An In-depth Investigation of VMAT2 and the FunVax Vaccine

 Vmat2, FunVax Vaccine and Adrenochrome – a tentative investigation

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FunVax Quarterly Report 06/01/07The objective of this phase of project 149AZ2 is to prepare a viral vector that
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  1. 1. 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 cells and 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 system as well as the brain. The newly-designed virus contains the typical VSV genome, a homologous region to VMAT2 and a gene from Adenovirus that 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 across the blood-brain barrier to infect brain cells. VSV287 had the least amount of endogenous VMAT2 protein and will be further tested to verify that it is the most efficient of the 600 strains.
  2. 2. 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 monoamines into 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 VMAT2 deficient monkey should have expression of VMAT2 80-95% lower than a wild-type monkey. The VMAT2 deficient monkey should produce results by July 2007
  3. 3. Summary of recommendation 1. Quantitative PCR of all 600 animal subjects should be done to ensure that the data from the ELISA experiments, which showed a decrease in endogenous VMAT2 is 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 VSV287 or similar strains should allow the subject to breathe in the virus rather 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
  4. 4. Vesicular monoamine transporters (VMATs) mediate accumulation of monoamines such as serotonin, dopamine, adrenaline, noradrenaline, and histamine from the cytoplasm into storage organelles Association study of the vesicular monoamine transporter 1 (VMAT1) gene with schizophrenia in a Japanese population Misty Richards1,2, Yoshimi Iijima1, Hitomi Kondo1, Tomoko Shizuno1, Hiroaki Hori1, Kunimasa Arima3, Osamu Saitoh3 and Hiroshi Kunugi*1 VMAT1 is expressed primarily in neuroendocrine cells such as the adrenal medulla and pineal gland, while VMAT2 is expressed in all aminergic neurons in 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
  5. 5. 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. Organophosphates disrupt the mechanism by which nerves transfer messages to organs. The disruption is caused by blocking acetylcholinesterase (Ach) in the synapse, an enzyme that normally destroys acetylcholine, a neurotransmitter. Ach gives the signal for muscles to contract, preventing them from relaxing. 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.
  6. 6. 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. Pralidoxime blocks the binding of the nerve agent to the acetylcholine. In the treatment of nerve agent poisoning, atropine is most often administered along with pralidoxime, which reactivates acetylcholinesterase which 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 reactivating acetylcholinesterase inhibited by some older nerve agents such as soman or the Novichok nerve 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. However large 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
  7. 7. Novichok – Choline esterase inhibitors – Acetylcholine blocker (Novichok 5 and Novichok 7) – A-232 had 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 Soman by 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 form by using acetonitrile and an organic phosphate compound. 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. Pralidoxime blocks the binding of the nerve agent to the acetylcholine. 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.
  8. 8. Carbamates were used as insecticides. Their poisoning process differs from V-agents’ as binding of carbamate and acetylcholine 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 oxime reactivators of the inhibited enzyme activity 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.
  9. 9. 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 the piloerector muscle of the sympathetic autonomic nervous system. In the 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 Since carbamates, as well as Organophosphorus compounds (OPs) , are AChE inhibitors, 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. Carbamates are considered to be safer than OP insecticides that irreversibly inhibit AChE causing more severe cholinergic poisoning 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 malaise has also been reported
  10. 10. Organophosphorus Nerve Agents/Gases Nerve agents of OP group include tabun, sarin, soman, cyclosarin and VX. Sarin, soman and cyclosarin are phosphonofluoridates, 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 failure seems 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 Seizures are 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.
  11. 11. 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 among many 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. The Adrenochrome Hypothesis of Schizophrenia Revisited A. Hoffer, M.D., Ph.D.1 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 acid is 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 nicotinamide increase the auto-oxidation of noradrenaline but the complex reacts differently with ascorbic acid. ORTHOMOLECULAR PSYCHIATRY, VOLUME 10, NUMBER 2, 1981, Pp. 98-118
  12. 12. Vitamin B-3 (Niacinamide) and B-6 (pyridoxine) dependency should be accounted for and the hypothesis should try to account for the therapeutic value of other orthomolecular treatments such as ascorbic acid For excess adrenochrome for any reason whatever would flow into adrenolutin which like adrenochrome is an hallucinogen. Leukoadrenochrome is 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 expressions of allergy such as asthma, hay fever; adrenochrome has antimitotic properties. This suggests schizophrenics should be less subject to attack by cancer and 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: Stress is harmful for two reasons. 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. A decrease in ascorbic acid will increase reaction 5.
  13. 13. Early in our research we found that adrenochrome injected intravenously into known epileptics greatly 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 nicotinamide adenine dinucleotide (NAD). A deficiency of NAD is present in the Vitamin B3 deficiency disease pellagra. Pellagra is caused by a diet deficient in Vitamin B3, by a diet which is too low in tryptophan, too low in absorbable Vitamin B3 and too rich in leucine. Adequate amounts of NAD at the same receptor would decrease the formation of the aminochrome and thus protect the receptor. This idea has not been tested. This hypothesis suggests that ascorbic acid and Vitamin B-3 in adequate quantities should protect dopamine receptors against TOPA The well-known toxicity of 6 hydroxy dopamine (also called tri-hydroxyindole or TOPA) is due to its conversion to an aminochrome
  14. 14. Some patients will be psychotic because they require large amounts of Vitamin B3 and B6 to keep the adrenochrome/adrenolutin reaction 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 day of nicotinic acid However, ascorbic acid which in the brain is as active as Haldol (which is not a tranquilizer). It has been valuable in controlling anxiety in some schizophrenics and has “cured” a few schizophrenics when 20 grams per day was used. A deficiency of serotonin would increase the conversion of dopamine to aminochrome. 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 catecholamines into other pathways including the aminochrome pathway. The adrenochrome hypothesis immediately called attention to Vitamin B3 and 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.
  15. 15. 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 vitamins are important components in orthomolecular treatment as applied to schizophrenics. Finally it helped point to the catecholamines as significant factors in the etiology of schizophrenia.
  16. 16. However, urinary neurotransmitter testing revealed elevated levels of monoamine metabolites 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 dopamine and serotonin into synaptic vesicles and 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]). VMAT2 also functions at sites outside the central nervous system, including the peripheral nervous system, adrenal medulla, and platelets Prolineto-leucine substitutions are generally considered to be deleterious to organismal fitness, A complete knockout of Slc18a2 in 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.
  17. 17. High regulatability favours genetic selection in SLC18A2, a vesicular monoamine transporter essential for life SLC18A2 encodes 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
  18. 18. 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). 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. Zucker M, 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) High regulatability favors genetic selection in SLC18A2, a vesicular monoamine transporter essential for life 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
  19. 19. Acting to remove cytosolic monoamines [dopamine (DA), serotonin, norepinephrine, and histamine] by uptake into intracellular vesicles and to discharge the monoamines into extracellular space, VMAT2 prevents neurotoxicity of these monoamines in the cytosol and regulates neurotransmission. 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 substantia nigra neurons. Neuron. 2009;62:218–229. [PMCID: PMC2677560] [PubMed: 19409267] Data from knockout mice indicate that expression of VMAT2 is essential for survival and 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 VMAT2 gene, 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)
  20. 20. 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 SLC18A2 regulatability is critical for survival during human evolution, which is consistent with variations in VMAT2 expression influencing brain integrity, function, and behaviours in knockout mice. In particular, the high regulatability may underlie forms of synaptic plasticity in which regulation of neurotransmission relies on variation in VMAT2-facilitated quantal size of transmitter release SLC18A2 promoter haplotypes and identification of a novel 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
  21. 21. 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 alcoholism that 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 VMAT2 reduced 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 supersensitivity to cocaine and amphetamine. Neuron, 19, 1285–1296.
  22. 22. 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 supersensitivity to cocaine and amphetamine. Neuron, 19, 1285–1296. 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) Gain-of-function haplotypes in 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, histamine and dopamine . VMAT2 is the single transporter in the central nervous system available for packaging biogenic amines into synaptic vesicles (3). 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] SLC18A2, the gene for VMAT2, is located on chromosome 10q25 and is a candidate gene for Parkinson Disease (PD). We have previously screened the coding sequence of SLC18A2 in a non-PD population 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 neurotransmission and behaviour
  23. 23. 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 probands is greater than that for females, suggesting that shared genetic factors may exert different degrees of risk or protection on men versus women 20. Wooten GF, Currie LJ, Bovbjerg VE, Lee JK, Patrie J. Are men at greater risk for Parkinson’s disease than women? J Neurol Neurosurg Psychiat. 2004; 75(4):637–639. [PubMed: 15026515] 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 (27) After standardization, men had PD with and without dementia more frequently than did women. The major difference between patients with and without dementia was 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 nonwhites. Moreover, dementia in patients with PD is more frequent than previously recognized and 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 found gender differences in the symptom profile of PD 25. Risk factors for Parkinson’s disease. Hubble JP1, Cao T, Hassanein RE, Neuberger JS, Koller WC.
  24. 24. The brain synaptic vesicular amine transporter SLCA18A2 is a key component for the uptake of monoamines like dopamine or serotonin into vesicles. We have analyzed seven 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. 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, Matyas Trixler, Michael Knapp, Wolfgang Maier and Dieter B Wildenauer The brain reward circuits include dopamine projections from the ventral tegmental area and substantia nigra 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 (Robinson and 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 striatum may attribute incentive salience to alcohol-associated stimuli.
  25. 25. 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 the mesolimbic 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. Dopamine is 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 dopamine in 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, Dopamine can increase in the nucleus accumbens in people with post-traumatic stress disorder when they are experiencing heightened vigilance and paranoia. 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. Dopamine is involved in the frontal cortex in . executive functions like attention In 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 neurons in an area of the brain called the substantia nigra is what produces the symptoms of Parkinson’s disease (PD)
  26. 26. One of the proteins necessary for a proper functioning of monoaminergic neurons is the brain vesicular amine transporter (SLC18A2, VMAT2). This transporter uses energy from 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, serotonin and 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, the chromosomal 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 analyzed SNPs are known to cause functional changes of the protein encoded by the SLC18A2 gene. 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 ethanol consumption 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-dependent individuals mainly contributing to the observed association
  27. 27. Experimenting with Spirituality: Analyzing The 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 Zheng et al., 2006). Thus, an alteration in the transporter could potentially affect the levels of multiple types of neurotransmitters, resulting in altered brain function. Changes in this monoamine transporter’s sequence or expression have 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), both of 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 schizophrenia became available. In Talkowski et 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 schizophrenia in two populations; none of these were those SNPs studied in the God Gene project.
  28. 28. *Vesicular monoamine transporter 2 (VMAT2) is the protein responsible for transporting both dopamine and serotonin into synaptic vesicles *F-AV133 Cerebral VMAT2 Binding Correlated with α-synuclein Spliced Variants in Parkinson’s Disease Journal of Neuroimaging in Psychiatry & Neurology 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 substantia nigra, and A10 in the ventral tegmental area. The neurons arising from A8 and A9 ascend to the striatum, forming part of the extrapyramidal system, and are involved in initiating and coordinating movement. The neurons of the A10 area 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, schizophrenia and substance abuse. Much attention has focused recently on the interaction between dopamine and serotonin neurons in mediating psychosis, negative symptoms and the extrapyramidal side effects of neuroleptics. Serotonin can inhibit the firing of dopaminergic neurons that project to the striatum. Serotonin reuptake inhibitors used to treat depression occasionally can produce extrapyramidal side effects, and the lesioning of 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
  29. 29. The major finding of our study is the confirmation of association between the gene encoding the Vesicular Monamine Transporter 2 (VMAT2) and alcohol dependence. The analysis of eight single 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) Association of VMAT2 gene polymorphisms with alcohol dependence
  30. 30. 1Trans wellbeing and healthcare A Guide to Hormone Therapy for Trans People Will I always have to take hormones? Yes, you will need to take hormones for the rest of your life if you want to maintain the feminising effects of oestrogen or the masculinising effects of testosterone. Will I still be able to have children? Hormone treatment will make you infertile after a while. This is true for trans men and for trans women. If you are a trans woman, it may be possible to restore your sperm production by stopping hormone therapy. It is not known how long it takes for treatment to make you infertile and it will vary from person to person. If you have your testes removed, you will be permanently infertile If you are a trans man, testosterone treatment appears to take much longer to make you permanently infertile, but again, it is not known how long. Medication to reduce testosterone effects – Cyproterone acetate (50-100mg daily) is preferred by some service users because it is in pill form and is therefore easily administered. It works by blocking testosterone receptors, and it is effective against androgen (testosterone) produced by the adrenal glands, as well as that produced by the testes. Heavy alcohol use reduces its effectiveness. Spironolactone (taken in pill form 100-300mg daily) is a well-tolerated and effective anti- androgen (feminizing agent) Goserelin or Leuprorelin is administered subcutaneous depot injection but does not block testosterone produced by the adrenal gland. 1 Produced by COI for the Department of Health
  31. 31. BICALUTAMIDE- Bicalutamide tablet: Initial U.S. Approval: 1995 (Intas Pharmaceuticals Limited) Bicalutamide tablets 50 mg is a a non-steroidal androgen receptor inhibitor with no other known endocrine activity and is indicated for use in combination therapy with a luteinizing hormone-releasing hormone (LHRH) analog for the treatment of Stage D metastatic carcinoma of the prostate. It competitively inhibits the action of androgens by binding to cytosol androgen receptors in the target tissue (prostatic carcinoma). R-bicalutamide is an inhibitor of CYP3A4 Pregnancy 8.1 Bicalutamide is contraindicated for use in pregnant women because it can cause fetal harm. Bicalutamide is not indicated for use in females. There are no human data on the use of bicalutamide in pregnant women. In animal reproduction studies, oral administration of bicalutamide to pregnant rats during organogenesis caused abnormal development of reproductive organs in male fetuses at exposures approximately 0.7 to 2 times the human exposure at the recommended dose Bicalutamide can cause fetal harm when administered to a pregnant woman Based on animal studies, bicalutamide can lead to inhibition of spermatogenesis and may impair fertility in males of reproductive potential. The long-term effects of bicalutamide tablets on male fertility have not been studied. Anti-androgen therapy may cause morphological changes in spermatozoa
  32. 32. Congenital Adrenal Hyperplasias or CAH Caused by a lack in the production of cortisol in both the salt-losing and simple- virilizing forms. Most of the problems caused by classic CAH are related to a lack of cortisol, which plays an important role in regulating your blood pressure, maintaining blood sugar and energy levels, and protecting your body against stress. Virilization or masculinization (Undervirilization and more feminine oestrogen) is the biological development of sex differences, changes that make a male body different from a female body. Most of the changes of virilization are produced by androgens. Excess production of the male sex hormones (androgens such as testosterone) – This can result in short height, early puberty and in females, abnormal genital development while in the womb. 21-hydroxylase deficiency is an inherited disorder that affects the Adrenal Glands which produce excess androgens, which are male sex hormones. “The adrenal gland is an intricate part of the HPA (Hypothalamus, Pituitary, adrenal) Axis,” Dr. Mark Engelman, permanent Clinical Consultant for , told Live Science. “This intimate physiological relationship is fundamental and critical to our wellbeing.”
  33. 33. Endocrine intervention for transsexuals Clinical Endocrinology (2003) 59, 409–418 Diagnosis Gender dysphoria is a self-diagnosis, often supported by friends and family, with no supporting tests other than persistence of dysphoria for at least 2 years alleviated by cross-gender identification Female-to-male. For female-to-male transsexuals, the usual hormone treatment is Sustanon 250 mg intramuscularly (i.m.) every 2 weeks, or testosterone enanthate (Primoteston Depot) if the patient is sensitive to peanut (arachis) oil. In a retrospective study of 303 male-to-female transsexuals treated with cross-sex hormones, the death rate due to increased numbers of suicide and death of unknown cause was five times that of a reference population (Asscheman et al ., 1989). Depressive mood changes were 15 times more common and thromboembolic events were increased 45-fold compared to the general population (Asscheman et al ., 1989). In male-to-female transsexuals there was an increased risk of suicide and HIV infection (a nine- fold and six-fold increase in risk, respectively: n = 13) In a study of 20 male-to-female transsexuals exposed to cross-sex hormones for 28–63 months, oestrogen treatment (ethinylestradiol 100 μg daily with cyproterone acetate 100 mg/day (50–100 μg oestradiol without cyproterone after gonadectomy)) prevented bone mineral density loss induced by testosterone privation (van Kesteren et al ., 1998), suggesting that oestrogen treatment for 2 years or more maintains or increases bone mineral density
  34. 34. Increase in breast size usually begins 2–3 months after the start of female sex hormone treatment in male-to-female transsexuals and continues for 2 years (Meyer et al ., 1986; van Kesteren, 2002). Unfortunately, only one-third of transsexuals achieve more than a B cup and with 45% not advancing beyond an A cup, at least 60% require breast augmentation to achieve the appearance they desire or at least find acceptable. There are case reports of breast carcinoma in hormonally treated male-to-female transsexuals (Symmers, 1968; Ganly & Taylor, 1995) Female sex hormones adversely affect fertility in most XY individuals (Thiagaraj et al ., 1987; Venizelos & Paradinas, 1988; Lubbert et al ., 1992; Handelsman et al ., 2000). As far as freezing unfertilized ova in female-to-male transsexuals before treatment, the chance of a successful pregnancy resulting from the use of such ova is currently believed to be very low. The rate of development, extent and potential reversibility of fertility reduction in female-to-male transsexuals receiving testosterone is also unclear at the present time. (2003) Adipose tissue Magnetic resonance imaging (MRI) analysis of regional fat deposition in 20 male-to-female transsexuals before and 1 year after cross-sex hormone treatment demonstrates a significant increase in subcutaneous and visceral fat depots and a decrease in thigh muscle area (Elbers et al., 1999). Many male-to-female transsexuals report that oestrogen treatment is associated with a calming, almost anti-depressive effect (Reid, 2002) Summary and conclusions In summary, transsexuals have persistent cross-gender identities, usually without any predisposing factors. Self-diagnosis is confirmed by psychological assessment, which includes a trial period living in the chosen gender before consideration of hormonal treatment and surgery
  35. 35. Trans women – SRS – Sex Reassignment Surgery (or Gender Confirmation Surgery) This is the most often heard of surgery for MTFs, euphemistically referred to as “the operation”, but it is not a necessary prerequisite for “becoming a woman” Orchidectomy is the removal of the testes. This operation means that testosterone will no longer be naturally produced in the body and therefore you can do without your testosterone blocker. Some trans women may even feel that having a penis is important for their sexuality, but will have an orchidectomy for hormonal reasons. Androgen receptor blockers: These drugs attach to the same site as the male hormones, effectively denying the androgen access to the receptor and causing the androgens to be ineffective. Examples of these drugs include: Cyproterone, flutamide (Eulexin), and Spironolactone (Aldactone) 5 alpha-reductase inhibitors: These drugs act on an enzyme that converts androgens to their active form. If you limit the enzyme, you prevent the conversion of testosterone to its more potent form. Here are some examples: Finasteride (Proscar), Eflornithine Hydrochloride (Vaniqa).
  36. 36. OESTRADIOLS Trans women – Feminising medication – -Oestrogen – Oestradiol-based formulations are naturally occurring oestrogen Oestradiol). Topic to discuss – Oestradiol (in men) This gathers in the intestines and can cause that middle-aged spread or excess Estradiol accumulation due to less Testosterone (due to conversion) The most serious risks when taking oestrogens are: • thrombosis, deep vein thrombosis (DVT), stroke – pulmonary embolism (block in a blood vessel in the lungs) altered liver function. There are far fewer risks of thrombosis with oestradiol, which is the product most widely prescribed for trans women now. THYROID The thyroid also secretes a range of hormones that affect the whole body. “Thyroid hormones impact a host of vital body functions, including heart rate, skin maintenance, growth, temperature regulation, fertility and digestion,” said Dr. Jerome M. Hershman, a professor of medicine at the David Geffen School of Medicine at.. “In this way, the thyroid gland is the body’s master metabolic control center,” said Cindy Samet, a chemistry professor at Dickinson College in Carlisle, Pennsylvania. “Brain, heart and kidney function, as well as body temperature, growth and muscle strength — and much more — are at the mercy of thyroid function.”


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