infection • Measles, Mumps, Rubella, Herpes, Herpes Zostar (Chicken Pox) • Streptococcus, Herpes Virus, Measles Virus use the same receptor to get into cells • Increased susceptibility to all three • Viruses

glutamate

• Viruses

host MT proteins

bind metals inside virally infected cells

Consequences of Chronic Viral Infection Depletion of T cells TNF alpha glutamate Measles gut, neurotransmitters in gut Mumps hormones, neurotransmitters Rubella pancreas HHV-6 seizures glutamate gaba

MTHFr zmethylation zpurine metab. zpyrimidine metab. zT cells yHistamine zBH4 ztyrosine zN methyl nicotin (stress uv) zmyelination yprecursors glut/glycine z5 methyl C z1 methyl tryp. yIDO ztryptophan yyIL6 zmethylation yyTNF zmethylation zmyelination yTNF zmethylation yTNF zmethylation zVit. K yIL6 yIL6 zmethylation yIDO ztryptophan

Metals

Glutamate R

Strep

yglutamate yglutamate intelligence brain size zglutathione (yTNF) leaky gut yexcitotoxin dam yopiods zsleep zeye contact yreckless behavior yacetylcholine bladder strabismus pupils contract zgaba zlanguage yanxiety zbowel function yaggression zVit. K (calcium) yglutamate yglutamate yglutamate

zglutathione (yTNF) yglutamate yglutamate zglutathione (yTNF) yTNF ystreptokinase yTNF yIL6 binds CD26 zCD26 (DPPIV) gluten casein T cell memory yneuraminidase yNADase zNAD ztryp. yinflamm. mediator leaky gut deplete T & B cells chronic infection yH202 ztyrosine yT3/T4 zdopa. z?Vit. K (antibiotics) y?niacinamide ztryptophan zserotonin zmelotonin yquinolinic yglutamate yIDO depletion niacin. zT cells

y glutamzglutath z glutathy TNF

Virus/Vaccine zzzT cells By zzz T cells B only response chronic viral metals nature RV vaccine eff. yTNF yMT (own use) Measles gut yinflamm. Mumps horomones neurotrans zpain Rubella pancreas zGABA zsecretin zNYP1 zVit. K (IL6) zgastrin yhistamine yacid zCCK yopiods zpain zbile yanxiety WI 38 yIFN-G yIDO yIDO yneuraminidase yIDO yMT

Blood/HLA ABO/strep auto antibodies NAC gluc zgags gut basal ganglia yIDO yIDO z1 methyl tryp

DPT

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection Chronic Viral Infection

Heavy Metal Burden HLA Type Relationship to Asthma DPT Vaccine

Role of Metals • Relationship between metals and methylation • Specifics to be discussed tomorrow • Relationship between Microbes and Metals

“In the absence of glutamate, neurons are unaffected by acute exposure to mercury, suggesting that neuronal dysfunction is secondary to disturbances in astrocytes.” “Coapplication of nontoxic concentrations of MeHG and glutamate leads to the typical appearance of neuronal lesions associated with excitotxin stimulation”

Brookes, 1992/Matyja and Albrecht, 1993

Methylation in Arsenic Detoxification Arsenite methylation by methylvitamin B12 and glutathione does not require an enzyme. Toxicol Appl Pharmacol. 1999 Feb 1;154(3):287-91. Zakharyan RA, Aposhian HV. Department of Molecular and Cellular Biology, The University of Arizona, Tucson, Arizona, 85721-0106, USA.

Although inorganic arsenic is methylated enzymatically by arsenic methyltransferases, which have been found in many mammalian livers, the detection of such enzymes has not been successful in surgically removed human livers. Results of the present experiments demonstrated that methylvitamin B12 (methylcobalamin, CH3B12) in the presence of thiols and inorganic arsenite can produce, in vitro, substantial amounts of monomethylarsonic acid (MMA) and small amounts of dimethylarsinic acid (DMA) in the absence of enzymes. Furthermore, this nonenzymatic methylation of inorganic arsenite by CH3B12 was increased substantially by the presence of dimercaptopropanesulfonate (DMPS) and/or sodium selenite. The actions of DMPS and selenite together were additive. The methylation by CH3B12 was neither inhibited nor stimulated by human liver cytosol. Since the amount of MMA produced by the in vitro system described in this study was not small, these results emphasize the need for a properly designed nutritional study in humans exposed to inorganic arsenic as to the relationship between vitamin B12, selenium, and the metabolism of carcinogenic inorganic arsenic. Copyright 1999 Academic Press.

Lipoic acid attached to pyruvate dehydrogenase (PDH)

The sulfhydryl groups of lipoic acid is the target for arsenic poisoning.Lipoic acid is a key component of two important enzymes: pyruvate dehydrogenase (PDH) and alpha-ketogluterate dehydrogenase (KDH). PDH converts the end product of glycolysis to acetyl coenzyme A, which can then enter the tricarboxylic acid (TCA) cycle. KDH is an enzyme with the TCA cycle. Lipoic acid is an 8-carbon dithiol that is covalently linked to one subunit of the PDH and KDH complex. In the presence of arsenic, the two sulfhydryl groups bind with arsenic to form a sixmembered ring.

Six-membered ring formed by arsenic and lipoic acid Domingo Tabangcura, Jr. and G. Patrick Daubert, MD

Proper methylation mitigates selenium toxicity

BH2

Inhibited by lead Inhibited by aluminum

BH4

serotonin

dopamine

Toxicol In Vitro. 2003 Oct-Dec;17(5-6):533-7 Altindag ZZ, Baydar T, Engin AB, Sahin G.

Effects of the metals on dihydropteridine reductase activity. Metals are the oldest toxins known to human. Particularly, occupational and environmental exposure to aluminium, lead, mercury, cadmium, and manganese cause serious health problems by interaction with biological systems. Cellular targets of these metals are mostly specific biochemical processes (enzymes) and/or membranes of cells and organelles. To prevent and/or reduce the untoward or irreversible toxic effects of the metals by using biomarkers are as important as to know and to understand of their toxicity mechanisms. Dihydropteridine reductase (DHPR), which possessed essential thiol groups at the activity site, plays a crucial role in the maintenance of tetrahydrobiopterin (BH4). BH4 is the cofactor in the synthesis and regulation of neurotransmitters. A limited number of the evidences have shown that DHPR may be a target for the metals. Therefore, the present study was designed to assess possible in vitro effects of the commonly exposed metals on the enzyme activity. It was found that aluminium, cadmium, mercury, di-phenyl mercury, lead, diethyl lead, in chloride forms, and manganese, in sulphate form, led to statistically significant decreases in DHPR activity, in a concentration-dependent manner, in vitro.

5 amino levulinic acid

Glutamic acid

heme

Inhibited by lead

red blood cells

cytochromes

The binding sites for Zn++ in the homooctomeric mammalian Porphobilinogen Synthase, which include cysteine S ligands, can also bind Pb++ (lead). Inhibition of Porphobilinogen Synthase by Pb++ results in elevated blood ALA, which may cause some of the neurological effects of lead poisoning. ALA (d-aminolevulinate) is toxic to the brain. This may be due in part to the fact that ALA is somewhat similar in structure to the neurotransmitter GABA (g-aminobutyric acid). In addition, reactive oxygen species (oxygen radicals) are generated during autoxidation of ALA.

lead BH4 anemia Cytochrome deficiency

glutamate

serotonin

+

even LOW doses of mercury

nerve damage

decreased B12

dopamine

Biol Trace Elem Res. 1997

Dietary nickel and folic acid interact to affect folate and methionine metabolism in the rat. Uthus EO, Poellot RA. United States Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, ND 58202-9034, USA.

Nickel caused a further increase in urinary FIGLU Nickel caused a further decrease in SAMe … Because of this, it is proposed that the physiological function of nickel is related to the common metabolism shared by SAM and FIGLU. Possibly the physiological function of

nickel could be related to the tissue concentration of 5-methyltetrahydrofolate (MTHF) or tetrahydrofolate (THF).

histidine

β alanine

1 methyl histidine

urocanate

carnosine 4 imidazolone 5 propionate anserine

FIGLU

tetrahydrofolate

If there is not sufficient THF then FIGLU as well as histidine can accumulate glutamate

Metals

myelination

methylation is necessary for myelination Metals

myelination

remyelination

lack of methylation

Metals

Myelination

Myelination ensures the rapid conduction of electrical impulses in the nervous system.

Neuregulins

Myelin wrapping

Neuregulin is the signal for the regulation of myelin sheath thickness Neuregulin also induces GABA receptors Constant activation of neuregulin may cause demyelination

Neuregulin Receptor Proteins • erbB2 and erbB3 • Expressed on cells that are myelinating • Mice with erbB2 have thin myelin sheaths and fewer wraps

• Estrogen helps to protect dopamine neurons • This protection by estrogen involves activation of the IGF system • Estrogen is also known to stimulate CCK • In animal models estradiol increased tyrosine hydroxylase expression.

•CCK Increases Fos expression. •Fos is a marker of neuronal activation in specific medullary and hypothalmic nuclei. •There is conflicting data that suggests that estrogens have a stimulatory effect on the actions of CCK.

• CCK coexists with dopamine in neurons • CCK had no effect alone, but modulated dopamine mediated behaviors • CCK is also colocalized with serotonin and GABA. • Norepinephrine and acetylcholine modulate CCK release. • Three out of four tyrosine residues of CCK contain sulfate groups. Sulfation is needed for correct sorting and processing of CCK.

CCK • CCK coexists with dopamine in neurons (Hokfelt, 1980) • CCK levels are altered by estrogen, caffeine, NMDA, opiates. (Micevych 1996) • Sulfation of CCK as well as gastrin is important for their activity. (Huang 1989) • In animal models, CCK when injected alone had no effect. CCK modulated dopamine mediated behavior ( Crawley 1994)

– CCK potentiated dopamine release via CCK 1 receptor – CCK inhibited dopamine release via CCK 2 receptor

CCK • CCK coexists and interacts with dopamine, serotonin, norepinephrine, GABA, acetylcholine and vasopressin. (Harro 1993, Copper 1991, Schafmayer 1988) • Norepinephrine may regulate the synthesis of CCK in the brain. ( Monstein 1990) • CCK containing dopaminergic neurons may be implicated in schizophrenia and Parkinson’s disease. ( Crawley 1990) • GABA appears to modulate the mechanism that controls CCK release in the forebrain. ( Harro 1991).

Reelin • DNA methylation • Decreased gaba • Relationship to Schizophrenia

Reelin • Methylation of the reelin gene is used to regulate its activity. • There are 100 potential sites for methylation on the reelin gene. • Lack of methylation may lead to the extra long versions of the reelin protein found by Kellers group. Nucleic Acids Research July 2002, page 2930, PNAS March 29, 2000, page 3556

On the epigenetic regulation of the human reelin promoter. Chen Y, Sharma RP, Costa RH, Costa E, Grayson DR. Nucleic Acids Res. 2002 Jul 1;30(13):2930-9.

• Reln mRNA and protein levels are reduced by approximately 50% in various cortical structures of postmortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. • These findings indicate that one determinant responsible for regulating reelin expression is the methylation status of the promoter. Our data also raise the interesting possibility that the down-regulation of reelin expression documented in psychiatric patients might be the consequence of inappropriate promoter hypermethylation.

Methylation Reelin

Reelin GAD enzyme

IUBMB Enzyme Nomenclature EC 2.1.1.126 Reaction:

S-adenosyl-L-methionine + [myelin basic protein]-arginine

S-adenosyl-L-homocysteine + [myelin basic protein]-Nw-methylarginine

Common name: [myelin basic protein]-arginine N-methyltransferase Other name(s): myelin basic protein methylase I; protein methylase I Systematic name: S-adenosyl-L-methionine:[myelin-basic-protein]-arginine Nwmethyltransferase

J Neurochem. 1982 Jul;39(1):244-7. The relationship of myelin basic protein (arginine) methyltransferase to myelination in mouse spinal cord. Crang AJ, Jacobson W. The relationship between the activity of myelin basic protein (arginine) methyltransferase and myelination in the mouse spinal cord has been examined. The activity of this methylase increases between 8 and 45 days postnatal age and correlates well with other parameters of myelination. A comparison of myelin basic protein methylase with histone methylase activity during development indicates that each is a distinct, specific enzyme activity. Together, these results are considered to establish myelin based protein methylase as a myelination-related enzyme. PMID: 6177833 [PubMed - indexed for MEDLINE]

Neurochem. 1984 Aug;43(2):494-8 Amur SG, Shanker G, Pieringer RA PMID: 6736963

Regulation of myelin basic protein (arginine) methyltransferase by thyroid hormone in myelinogenic cultures of cells dissociated from embryonic mouse brain. .The ontogenetic expression of myelin basic protein (arginine) methyltransferase in myelinogenic cultures of cells dissociated from embryonic mouse brain is highly dependent on the presence of thyroid hormone. Restoration of myelin basic protein methyltransferase to normal activities occurred 16 h after the addition of 100 nM L3,5,3'-triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event. On the other hand, histone (arginine) methyltransferase has a different temporal activity pattern, which is not coordinated with myelination, and is not influenced by the lack of thyroid hormone. These data, which suggest the existence of two methyltransferases, were substantiated by demonstrating that the total amount of methylation of added myelin basic protein and histone is the same whether they are incubated together or separately. The requirement of thyroid hormone for the expression of the myelin basic protein methyltransferase and not for histone methyltransferase suggests that thyroid hormone preferentially regulates myelin-associated events in these cultures.

Thyroid hormone is important for myelin basic protein methyltransferase activity.

Beta adrenergic stimulation of protein (arginine) methyltransferase activity in cultured cerebral cells from embryonic mice. • Adrenergic effectors were tested for their ability to regulate myelin basic protein methyltransferase. • Beta adrenergic agonists stimulated adenylate cyclase. • This led to an increase in cAMP • The increase in cAMP stimulated the methyltransferases. • Methylation of MBP appears to be mediated via cAMP production. • Methylation of MBP is also stimulated by thyroid hormone (J Neurochem. 1984 Aug;43(2):494-8.Regulation of myelin basic protein (arginine) methyltransferase by thyroid hormone in myelinogenic cultures of cells dissociated from embryonic mouse brain. Amur SG, Shanker G, Pieringer RA).

methylation

and

metals

myelination

Without myelin, axons will continue to grow and pruning will be compromised.

Proper myelination requires proper pruning

Nature Reviews Neuroscience May 2005

Nature Reviews Neuroscience May 2005

Consequences of Increased Metal Burden TNF alpha Glutathione Myelination and decreased pruning Memory Cognition Bridging of GSH : GSH

affect T cell activation

MTHFr zmethylation zpurine metab. zpyrimidine metab. zT cells yHistamine zBH4 ztyrosine zN methyl nicotin (stress uv) zmyelination yprecursors glut/glycine z5 methyl C z1 methyl tryp. yIDO ztryptophan yyIL6 zmethylation yyTNF zmethylation zmyelination yTNF zmethylation yTNF zmethylation zVit. K yIL6 yIL6 zmethylation yIDO ztryptophan

Metals

Glutamate R

Strep

yglutamate yglutamate intelligence brain size zglutathione (yTNF) leaky gut yexcitotoxin dam yopiods zsleep zeye contact yreckless behavior yacetylcholine bladder strabismus pupils contract zgaba zlanguage yanxiety zbowel function yaggression zVit. K (calcium) yglutamate yglutamate yglutamate

zglutathione (yTNF) yglutamate yglutamate zglutathione (yTNF) yTNF ystreptokinase yTNF yIL6 binds CD26 zCD26 (DPPIV) gluten casein T cell memory yneuraminidase yNADase zNAD ztryp. yinflamm. mediator leaky gut deplete T & B cells chronic infection yH202 ztyrosine yT3/T4 zdopa. z?Vit. K (antibiotics) y?niacinamide ztryptophan zserotonin zmelotonin yquinolinic yglutamate yIDO depletion niacin. zT cells

yTNF zglutathione yglutamate zmyelination zspeech zmemory zcognition yyMT chronic viral level GSH bridge GSH/GSH T cells thimerosal antimicrobial antiviral antiAIDS DNA/RNA TK enzyme zsynth zT cells horomones testosterone progesterone Adult Vaccination Mom Teacher healthcare

y glutamzglutath z glutathy TNF

Virus/Vaccine zzzT cells By zzz T cells B only response chronic viral metals nature RV vaccine eff. yTNF yMT (own use) Measles gut yinflamm. Mumps horomones neurotrans zpain Rubella pancreas zGABA zsecretin zNYP1 zVit. K (IL6) zgastrin yhistamine yacid zCCK yopiods zpain zbile yanxiety WI 38 yIFN-G yIDO yIDO yneuraminidase yIDO yMT

Blood/HLA ABO/strep auto antibodies NAC gluc zgags gut basal ganglia yIDO yIDO z1 methyl tryp

DPT

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection Chronic Viral Infection Heavy Metal Burden

HLA Type Relationship to Asthma DPT Vaccine

HLA Type

HLA Type •

Association of particular HLA types with celiac disease

•

Association of particular HLA types as over or under responders to measles vaccine

•

Association of particular HLA type with sensitivity to thimerosal

•

Association of particular HLA type with the ability of HIV to evade the immune system. (Nature Reviews Immunology May 2004)

•

Infection with virus that causes PERSISTENT infection modified host HLA antigens, while infection with the same virus under ACUTE conditions did not modify host HLA antigens. (Nature Reviews Immunology, April 2004)

Vaccines 97 Cold Spring Harbor Laboratory Press

•

Role of chronic viral infection in preventing rejection of “foreign” HLA types.

•

Role of elevated IDO in preventing rejection of “foreign” HLA types.

•

Role of decreased Tcell activity in preventing rejection of “foreign” HLA types.

•

Finding that newly formed viruses should bear the HLA proteins from the people they are infecting at the time. If the virus then moves into a second person with dissimilar HLAs, the second person should mount an immune response to the virus. (Gould, S. et al. The Trojan Exosome Hypothesis. PNAS 2003;100(19):10592.)

•

Question of relationship between HLA type of WI 38cells used to make vaccine and HLA type of autistic individuals.

Host cell same HLA type as WI38 cells

IDO T cells

Chronic Viral Infection

HLA

MTHFr zmethylation zpurine metab. zpyrimidine metab. zT cells yHistamine zBH4 ztyrosine zN methyl nicotin (stress uv) zmyelination yprecursors glut/glycine z5 methyl C z1 methyl tryp. yIDO ztryptophan yyIL6 zmethylation yyTNF zmethylation zmyelination yTNF zmethylation yTNF zmethylation zVit. K yIL6 yIL6 zmethylation yIDO ztryptophan

Metals

Glutamate R

Strep

yglutamate yglutamate intelligence brain size zglutathione (yTNF) leaky gut yexcitotoxin dam yopiods zsleep zeye contact yreckless behavior yacetylcholine bladder strabismus pupils contract zgaba zlanguage yanxiety zbowel function yaggression zVit. K (calcium) yglutamate yglutamate yglutamate

zglutathione (yTNF) yglutamate yglutamate zglutathione (yTNF) yTNF ystreptokinase yTNF yIL6 binds CD26 zCD26 (DPPIV) gluten casein T cell memory yneuraminidase yNADase zNAD ztryp. yinflamm. mediator leaky gut deplete T & B cells chronic infection yH202 ztyrosine yT3/T4 zdopa. z?Vit. K (antibiotics) y?niacinamide ztryptophan zserotonin zmelotonin yquinolinic yglutamate yIDO depletion niacin. zT cells

yTNF zglutathione yglutamate zmyelination zspeech zmemory zcognition yyMT chronic viral level GSH bridge GSH/GSH T cells thimerosal antimicrobial antiviral antiAIDS DNA/RNA TK enzyme zsynth zT cells horomones testosterone progesterone Adult Vaccination Mom Teacher healthcare

y glutamzglutath z glutathy TNF

Virus/Vaccine zzzT cells By zzz T cells B only response chronic viral metals nature RV vaccine eff. yTNF yMT (own use) Measles gut yinflamm. Mumps horomones neurotrans zpain Rubella pancreas zGABA zsecretin zNYP1 zVit. K (IL6) zgastrin yhistamine yacid zCCK yopiods zpain zbile yanxiety WI 38 yIFN-G yIDO yIDO yneuraminidase yIDO yMT

Blood/HLA ABO/strep auto antibodies NAC gluc zgags gut basal ganglia yIDO yIDO z1 methyl tryp WI 38 HLA Celiac Vaccine reaction Thimerosal HIV

Asthma yRNA viruses yacetylcholine zgastrin zmuscarinic strabismus yhistamine ypermeability

DPT

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection Chronic Viral Infection Heavy Metal Burden HLA Type

Relationship to Asthma DPT Vaccine

Genetic Predisposition • Association between autism and the presence of family members with asthma. • Asthma is related to imbalances in acetylcholine stimulation. • Retroviruses may be involved in triggering asthma

AHCY

adenosine

Adenosine and asthma relationship to drugs during labor

Supplements to decrease uric acid • Quercetin, inhibits xanthine oxidase, the enzyme which is involved in uric acid synthesis • Dandelion root decreases uric acid and increases bile • Pantothenic acid reduces uric acid and also supports adrenal function.

Issues with low uric acid • Low uric acid has been associated with MS • Decreased levels of molybdenum can contribute to low uric acid, as it is a cofactor for the enzyme xanthine oxidase.

adenosine stimulation

Fig. 3.— Diagram hypothesising the mechanism by which adenosine enhances histamine release from human airway mast cells in asthma. a) Normally, stimulation of the highaffinity A2A adenosine receptor by adenosine leads to the generation of large quantities of intracellular cyclic adenosine 5'-monophosphate (cAMP), which downregulates the biochemical pathways implicated in the release of histamine. b) Possible scenario in asthma, where in the presence of high concentrations of adenosine the relative importance of the low-affinity A2B receptor becomes greater with prevalent activation of regulatory subunit q of G-protein coupled receptor (Gq) proteins with significant mast-cell degranulation. In addition, downregulation of the subunit s of Gprotein coupled receptor (Gs) function leads to a reduction of cAMP generation with subsequent reduced negative modulation on histamine release.

adenosine stimulation

Synaptobrevin in involved in the affects of DTP Q75L80_HUMANClaudin-4 (Clostridium perfringens enterotoxin receptor) (CPE- receptor) (CPE-R) (Williams-Beuren syndrome chromosome region 8 protein). SwissProt:O14493Q75MD9_HUMANSyntaxin-1A (Neuron-specific antigen HPC-1). SwissProt:Q16623Q75ME3_HUMANPutative methyltransferase HUSSY-03 (EC 2.1.1.-) (Williams-Beuren syndrome chromosome region 22 protein). Swiss-Prot:O43709

SAMe

CH3

AHCY Homocysteine

SAH

Adenosine

Regulation of SAHHase activity (A) and mRNA (B and C) by aldosterone

Fig. 4. Regulation of SAHHase activity (A) and mRNA (B and C) by aldosterone. A, depleted cells ( ) were aldosterone- and serum-deprived for 48 h; repleted cells (+) were subsequently treated with aldosterone for 4 h; and DZA = assay performed in the presence of 10 µM inhibitor. *, p < 0.05 versus depleted; **, versus repleted. Shown in B is a typical Northern blot. GAPDH and SAHHase are mRNA species identified by specific probes. C, graphic representation of the relative density of SAHHase versus GAPDH mRNA in the absence and presence of aldosterone. Paired experiments from the same blots are indicated by connecting lines. Relative density for depleted and repleted were not different (p = 0.33).

Aldosterone increases the activity of SAHHase (AHCY) Stockand, J. D. et al. J. Biol. Chem. 1999;274:3842-3850

• Aldosterone is responsible for increasing the level of sodium uptake. • Aldosterone is known to affect the levels of SAH. • Protein methylation is essential for aldosterone induced sodium transport • Aldosterone increases protein methylation by regulating methyltransferase activity through its effects on SAH, as well as having an effect on the methylation of membrane proteins that are required for sodium transport.

Al-Baldawi et al. Am J Physiol Cell Physiol. 279:439, 2000

SAMe

CH3

AHCY

Homocysteine

SAH

Adenosine

EntryR04858

Reaction

Name S-Adenosyl-L-methionine:DNA (cytosine-5-)-methyltransferase Definition S-Adenosyl-L-methionine + Cytosine (in DNA) S-Adenosyl-L-homocysteine + 5-Methylcytosine (in DNA) EquationC00019 + C05522 C00021 + C05523 RPairRP: A00004 C00019_C00021 main RP: A04473 C05522_C05523 main RP: A08326 C00019_C05523 trans PathwayPATH: rn00271 Methionine metabolism SAH + cytosine-methyl group SAMe to cytosine

Implication that in order to reconvert SAH to SAMe you lose DNA methylation. Implies should add SAMe???

CH3 from DNA SAMe

CH3

AHCY

Homocysteine

SAH

Adenosine

Overview of one-carbon metabolism with emphasis on the reversible SAH hydrolase reaction. The hydrolysis of SAH is dependent on product removal of homocysteine and adenosine. In the absence of efficient product removal, SAH accumulation can inhibit methyltransferase reactions by high affinity binding to the enzyme active site. THF, tetrahydrofolate; 5CH3THF, 5-methyltetrahydrofolate; DMG, dimethylglycine

AHCY

Overview of one-carbon metabolism with emphasis on the reversible SAH hydrolase reaction. The hydrolysis of SAH is dependent on product removal of homocysteine and adenosine. In the absence of efficient product removal, SAH accumulation can inhibit methyltransferase reactions by high affinity binding to the enzyme active site. THF, tetrahydrofolate; 5CH3THF, 5-methyltetrahydrofolate; DMG, dimethylglycine

Predisposing Factors That Increase Susceptibility to Autism Methylation Cycle Mutations Increased Glutamate Receptors (total “Excitotoxin” level) Chronic Bacterial Infection Chronic Viral Infection Heavy Metal Burden HLA Type Relationship to Asthma

DPT Vaccine

The DPT vaccine is made with toxoid, which is toxin that has been inactivated.

Safety testing is done in animals toxoid safe

Immunocompetent animals

toxoid

? MTHFr mutation, T cells…

When testing is done in vitro (in a test tube) with NO immune system Paul-Ehrlich-Institute, Federal Agency for Sera and Vaccines, Langen, Germany

“Neurotransmitters are stored in synaptic vesicles at neuron terminals that connect other neurons. When the synaptic vesicle membrane fuses with the plasma membrane, the neurotransmitter is released into the synaptic cleft.”

18 JUNE 2004

VOL 304

SCIENCE www.sciencemag.org

• Tetanus toxin blocks the release of GABA and glycine • Tetanus toxin affects the hypothalamus, decreases hormone levels (such as testosterone), • Tetanus toxin causes excessive sympathetic discharge with urinary excretion of catecholamines (i.e.dopamine) and can cause chronic seizure activity. • Antibodies against the GAD enzyme (the enzymes that converts glutamate to GABA) have been reported in some cases of tetanus toxicity. • Tetanus toxin can also inhibit the release of norepinephrine, enkephalins and acetylcholine.

• Tetanus toxin can also inhibit the release of acetylcholinesterase. • Excess levels of acetylcholine are regulated by the enzyme acetylcholinesterase which causes the breakdown of acetylcholine • Lack of acetylcholinesterase will result in a down-regulation of muscarinic receptors. • Tetanus toxin is known to cause sympathetic overactivity.

The result is the unexpected outcome of high levels of acetylcholine, however with low levels of muscarinic (parasympathetic) receptors. Acetylcholine

Muscarinic receptors unchanged

Nicotinic receptors

This can result in excessive binding and activation of nicotinic receptors.

The Autonomic Nervous System Structure

Sympathetic Stimulation

Parasympathetic Stimulation

Iris (eye muscle)

Pupil dilation

Pupil constriction

Salivary Glands

Saliva production reduced

Saliva production increased

Oral/Nasal Mucosa

Mucus production reduced

Mucus production increased

Heart

Heart rate and force increased

Heart rate and force decreased

Lung

Bronchial muscle relaxed

Bronchial muscle contracted

Stomach

Peristalsis reduced

Gastric juice secreted; motility increased

Small Intestine

Motility reduced

Digestion increased

Large Intestine

Motility reduced

Secretions and motility increased

Liver

Increased conversion of glycogen to glucose

Kidney

Decreased urine secretion

Adrenal medulla

Norepinephrine and epinephrine secreted

Bladder

Wall relaxed Sphincter closed

Increased urine secretion

Wall contracted Sphincter relaxed

• Tetanus toxin binds irreversibly • Nerve function can only be returned by the growth of new terminals and synapses.

C.tetani is a slender, gram-positive, anaerobic rod that may develop a terminal spore, giving it a drumstick appearance. The organism is sensitive to heat and cannot survive in the presence of oxygen. The spores, in contrast, are resistant to heat and the usual antiseptics. They can survive autoclaving at 121°C for 10-15 minutes. The spores are also relatively resistant to phenol and other chemical agents.

http://www.kitasato.or.jp/rcb/eng/intro.html

http://www.klinik.uni-frankfurt.de/ni/forschung13.html

MTHFr zmethylation zpurine metab. zpyrimidine metab. zT cells yHistamine zBH4 ztyrosine zN methyl nicotin (stress uv) zmyelination yprecursors glut/glycine z5 methyl C z1 methyl tryp. yIDO ztryptophan yyIL6 zmethylation yyTNF zmethylation zmyelination yTNF zmethylation yTNF zmethylation zVit. K yIL6 yIL6 zmethylation yIDO ztryptophan

Metals

Glutamate R

Strep

yglutamate yglutamate intelligence brain size zglutathione (yTNF) leaky gut yexcitotoxin dam yopiods zsleep zeye contact yreckless behavior yacetylcholine bladder strabismus pupils contract zgaba zlanguage yanxiety zbowel function yaggression zVit. K (calcium) yglutamate yglutamate yglutamate

zglutathione (yTNF) yglutamate yglutamate zglutathione (yTNF) yTNF ystreptokinase yTNF yIL6 binds CD26 zCD26 (DPPIV) gluten casein T cell memory yneuraminidase yNADase zNAD ztryp. yinflamm. mediator leaky gut deplete T & B cells chronic infection yH202 ztyrosine yT3/T4 zdopa. z?Vit. K (antibiotics) y?niacinamide ztryptophan zserotonin zmelotonin yquinolinic yglutamate yIDO depletion niacin. zT cells

yTNF zglutathione yglutamate zmyelination zspeech zmemory zcognition yyMT chronic viral level GSH bridge GSH/GSH T cells thimerosal antimicrobial antiviral antiAIDS DNA/RNA TK enzyme zsynth zT cells horomones testosterone progesterone Adult Vaccination Mom Teacher healthcare

y glutamzglutath z glutathy TNF

Virus/Vaccine zzzT cells By zzz T cells B only response chronic viral metals nature RV vaccine eff. yTNF yMT (own use) Measles gut yinflamm. Mumps horomones neurotrans zpain Rubella pancreas zGABA zsecretin zNYP1 zVit. K (IL6) zgastrin yhistamine yacid zCCK yopiods zpain zbile yanxiety WI 38 yIFN-G yIDO yIDO yneuraminidase yIDO yMT

Blood/HLA ABO/strep auto antibodies NAC gluc zgags gut basal ganglia yIDO yIDO z1 methyl tryp WI 38 HLA Celiac Vaccine reaction Thimerosal HIV

Asthma yRNA viruses yacetylcholine zgastrin zmuscarinic strabismus yhistamine ypermeability

DPT zGABA zglycine zGAD zdopamine acetylcholine acetylcholinesterase muscarinic

Reglan : dopamine antagonist Rhogam : anti Rh antibody Rh transports CO2 and NH4 Pitocin : oxytocin Terbutaline (albuterol) : beta agonist

www.holistichealth.com www.holisticheal.com www.autismanswer.com Parents chat room

Additional Resources Specific information about supplements can be obtained at www.holisticheal.com Dr. Amy’s personal website is www.holistichealth.com Dr. Amy answers questions at www.autismanswer.com Videos/DVDs that are divided by topic: Glutamate and Gaba Neurological Inflammation, Video or DVD

Virus, Metals, Methylation

Austin Conference, November 2004 DVD Phoenix Conference, April 2005 DVD

Factors Contributing to Autism

Putting It All Together Parents Weekend, Video or DVD Boston Conference, August 2004 DVD The Puzzle of Autism, book Dr. Amy’s Supplement Video Autism Educational Starter Package

RNA

Boston Conference, August 2004, DVD Phoenix Conference, April 2005, DVD Heal Your Body Naturally, book RNA Educational Starter Package