FUNVAX VMAT 2 PARKINSONS, DRUG DEPENDENCY AND ADRENOCHROME

1. FUNVAX, VMAT-2 and SLC18A2 Adenoviruses and CAR (Coxsackie-Adeno-Receptor) Organophosphates, Carbomates and Agricultural Toxins and Drug dependence Parkinson’s Disease, Dopamine and addiction, Ascorbic Acid, Schizophrenia and The Adrenochrome Hypothesis

2. FunVax Quarterly Report 06/01/07 The objective of this phase of project 149AZ2 is to prepare a viral vector that Will inhibit/decrease the expression of VMAT2 within a human population. Abstract – Because of the Vesicular Stomatitis Virus’ ability to infect brain cellsand its two step life-cycle, cytolytic infections in mammals and transmission by insects. It provided a starting point to design an airborne virus that has the ability to infect the respiratory systemas well as the brain. The newly-designed virus contains the typical VSV genome, a homologous region to VMAT2and a gene from Adenovirusthat allows attachment to the coxsackie-adenovirus receptor(CAR) on host cells. This design allows the virus to infect the respiratory tract where cytolytic infection occurs and then subsequent diffusion acrosstheblood-brain barrier to infect brain cells. VSV287 had the least amount of endogenousVMAT2 protein and will be further tested to verify that it is the most efficient of the 600 strains.

3. VMAT2 Rhesus Monkey Knock-Out Mice (BALC) Abstract –VMAT2 homozygous knockout monkeys die within three days while the heterozygous monkey lives what appears to be normal life span. VMAT2 is responsible for packaging dopamine (DA) and other monoaminesinto vesicles that will be released at the synapse. Dopamine disruption has been shown to damage dopamine neurons. While the KO monkeys were alive, they did not feed and upon autopsy it was concluded that they died of starvation. It appears that they had no will to live. This same conclusion was found in VMAT2 KO mice in 1997. A VMAT2 deficient monkey was developed concurrently with the KO monkey. The VMAT2deficient monkey should have expression of VMAT2 80-95% lower than a wild-type monkey. The VMAT2 deficient monkey should produce results by July 2007

4. Summary of recommendation 1. Quantitative PCR of all 600 animal subjects should be done to ensure that the data from the ELISAexperiments, which showed a decrease in endogenous VMAT2is occurring because of viral insertion and not natural variation 2. Of the 600 variants of Vesicular Stomatitis Virus tested, VSV237 (237T) had the greatest decrease of endogenous VMAT2 within mice. However, this may not be the case for human subjects. All 600 strains of VSV should be retested on human subjects by the clinical group. 3. Bradford assays should be done on Infected subjects to determine endogenous VMAT2 concentrations before and after infection, not just after infection. 4. Mice or other subjects should not be injected with virus since this does not test the actual (aerosolised) dispersal method. Future experiments of VSV287or similar strains should allow the subject to breathe in the virusrather than being injected with it. As shown, VMAT2 KO experiment in mice, in 0.25% of subjects exposed to the vaccine there is a noticeable side effect – a benign blepharospasm. Tests need to be done on the human population .. If we assume that this side effect remains the same in humans

6. Association study of the vesicular monoamine transporter 1 (VMAT1) gene with schizophreniaina Japanese population Misty Richards1,2, Yoshimi Iijima1, Hitomi Kondo1, Tomoko Shizuno1, Hiroaki Hori1, Kunimasa Arima3, Osamu Saitoh3 and Hiroshi Kunugi*1 Vesicular monoamine transporters (VMATs) mediate accumulation of monoamines such as serotonin, dopamine, adrenaline, noradrenaline, and histaminefrom the cytoplasm into storage organelles VMAT1is expressed primarily in neuroendocrine cells such as the adrenal medullaand pineal gland, while VMAT2is expressed in all aminergic neuronsin the mammalian CNS In addition, with respect to the human VMAT2 gene, we failed to obtain evidence for a significant association of the detected polymorphisms with schizophrenia

7. Today's nerve gases are made of chemicals called organophosphorous compounds. They inhibit the action of a substance called acetylcholinesterase, which plays a vital role in the transmission of nerve impulses. Organophosphatesdisrupt the mechanism by which nerves transfer messages to organs. The disruption is caused by blocking acetylcholinesterase(Ach) in the synapse, an enzymethat normally destroys acetylcholine, a neurotransmitter.Achgives the signal for muscles to contract, preventing them from relaxing. The disruption is caused by blockingacetylcholine, which is a neurotransmitter. When acetylcholine transmitted the signal down, normally it is degraded into choline and acetic-acid by acetylcholine-estherase. This regenerates the receptor and renders it active again. Nerve agents act by inhibiting of hydrolysis of acetylcholine by acetylcholine estherase. They bind chemically to the acetylcholineso it is unable to deactivate acetylcholineresulting in persistent and uncontrolled stimulation of receptor. Poisoning by a nerve agentleads to contraction of pupils, profuse salivation, convulsions, involuntary urinationand defecation and death by asphyxiationas control is lost over the respiratory muscles.

8. Two drugs: atropine andpralidoxime-chloride have been used as antidotes for nerve agents poisoning.Atropineblocks one type of acethylcholine receptor so then the acetylcholine cannot work in the synapse. Pralidoximeblocks the binding of the nerve agent to the acetylcholine. In the treatment of nerve agent poisoning, atropine is most often administered along withpralidoxime, whichreactivates acetylcholinesterasewhich has been inactivated by phosphorylation by an organophosphorus nerve agent and relieves the respiratory muscle paralysis caused by some nerve agents. Pralidoxime is not effective in reactivatingacetylcholinesteraseinhibited by some older nerve agents such as somanor the Novichoknerve agents, described in the literature as being up to 8 times more toxic than nerve agent VX The Moscow Theatre Hostage Crisis Nerve Agent Used The incapacitating agent (Kolokol-1) used in the crisis probably was a derivative of fentanyl; this opium-based drug releases pain-killing endorphins inducing a state of euphoria. They can also induce sleep or unconsciousness. Howeverlarge doses of fentanyl and its derivatives can cause respiratory depression.It can starve the brain of oxygen causing permanent brain damage when prolonged. Effective antidote to opiates is widely available in the form of naloxon (Narcan). 3-methylfentanyl, a super potent fentanyl analogue(carfentanil), that is about 1000 times potent than morphine. It takes effect very quickly rendering its victims remain unconscious for two to six hours

9. Novichok– Choline esterase inhibitors – Acetylcholine blocker (Novichok 5and Novichok 7) - A-232had been an advantage: it could be used in cold temperatures and wouldn’t freeze on the battlefield(See FLUENZ TETRA and ‘cold-adapted’) Novichok 5 exceeds effectiveness of Somanby 10 times and of VX by 5 to 8 times. Novichok 5 (Substance A-232) and its ethyl-analog (Substance A-234) can be produced in binary formby using acetonitrile and an organic phosphate compound. Chemically they are organophosphates. They disrupt the mechanism by which nerves transfer messages to organs. The disruption is caused by blocking acetylcholine, which is a neurotransmitter. When acetylcholine transmitted the signal down, normally it is degraded into choline and acetic-acid by acetylcholine estherase. This regenerates the receptor and renders it active again. Nerve agents act by inhibiting of hydrolysis of acetylcholine by acetylcholine estherase. They bind chemically to the acetylcholine so it is unable to deactivate acetylcholine resulting in persistent and uncontrolled stimulation of receptor. Poisoning by a nerve agent leads to contraction of pupils, profuse salivation, convulsions, involuntary urination and defecation and death by asphyxiation as control is lost over the respiratory muscles. Two drugs: Atropine and Pralidoxime-chloride have been used as antidotes for nerve agents poisoning. Atropine blocks one type of acethylcholine receptor so then the acetylcholine cannot work in the synapse. Pralidoximeblocks the binding of the nerve agentto the acetylcholine.

10. Carbamateswere used asinsecticides. Their poisoning process differs from V-agents’ as binding of carbamate andacetylcholine can be very slowly hydrolysed, the hydrolysing process cannot be facilitated by enzyme regenerators, so that 2-PAM is ineffective in carbamate poisoning. Carbamates are in solid state, they are stabile, slowly disintegrating compounds. They have been used in vapour form.They are suitable for persistent poisoning of water and food. Some of them are more toxic than VX. The mechanism of action of binary chemical weapons consists in synthesizing the toxic agent from two or more non-toxic components(precursors) in the process of firing of a projectile(aircraft?), launching a missile, dropping an aerial bomb (Geo-Engineering and aerosolised spraying of the atmosphere) Acetylcholinesterase Inhibitors: Pharmacology and Toxicology Mirjana B. Colovic , Danijela Z. Krstic, Tamara D. Lazarevi-Pasti, Aleksandra M. Bondzic and Vesna M. Vasic Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oximereactivators of the inhibited enzymeactivity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system.

11. Since carbamates, as well as Organophosphorus compounds (OPs) , are AChEinhibitors, both compounds cause similar toxic acute effects and symptoms derived from poisoning. Carbamate compounds are applied as fungicides, insecticides and herbicides in agriculture, and belong to the second group of pesticides inhibiting cholinesterases. Carbamatesare considered to be saferthanOP insecticides that irreversibly inhibit AChEcausing moresevere cholinergicpoisoning OP pesticides can be absorbed by all routes, including inhalation, ingestion, and dermal absorption. Their toxicity is not limited to the acute phase, but chronic effects have long been noted. Actually, repeated or prolonged exposure to OPs may result in the same effects as acute exposure including the delayed symptoms. The effects, reported in workers repeatedly exposed, include impaired memory and concentration, disorientation, severe depressions, irritability, confusion, headache, speech difficulties, delayed reaction times, nightmares, sleepwalking and drowsiness or insomnia. Influenza-like condition with headache, nausea, weakness, loss of appetite, and malaisehas also been reported In the autonomic nervous system, ACh is the neurotransmitter in the preganglionic sympathetic and parasympathetic neurons, as well as at the adrenal medulla and serves as the neurotransmitter in all the parasympathetic innervated organs. ACh is also the neurotransmitter at the sweat glands, and at thepiloerector muscle of the sympathetic autonomic nervous system. Inthe peripheral nervous system, ACh is the neurotransmitter at the neuromuscular junction between the motor nerve and skeletal muscle The severity of cognitive impairment in Alzheimer's Disease (AD) correlates with loss of nicotinic receptors

12. Organophosphorus Nerve Agents/Gases Nerve agents of OP group include tabun, sarin, soman, cyclosarinand VX. Sarin, somanand cyclosarinarephosphonofluoridates, and VX is a phosphonothioate Based on the acute toxicity, VX is the most toxic compound among all the nerve agents . The developing and production of these extremely toxic nerve agents started in the 1930s, and later used in wars and by terrorists on several occasions. As chemical weapons, they are classified as weapons of mass destruction by the United Nations, and their production and stockpiling was outlawed by the Chemical Weapons Convention. Some nerve agents are readily vaporized or aerosolized and the primary portal of entry into the body is the respiratory system. Nerve agents can also be absorbed through the skin, requiring that those exposed to such agents wear a full body suit in addition to a respirator.Moreover, the effects of nerve agents are very long lasting and cumulative (increased by successive exposures), and survivors of nerve agent poisoning usually suffer chronic neurological damage that can lead to continuing psychiatric effects (Skripals, 2018 and Novichok nerve agent) Respiratory failure may also occur many hours later, either separated in time from the cholinergic crisis(intermediate syndrome) or merged into the acute cholinergic crisis. The pathophysiology of this late respiratory failureseems to involve down-regulation of nicotinic acetylcholine receptors. Intermediate syndrome is particularly important since people who are apparently well can progress rapidly to respiratory arrest Seizuresare believed to be initiated by excess acetylcholine in the brain after inhibition of acetylcholinesterase, with subsequent disruption of other neurotransmitter systems such as glutamate and catecholamines.

13. Brain Dopamine–Serotonin Vesicular Transport Disease and Its Treatment Jennifer J. Rilstone, B.Sc., Reem A. Alkhater, M.D., and Berge A. Minassian, M.D. VMAT2 translocates dopamineand serotonininto synaptic vesiclesand is essential for motor control, stable mood, and autonomic function. Evidence supporting its causation by a mutation in SLC18A2 (which encodes vesicular monoamine transporter 2 [VMAT2]). However, urinary neurotransmitter testing revealed elevated levels of monoamine metabolites VMAT2 also functions at sites outside the central nervous system, including the peripheral nervous system, adrenal medulla, and platelets Prolineto-leucinesubstitutions are generally considered to be deleterious to organismal fitness, A complete knockout of Slc18a2in mice results in a lack of exocytotic monoamine neurotransmission; the mutant animals feed poorly and die within days after birth. By contrast, mice that express just 5% native Vmat2 levels live to adulthood and have minor age-related motor deficits over time. Although the improvement in the patients (5 of 8) in this study was striking, it was not complete, probably because of monoamine deficiency during development and also because of on-going deficiencies of the non-dopamine amines and impairment in regulated release of dopamine.

14. High regulatability favours genetic selection in SLC18A2, a vesicular monoamine transporter essential for life SLC18A2encodes the vesicular monoamine transporter 2 protein that regulates neurotransmission and reduces cytosolic toxicity of monoamines. Deletion of this gene causes lethality in mice, and DNA sequence variation in this gene is associated with alcoholism and Parkinson’s disease, among other disorders

15. High regulatability favors genetic selection in SLC18A2, a vesicular monoamine transporter essential for life SLC18A2, the human Vesicular Monoamine Transporter 2 gene, is associated with a number of brain disorders, including alcoholism, Parkinson’s disease(PD), and schizophrenia (1,2,3,4,5,6). The vesicular monoamine transporter 2 (VMAT2) is an important molecule for the function of monoaminergic neurons that are key participants in locomotion, reward, working memory, and mnemonic brain systems 1. Zubieta J K, Taylor S F, Huguelet P, Koeppe R A, Kilbourn M R, Frey K A. Vesicular monoamine transporter concentrations in bipolar disorder type I, schizophrenia, and healthy subjects. Biol Psychiatry. 2001;49:110–116. [PubMed: 11164757] (Unavailable to read) 2. ZuckerM, Valevski A, Weizman A, Rehavi M. Increased platelet vesicular monoamine transporter density in adult schizophrenia patients. EurNeuropsychopharmacol. 2002;12:343–347. [PubMed: 12126874] (Unavailable to read) 3. Lin Z, Walther D, Yu X Y, Li S, Drgon T, Uhl G R. SLC18A2 promoter haplotypes and identification of a novel protective factor against alcoholism. Hum MolGenet. 2005;14:1393–1404. [PubMed: 15829504] (Available to read) 4. Glatt C E, Wahner A D, White D J, Ruiz-Linares A, Ritz B. Gain-of-function haplotypes in the vesicular monoamine transporter promoter are protective for Parkinson disease in women. Hum Mol Genet. 2006;15:299–305. [PMCID: PMC3643966] [PubMed: 16339215] (Available to read) 5. Schwab S G, Franke P E, Hoefgen B, Guttenthaler V, Lichtermann D, Trixler M, Knapp M, Maier W, Wildenauer D B. Association of DNA polymorphisms in the synaptic vesicular amine transporter gene (SLC18A2) with alcohol and nicotine dependence. Neuropsychopharmacology. 2005;30:2263–2268. [PubMed: 15988470] (Available to read) 6. Talkowski M E, Kirov G, Bamne M, Georgieva L, Torres G, Mansour H, Chowdari K V, Milanova V, Wood J, McClain L, Prasad K, Shirts B, Zhang J, O'Donovan M C, Owen M J, Devlin B, Nimgaonkar V L. A network of dopaminergic gene variations implicated as risk factors for schizophrenia. Hum MolGenet. 2008;17:747–758. [PMCID: PMC3777405] [PubMed: 18045777] (Available to read)

16. Acting to remove cytosolic monoamines [dopamine(DA), serotonin, norepinephrine, and histamine] by uptake into intracellular vesicles and to discharge the monoamines into extracellular space, VMAT2prevents neurotoxicity of these monoamines in the cytosol and regulates neurotransmission. Data from knockout mice indicate that expression of VMAT2 isessential for survivaland that different expression levels have altered behavioural consequences. Homozygous (−/−) knockout mice survive only about 1 postnatal week because of developmental defects, which is in contrast to other monoamine transporters, for which homozygous deletions (−/−) cause no lethality (14). Notably, environmental factors can regulate expression of the VMAT2gene, including stress, clozapine, and environmental contaminants (15,16,17). Expression systems for regulation study To study regulation of the SLC18A2 promoter, human cell lines that express endogenous SLC18A2 were searched among 5 human cell lines, including 4 DA cell lines [SH-SY5Y, IMR-32, SK-N-AS, and BE(2)-M17] and a non-neuronal cell line (HEK293T) by using qRT-PCR. These data on more regulations of A Haplotype (Caucasian) are consistent with the fact that A is recognized by more nuclear proteins than the other haplotypes (C, G and T) 10. Mosharov E V, Larsen K E, Kanter E, Phillips K A, Wilson K, Schmitz Y, Krantz D E, Kobayashi K, Edwards R H, Sulzer D. Interplay between cytosolic dopamine, calcium, and α-synuclein causes selective death of substantianigra neurons. Neuron. 2009;62:218–229. [PMCID: PMC2677560] [PubMed: 19409267]

17. High SLC18A2 regulatability is critical for survival during human evolution

18. Haplotype A has been heavily favoured during human biological history; Haplotype T is relatively close to A but is deselected These data imply that high SLC18A2regulatability is critical for survival during human evolution, which is consistent with variations in VMAT2 expression influencing brain integrity, function, and behavioursin knockout mice. In particular, the high regulatability may underlie forms of synaptic plasticityin which regulation of neurotransmission relies on variation in VMAT2-facilitated quantal size of transmitter release SLC18A2 promoter haplotypes and identification of anovel protective factor against alcoholism Zhicheng Lin*, Donna Walther, Xiao-Ying Yu, Suxia Li, Tomas Drgon and George R. Uhl Evidence is provided here that the promoter region has blocks of restricted diversity that lead to the formation of a major haplotype and a few minor ones

19. Genetic analysis of nucleotide diversity indicates strong positive selection of SLC18A2 promoter haplotypes. Cladistic analysis of the haplotypes reveals a large distance between the two most frequent haplotypes A and B. Evidently, A has been heavily favoured during human biological history Significant association between SLC18A2 promoter and alcoholismthat our data suggest here is consistent with several lines of evidence pointing to an association between SLC18A2 and different diseases including addiction. It has been found that VMAT2reduced expression levels in cocaine abusers These data are consistent with the observations that the heterozygous VMAT2 knockout mice gained significant sensitivity to drugs such as cocaine, ethanol and amphetamine (6,11)suggesting that alterations in SLC18A2 expression may underlie different human diseases. 11. Wang,Y.M., Gainetdinov, R.R., Fumagalli, F., Xu, F., Jones, S.R., Bock, C.B., Miller, G.W., Wightman, R.M. and Caron, M.G. (1997) Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivityto cocaine and amphetamine. Neuron, 19, 1285–1296.

20. Gain-of-function haplotypesin the vesicular monoamine transporter promoter are protective for Parkinson disease in women The brain form of the vesicular monoamine transporter (VMAT2) is an essential molecule for synaptic transmission of all of the biogenic amines: serotonin, norepinephrine, histamineand dopamine. VMAT2 is the single transporter in the central nervous system available for packaging biogenic aminesinto synaptic vesicles (3). SLC18A2, the gene for VMAT2, is located on chromosome 10q25 and is a candidate genefor Parkinson Disease (PD). We have previously screened the coding sequence of SLC18A2 in a non-PDpopulation sample and a large sample of PD patients. In both of these studies, we found that polymorphisms in coding sequence which predict alterations in the amino acid structure of VMAT2 are very rare and therefore cannot contribute in a substantial way to the population prevalence of sporadic PD Nonetheless, quantitative reduction of VMAT2 activity in genetically modified mice results in marked effects on neurotransmissionand behaviour 3. Peter D, Liu Y, Sternini C, de Giorgio R, Brecha N, Edwards RH. Differential expression of two vesicular monoamine transporters. J Neurosci. 1995; 15(9):6179–6188. [PubMed: 7666200] 4. Glatt C E, Wahner A D, White D J, Ruiz-Linares A, Ritz B. Gain-of-function haplotypes in the vesicular monoamine transporter promoter are protective for Parkinson disease in women. Hum Mol Genet. 2006;15:299–305. [PMCID: PMC3643966] [PubMed: 16339215] (Available to read) 11. Wang,Y.M., Gainetdinov, R.R., Fumagalli, F., Xu, F., Jones, S.R., Bock, C.B., Miller, G.W., Wightman, R.M. and Caron, M.G. (1997) Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivityto cocaine and amphetamine. Neuron, 19, 1285–1296.

21. Although the association we have identified is of only nominal statistical significance, the degree of the protective effect of homozygous gain-of-function haplotypes for women is large A number of epidemiological studies have found increased rates of PD in men (25–28). A recent meta-analysis found that PD is roughly 1.5 times more prevalent in men than in women (20). Further, the relative risk for male first-degree relatives of PD probandsis greaterthan that for females, suggesting that shared genetic factors may exert different degrees of risk or protection on men versus women (27) After standardization, men had PD with and without dementia more frequently than did women. The major difference between patients with and without dementiawas a later estimated age at onset of motor manifestations. We conclude that PD is a frequent disorder in the elderly population that affects men and whites (haplotype A) more frequently than women and non-whites. Moreover, dementia in patients with PD is more frequent than previously recognizedand is strongly related to the age at onset of motor manifestations. (25) Pesticide use (distinguishable from rural living) can be considered a risk factor for the development of PD, with family history of neurologic disease and history of depression serving as weaker predictors of PD. These findings are consistent with a study that foundgender differences in the symptom profile of PD 20. Wooten GF, Currie LJ, Bovbjerg VE, Lee JK, Patrie J. Are men at greater risk for Parkinson’s disease than women? J NeurolNeurosurgPsychiat. 2004; 75(4):637–639. [PubMed: 15026515] 25. Risk factors for Parkinson's disease. Hubble JP1, Cao T, Hassanein RE, Neuberger JS, Koller WC. 27. A Population-Based Investigation of Parkinson's Disease With and Without Dementia Relationship to Age and Gender Richard Mayeux, MD; Jean Denaro, MS; Nancy Hemenegildo, MD; et al

22. Association of DNA Polymorphisms in the Synaptic Vesicular Amine (2005) Transporter Gene (SLC18A2) with Alcohol and Nicotine Dependence Sibylle G Schwab, Petra E Franke, Barbara Hoefgen, Vera Guttenthaler, Dirk Lichtermann, MatyasTrixler, Michael Knapp, Wolfgang Maier and Dieter B Wildenauer The brain synaptic vesicular amine transporter SLCA18A2 is a key component for the uptake of monoamines like dopamine or serotonin into vesicles. We have analyzedseven DNA polymorphisms located in the genomic region of SLC18A2 for association with alcohol- and nicotine dependence, using a family-based design. Our sample comprised 131 families with alcohol-dependent offspring and 96 families with at least one nicotine-dependent offspring. For the alcohol-dependent sample, we found statistical significant association for two single markers (rs363387, P¼0.03; rs363333, P¼0.0066)…We conclude that DNA polymorphisms located in SLC18A2 might contribute to the development of substance dependence. The brain reward circuits include dopamine projections from the ventral tegmental area and substantianigra to the nucleus accumbens and striatum, as well as glutamate inputs from the prefrontal cortex, amygdala, and hippocampus. Drug sensitization-related changes have been described in many neurotransmitter systems that are integral to the function of the reward system, that is,dopamine, serotonin, norepinephrine, acetylcholine, opioid, and GABA systems (Robinsonand Berridge, 2003). Recently, a report by Heinz et al (2004) described reduced dopamine D2 receptor availability in detoxified alcoholics. These authors suggested that dopaminergic dysfunction in the ventral striatummay attribute incentive salienceto alcohol-associated stimuli.

23. Dopamine: 5-7 known receptor types D1 – D5 Dopamine: the molecule behind all our most sinful behaviours and secret cravings. Dopamine is love. Dopamine is lust. Dopamine is adultery. Dopamine is motivation. Dopamine is attention. Movement, motivation, attention, psychosis.Dopamine is feminism. Dopamine is addiction.. When most people talk about dopamine, particularly when they talk about motivation, addiction, attention, or lust, they are talking about the dopamine pathway known as themesolimbic pathway, which starts with cells in the ventral tegmental area, buried deep in the middle of the brain, which send their projections out to places like the nucleus accumbens and the cortex. Dopamineis signalling feedback for predicted rewards. If you, say, have learned to associate a cue (like a crack pipe) with a hit of crack, you will start getting increases in dopaminein the nucleus accumbens in response to the sight of the pipe, as your brain predicts the reward. But if you then don’t get your hit, well, then dopamine can decrease, Dopaminecan increase in the nucleus accumbensin people with post-traumatic stress disorder when they areexperiencing heightened vigilanceandparanoia. So you might say, in this brain area at least, dopamine isn’t addiction or reward or fear. Instead, it’s what we call salience. Salience is more than attention: It’s a sign of something that needs to be paid attention to, something that stands out. This may be part of the mesolimbic role in attention deficit hyperactivity disorder and also a part of its role in addiction. Dopamineis involved in the frontal cortex inexecutive functions like attentionIn the rest of the body, dopamine is involved in nausea, in kidney function, and in heart function.Dopamine plays a big role in starting movement, and the destruction of dopamine neuronsin an area of the brain called the substantianigrais what produces the symptoms of Parkinson’s disease (PD)

24. One of the proteins necessary for a proper functioning of monoaminergicneurons is the brain vesicular amine transporter(SLC18A2, VMAT2). This transporter uses energyfrom vesicular proton gradients to accumulate monoamine neurotransmitters from the neuronal cytoplasm into synaptic vesicles(Johnson, 1988; Henry et al, 1994). Predominant substrates for SLC18A2 are, among others, serotoninand dopamine; therefore, making it an ideal candidate gene for analysis of association with substance dependence. Moreover, Straub et al (1999) published a genome scan on nicotine addiction, with chromosome 10q26, thechromosomal location of SLC18A2, being one of the putatively linked regions. A follow-up of this study, using a computational approach, which allows searching for epistatic loci, identified SLC18A2 as a prime candidate in substance dépendance (Sullivan et al, 2004). Combining alcohol- and nicotine-dependent families again revealed association with the same two SNPs. None of the analyzedSNPs are known to cause functional changes of the protein encoded by the SLC18A2gene. Since mice lacking the neuronal isoform of the vesicular monoamine transporter do not survive for more than a few days (Uhl et al, 2000), quantitative changes in function of SLC18A2 are more likely to be involved in drug dependence. Hall et al (2003) have studied sex-dependent modulation of ethanolconsumption in SLC18A2 heterozygous knockout mice. They were able to show that male SLC18A2 heterozygous knockout mice consumed larger amounts of higher concentrations of ethanol as compared to wild-type mice. This appears to be consistent with our finding of male alcohol-dependentindividuals mainly contributing to the observed association

25. Experimenting with Spirituality: AnalyzingThe God Gene in a Non-majors Laboratory Course Linda A. Silveira VMAT2 encodes a transporter protein that imports several monoamine neurotransmitters into vesicles in the brain(reviewed in Zhenget al., 2006). Thus, an alterationin the transportercould potentially affect the levels of multiple types of neurotransmitters, resulting in altered brain function. Changes in this monoamine transporter’s sequence or expressionhave been associated with substance abuse and Parkinson’s disease (Lin et al., 2005; Schwab et al., 2005; Glatt et al., 2006; Yamamoto et al., 2006). For more detailed information about VMAT2, students might read Wang et al. (1997) or Takahashi et al. (1997), bothof which describe the phenotype of VMAT2 knockout mice. Both papers detail similar observations; the Wang paper, published second of the two, has more extensive background information that might be helpful to students. Lin et al. (2005) is an example of a published study linking VMAT2 single-nucleotide polymorphisms (SNPs) to a complex trait, describing the effects of variations in the VMAT2 promoter on alcoholism After this manuscript was submitted, two reports that investigated correlation between VMAT2 variations and schizophreniabecame available. In Talkowskiet al. (epub ahead of print, Nov. 27, 2007, Hum. Mol. Genet.) a series of genes involved in dopamine synthesis or function were investigated, including VMAT2.Three polymorphisms in VMAT2 (rs363393, rs363338, and rs363227) were correlated with schizophreniain two populations; none of these were those SNPsstudied in the God Gene project.

26. *F-AV133 Cerebral VMAT2 Binding Correlated with α-synucleinSpliced Variants in Parkinson’s Disease Journal of Neuroimaging in Psychiatry & Neurology *Vesicular monoamine transporter 2 (VMAT2) is the proteinresponsible for transporting both dopamine and serotonin into synaptic vesicles The Dopaminergic System In the brain, the principal dopamine systems arise from cells in the midbrain and the hypothalamus.The cells in the midbrain can be divided into three groups: A8 in the retro-rubral field, A9 in the substantianigra, and A10in the ventral tegmental area. The neuronsarising from A8 and A9 ascend to the striatum, forming part of the extrapyramidal system, and are involved in initiating and coordinating movement. The neuronsof the A10area project to the limbic and cortical areas and are referred to as the mesolimbic and mesocortical tracts, respectively. Researchers believe that these neurons are involved in emotional expression and cognitive function, and this system may be involved in the pathophysiology of mood disorders, schizophreniaand substance abuse. Much attention has focused recently on the interaction between dopamine and serotonin neurons in mediating psychosis, negative symptoms and the extrapyramidal side effectsof neuroleptics. Serotonincan inhibit the firing of dopaminergic neurons that project to the striatum. Serotoninreuptake inhibitors used to treat depression occasionally can produce extrapyramidal side effects, and the lesioningof serotonergic neurons in the dorsal raphe can diminish haloperidol-induced catalepsy. Serotonin also can inhibit the firing of dopaminergic neurons in limbic structures such as the nucleus accumbens http://www.psychiatrictimes.com/neuropsychiatry/dopamine-receptors-human-brain

27. Association of VMAT2 gene polymorphisms with alcohol dependence The major finding of our study is the confirmation of association between the gene encoding theVesicular MonamineTransporter 2 (VMAT2)andalcohol dependence. The analysis of eightsingle nucleotide polymorphisms (SNPs) in the gene locus SLC18A2 encoding the VMAT2 transporter gene demonstrated a significant allelic and genotype association of rs363387 with alcohol dependence. The presence of the rare rs363387 G allele and especially the rs363387 G/G genotype corresponded to a 50 % reduction in development of the phenotype. Thus, both rs363387 GT and GG genotypes may be protective against alcohol dependence. (G protects. T suggests risk)

28. The Adrenochrome Hypothesis, Ascorbic Acid and Schizophrenia

29. ORTHOMOLECULAR PSYCHIATRY, VOLUME 10, NUMBER 2, 1981, Pp. 98-118 The Adrenochrome Hypothesis of Schizophrenia Revisited A. Hoffer, M.D., Ph.D.1 Vitamin B-3 appears to protect patients against tardive dyskinesia. Hawkins in a personal communication reported that over fifteen years he has not seen any cases amongmany thousands treated. In my own practice I have not seen any develop in twenty years. It may well be some of the protective effect is due to the lower doses of tranquilizer required but there is a direct protective effect as well, probably at the synapse. There the medulla, containing noradrenaline and adrenaline, is surrounded by the adrenal cortex which is very rich in ascorbic acid. This stabilizes the amines as do other natural anti oxidants. Manganese may have a protective effect by inhibiting aminochrome formation But since ascorbic acidis as active as Haldol (one of the most powerful dopamine receptor Blockers) it would be worthwhile to determine whether pregnant women given optimum doses of ascorbic acid would have children who would eventually be less apt to develop schizophrenia. Galzigna(1970) suggested a relationship between acetylcholine, a neurotransmitter, and catecholamines. Acetylcholine interacts with oxidized noradrenaline, yielding a complex which does not change to adrenolutin in ascorbic acid medium. It reacts similarly with dopamine. Both acetylcholine and nicotinamideincrease the auto-oxidation of noradrenalinebut the complex reacts differently withascorbic acid.

30. Vitamin B-3 (Niacinamide) and B-6 (pyridoxine) dependency should be accounted for and the hypothesis should try to account for thetherapeutic value of other orthomolecular treatments such as ascorbic acid For excess adrenochromefor any reason whatever would flow into adrenolutin which like adrenochrome is an hallucinogen. Leukoadrenochromeis not an hallucinogen. Any quantity of adrenochrome could be neutralized by diverting it into its leuko derivative. The same reasoning applies to every amine which is oxidized in the body into an aminochrome. If there is an increased production of adrenochrome, properties of adrenochrome should be conferred upon the patient. Thus it is known that adrenochrome has antihistamine properties; schizophrenia should then impart antihistaminic properties, i.e. they should be less subject to physical expressionsof allergysuch as asthma, hay fever; Adrenochrome has antimitotic properties. This suggests schizophrenicsshould be less subject to attack by cancerand should have lesser growth rates of rapidly growing tissues such as hair and nails. A few studies suggest that both these conclusions are true. The role of stress: Stressis harmful for tworeasons. The increase in the production of noradrenaline and adrenaline will lead to an increase in adrenochrome and in those genetically disposed to reaction 7 instead of 6 this will increase the amount of adrenolutin which is toxic. Secondly any stress decreases the amount of ascorbic acid in the body. Most people are on very low ascorbic acid intake and can ill afford any losses. Adecrease in ascorbic acid will increase reaction 5. (see next slide)

32. Early in our research we found that adrenochrome injected intravenously into known epilepticsgreatly worsened the EEG abnormality. One young patient with no previous psychotic episodes was given adrenochrome. Within a few minutes her EEG became more pathological, she became morose and quiet. A few days later she had to be admitted to a psychiatric ward for treatment of her first psychosis. It is clear nicotinic acid can quickly reverse the adrenochrome-induced EEC pathology Pyridoxine (vitamin B6) was established as an important treatment for autism by Rimland (1978); Rim land, Callaway and Dreyfus (1978) and by Lelord et al. (1978,1979) It is essential for the conversion of tryptophan into nicotinamideadenine dinucleotide (NAD). A deficiencyof NADis present in the Vitamin B3 deficiency disease pellagra. Pellagrais caused by a diet deficient in Vitamin B3, by a diet whichis too low in tryptophan, too low in absorbable Vitamin B3 and too rich in leucine. Adequate amounts of NAD at the same receptor woulddecrease the formation of the aminochromeand thus protect the receptor. This idea has not been tested. This hypothesis suggests that ascorbic acid and Vitamin B-3 in adequate quantitiesshould protect dopamine receptorsagainst TOPA The well-known toxicityof 6 hydroxydopamine (also called tri-hydroxyindoleor TOPA) is due to its conversion to an aminochrome

33. Some patients will be psychotic because they require large amounts of Vitamin B3and B6 to keep the adrenochrome/adrenolutinreaction under control, others will require an allergy-free environment (including food) while many will require both. My experience suggests that patients free of cerebral allergies can seldom tolerate more than 6 grams per dayof nicotinic acid However, ascorbic acid which in the brainis as active as Haldol (which is not a tranquilizer). It has been valuable in controlling anxiety in some schizophrenicsand has "cured" a few schizophrenics when 20 grams per day was used. A deficiencyof serotonin would increase the conversion of dopaminetoaminochrome. Smythies (1976) elaborated the dopamine hypothesis by involving serotonin. He suggests there is an imbalance between dopamine, which is too active, and serotonin, which is not active enough Glutathione protects the enzyme monoamine Oxidase (Klemm and Baumgarten, 1978). Inhibiting monoamine oxidase will drive more of the catecholaminesinto other pathways including the aminochrome pathway. The adrenochrome hypothesis immediately called attention to Vitamin B3and ascorbic acid as potential treatments for schizophrenia. The fact that we have indeed found these substances so helpful is a plus for the usefulness of the hypothesis. Osmond and I did not foresee that these vitamins would have a specific reaction with brain receptors.

34. Conclusion The adrenochrome hypothesis accounts for the syndrome schizophrenia more accurately than do any of the competing hypotheses. It helped originate the use of large doses of Vitamin B3 for treating schizophrenia patients and for alleviating the symptoms created by LSD. It also predicted the therapeutic use of ascorbic acid, again in large doses. Both these vitaminsare important components in orthomolecular treatment as applied to schizophrenics. Finally it helped point to the catecholaminesas significant factors in the etiology of schizophrenia.