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Showing posts with label Methylation. Show all posts
Showing posts with label Methylation. Show all posts

Monday 23 March 2015

“Epigenopathies” in Autism and Epigenetic Therapy in Current Use - Part 1








Today’s post is about epigenetics, a complex area of science, that has been touched upon in previous posts.

Since none of us are experts in genetics we will focus on the application of epigenetics rather than going into the excruciating details.  Skip over any parts that get too technical. Some of the interesting studies, that are of more academic interest, I will put in a later post.

Epigenetics is just one way in which gene expression (whether genes are turned on or off) can be altered.  There are other ways, which may be equally important. It is evident that epigenetics plays a role in many conditions including autism, schizophrenia, inflammation, asthma, COPD and cancer.

Even based on today’s highly superficial review, there is an immediate, practical, therapeutic prospect, worthy of investigation.  Thanks to Professor Peter Barnes in London and again those irrepressible researchers in Tehran, who were actually trialing theophylline for entirely different reasons.

You do need some basic definitions to understand what is going on in epigenetics, but in essence epigenetic changes are just like bookmarks.


DNA

DNA is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms.

The problem with DNA is that there is a lot of it.  It has to be very tightly packed since it has to fit inside every cell in your body.  In order to tightly fold up all that DNA you need Chromatin.


Chromatin

Chromatin is a complex of macromolecules found in cells, consisting of DNA, protein and RNA. The primary functions of chromatin are to:-

1) Package DNA into a smaller volume to fit in the cell
2) Reinforce the DNA macromolecule to allow mitosis
3) Prevent DNA damage
4) Control gene expression and DNA replication.

The primary protein components of chromatin are histones that compact the DNA.


Epigenetics – book marks on your DNA

Rather like you might stick post-it notes on your cookery book, or science text book, your body has various mechanism to highlight specific genes.  In effect these bookmarks turn on, or turn off that gene.

So in the jargon:-

Epigenetic changes involve non genetic changes in chromatin structure resulting in changes in gene expression

The important thing to note is that we are not talking about genetic defects, mutations, CNVs etc. which are usually what you might think about.

We all have these epigenetic markers and they are subject to change. Some of these markers become fixed and can then be inherited.  So if your ancestor lived/worked in a highly polluted place, you might have inherited some of his/her DNA tags/bookmarks, this would affect how your genes are expressed today.

The problem occurs when these markers get stuck, or are in the wrong place.  Imagine having a bookmark to remind you how long to roast your chicken and instead it takes you to the page with the recipe for pancakes.

In some inflammatory diseases, like COPD, the “good” genes are turned off and the “bad” genes have got stuck turned on.


Epigenetic change is reversible

Whereas genetic defects are irreversible, epigenetic changes are potentially reversible.  You just need to figure out how to rub them out.


Epigenetic Mechanisms

Just as you might use a variety of objects to mark pages in a book, so nature employs multiple methods to tag your DNA.


1.     DNA methylation

In this process the tag is a methyl group (CH3); to silence the bad gene you add more tags (Stimulate methylation).  To reverse a good gene that has been silenced, remove the tags (use a DNA methyltransferase inhibitors e.g. azacytidine)

Applicable to lung cancer & inflammation
Problems of specificity and targeting


I could only find a current methylation epigenetic therapy for schizophrenia:-




Recently, Satta et al. reported that nicotine decreases DNMT1 expression in GABAergic mouse neurons leading to decreased methylation at the GAD67 promoter and increased GAD67 protein expression. This effect was found to occur as a result of nicotinic receptor agonism. These improve cognitive functioning in schizophrenia, and may suggest in part why 80% of schizophrenia patients use tobacco. The specific nicotinic receptors that mediate this improved cognition have yet to be established. However, an alpha7-nicotine receptor agonist has been shown in small studies to improve cognition in schizophrenia subjects.



2. Histone modification 

Histones are the chief protein components of chromatin, acting as spools around which DNA winds.

There are several types of histone modification, that act as tags on your DNA:-

·        Lysine methylation
·        Arginine methylation
·        Lysine acetylation
·        Serine/Threonine/Tyrosine phosphorylation


The most studied variant is acetylation, this involves the addition or removal of acetyl groups (O=C-CH3)


INHIBITORS

In medicine a group of drugs already exists, called Histone Deacetylase inhibitors (HDAC inhibitors, HDIs).  HDIs are a class of compounds that interfere with the function of histone deacetylase.

HDIs have a long history of use in psychiatry and neurology as mood stabilizers and anti-epileptics. More recently they are being investigated as possible treatments for cancers, parasitic and inflammatory diseases.

The prime example of this is valproic acid, marketed as a drug under the trade names Depakene, Depakote, and Divalproex. In more recent times, HDIs are being studied as a mitigator for neurodegenerative diseases such as Alzheimer's disease and Huntington's disease.  Enhancement of memory formation is increased in mice given the HDIs sodium butyrate or SAHA.  While that may have relevance to Alzheimer's disease, it was shown that some cognitive deficits were restored in actual transgenic mice that have a model of Alzheimer's disease (3xTg-AD) by orally administered nicotinamide, a competitive HDI of Class III sirtuins.



Autism and HDIs 

There is research in mouse models showing that HDIs can improve autism.

Readers of this blog who are using the Supersprouts broccoli powder may not realize that the Sulforaphane produced, is a potent HDI (Histone Deacetylase Inhibitor).

In the autism world, the HDI research is still generally on mice, where social cognition is seen to improve.




Follow up study by Foley:-


In utero exposure of rodents to valproic acid (VPA) has been proposed to induce an adult phenotype with behavioural characteristics reminiscent of those observed in autism spectrum disorder (ASD). Our previous studies have demonstrated the social cognition deficits observed in this model, a major core symptom of ASD, to be ameliorated following chronic administration of histone deacetylase (HDAC) inhibitors. Using this model, we now demonstrate pentyl-4-yn-VPA, an analogue of valproate and HDAC inhibitor, to significantly ameliorate deficits in social cognition as measured using the social approach avoidance paradigm as an indicator of social reciprocity and spatial learning to interrogate dorsal stream cognitive processing. The effects obtained with pentyl-4-yn-VPA were found to be similar to those obtained with SAHA, a pan-specific HDAC inhibitor. Histones isolated from the cerebellar cortex and immunoblotted with antibodies recognising lysine-specific modification revealed SAHA and pentyl-4-yn-VPA to enhance the acetylation status of H4K8. Additionally, the action of pentyl-4-yn-VPA, could be differentiated from that of SAHA by its ability to decrease H3K9 acetylation and enhance H3K14 acetylation. The histone modifications mediated by pentyl-4-yn-VPA are suggested to act cooperatively through differential acetylation of the promoter and transcription regions of active genes.



ACTIVATORS

Histone modification is also implicated in inflammation.  We know that Autism is an inflammatory condition and of course we know which are the much better studied inflammatory conditions.

In terms of the brain, schizophrenia and even sometimes ADHD are better studied.

In the rest of the body arthritis, asthma and COPD are interesting.  Thanks to Peter Barnes at Imperial College, the COPD research is again leading the way.

COPD is like a severe drug resistant form of asthma.  Barnes has almost completely figured out the mechanism and how to best treat it.  One of the findings is to use a common drug called Theophylline in low doses as a HDAC activator.



The usual modes of action of Theophylline are:-

1.     competitive nonselective phosphodiesterase inhibitor, which raises intracellular cAMP, activates PKA, inhibits TNF-alpha  and inhibits leukotriene  synthesis, and reduces inflammation and innate immunity

2.     nonselective adenosine receptor antagonist


The usual dosage involves concentration of 10-20 micrograms/mL blood.  At this level there can be some side effects.

Barnes found that at sub-therapeutic doses (<8 micrograms/mL) , Theophylline actually has a different mode of action; it behaves as a HDAC activator; because the other modes of action were no longer present, no longer were their side effects.  He also showed that as the dose increases, the HDAC activation actually fades.  Another case of less being more.

Once deacetylated, DNA is repackaged so that the promoter regions of inflammatory genes are unavailable for binding of transcription factors such as NF-κB that act to turn on inflammatory activity. It has recently been shown that the oxidative stress associated with cigarette smoke can inhibit the activity of HDAC2, thereby blocking the anti-inflammatory effects of corticosteroids.)

Theophylline is a novel form of adjunct therapy in improving the clinical response to steroids in smoking asthmatics and people with COPD (some of whom do never smoked).


By using a low dose of Theophylline, steroid medication became much more effective allowing lower doses of steroids to be used.

Below is a presentation and one of Barnes’ many papers on this subject:-



Targeting the epigenome in the treatment of asthma and chronicobstructive pulmonary disease.

Abstract

Epigenetic modification of gene expression by methylation of DNA and various post-translational modifications of histones may affect the expression of multiple inflammatory genes. Acetylation of histones by histone acetyltransferases activates inflammatory genes, whereas histone deacetylation results in inflammatory gene repression. Corticosteroids exert their anti-inflammatory effects partly by inducing acetylation of anti-inflammatory genes, but mainly by recruiting histone deacetylase-2 (HDAC2) to activated inflammatory genes. HDAC2 deacetylates acetylated glucocorticoid receptors so that they can suppress activated inflammatory genes in asthma. In chronic obstructive pulmonary disease (COPD), there is resistance to the anti-inflammatory actions of corticosteroids, which is explained by reduced activity and expression of HDAC2. This can be reversed by a plasmid vector, which restores HDAC2 levels, but may also be achieved by low concentrations of theophylline. Oxidative stress causes corticosteroid resistance by reducing HDAC2 activity and expression by activation of phosphoinositide-3-kinase-delta, resulting in HDAC2 phosphorylation via a cascade of kinases. Theophylline reverses corticosteroid resistance by directly inhibiting oxidant-activated PI3Kdelta and is mimicked by PI3Kdelta knockout or by selective inhibitors. Other treatments may also interact in this pathway, making it possible to reverse corticosteroid resistance in patients with COPD, as well as in smokers with asthma and some patients with severe asthma in whom similar mechanisms operate. Other histone modifications, including methylation, tyrosine nitration, and ubiquitination may also affect histone function and inflammatory gene expression, and better understanding of these epigenetic pathways could led to novel anti-inflammatory therapies, particularly in corticosteroid-resistant inflammation.


COPD and Autism

COPD is not autism, but there are some similarities.  Both conditions are associated with chronic oxidative stress and inflammation.

The antioxidant NAC is effective in both conditions.

The Nrf2 activator Sulforaphane (from broccoli) is being trialed for both conditions and is shown effective in much autism.

Inhaled steroids keep people with COPD alive, and oral steroids are beneficial to many people with autism.  Their use in autism is severely limited by side effects of long term oral steroid use.

Some HDI drugs improve autism and some HDI drugs improve COPD.

It would seem that the Epigenopathies of autism and COPD may well overlap.  Could the COPD epigenetic therapy be effective in some autism?

  

Theophylline for Neurological Disorders?

You might have realized that epigenetic therapy should be highly focused, since some genes need to be switched on while others need to be switch off.

Nonetheless that natural question to ask is what is the effect of Theophylline on neurological disorders like autism.

I cannot answer that question; but we can see the effect on ADHD (autism-lite).

It should be noted that the below trial was nothing related to epigenetics and the dosage was the more typical high dosage.  The histone modifying (epigenetic) effect would have been greater at a slightly lower dosage.

At these doses Theophylline would act as a mild stimulant;  note that Theophylline is very closely related to caffeine.  Somewhat counter-intuitively, psychiatrists treat hyperactive people with stimulants.




A total of 32 children with ADHD as defined by DSM IV were randomized
to theophylline and methylphenidate dosed on an age and weight-adjusted basis at 4 mg/kg/day (under 12 years) and 3 mg/kg/day theophylline
(over 12 years) (group 1) and 1 mg/kg/day methylphenidate
(group 2) for a 6-week double-blind and randomized clinical trial. The principal measure of the outcome was the Teacher and Parent ADHD Rating Scale. Patients were assessed by a child psychiatrist, at baseline and at 14, 28 and 42 days after start of the medication.

The results suggest that theophylline may be a useful for the treatment of ADHD. In addition, a tolerable side-effect profile is one of the advantages of theophylline in the treatment of ADHD.



In autism it would be nice if somebody made a trial with 2mg/kg


Let us digress a little and see just what is Theophylline:-


Theophylline is naturally found in cocoa beans. Amounts as high as 3.7 mg/g have been reported in Criollo cocoa beans.

Trace amounts of theophylline are also found in brewed tea, although brewed tea provides only about 1 mg/L, which is significantly less than a therapeutic dose.


As a member of the xanthine family, it bears structural and pharmacological similarity to theobromine and caffeine


Derivatives of xanthine (known collectively as xanthines) are a group of alkaloids commonly used for their effects as mild stimulants and as bronchodilators, notably in the treatment of asthma symptoms. In contrast to other, more potent stimulants like sympathomimetic amines, xanthines mainly act to oppose the actions of the sleepiness-inducing adenosine, and increase alertness in the central nervous system. They also stimulate the respiratory centre, and are used for treatment of infantile apnea. Due to widespread effects, the therapeutic range of xanthines is narrow, making them merely a second-line asthma treatment. The therapeutic level is 10-20 micrograms/mL blood; signs of toxicity include tremor, nausea, nervousness, and tachycardia/arrhythmia.


Theophylline degrades to caffeine.



Inhibitor or Activator of HDAC ?

You may be wondering why we would want an HDAC activator for autism, if we know that Sulforaphane (broccoli) does just the opposite; it is an inhibitor.  The reason is that we have made a few simplifications in the science; there are many types of HDAC, and you might need an inhibitor of one type of HDAC and an activator of another.  Worse still, you might need something on one part of your body and something quite different in another part.

The HDACs can be divided into 3 classes based on their structure and sequence homology: class I consists of HDACs 1, 2, 3, 8, and 11; class II includes HDACs 4, 5, 6, 7, 9, and 10; and class III enzymes are HDACs originally found in yeast and include Sir2-related proteins. Increased HDAC activity and expression are common in many cancers and can result in repression of transcription that results in a deregulation of differentiation, cell cycle, and apoptotic mechanisms. Moreover, tumor suppressor genes, such as p21 appear to be targets of HDACs and are “turned off” by deacetylation. Prostate cancer cells also exhibit aberrant acetylation patterns. The use of class I and class II HDAC inhibitors in cancer chemoprevention and therapy has gained substantial interest.

   
Epigenopathies

When the epigenetic bookmarks appear in the wrong place, trouble will follow.  Genes that should be “off” are turned on and vice versa.

These events have recently been a new name “Epigenopathies”

Just as we can look at many dysfunctions in autism as Channelopathies; those dysfunctions in ion channels and ion transporters, we will be able to consider others as Epigenopathies.

Who first came up with this terminology is not certain, but it might be a clever Frenchman called Mark Millan who works at the Unit for Research and Discovery in Neuroscience, Institut de Recherches Servier, beside the river Seine.

The good news is that here is a very clever neuroscientist with an interest in autism, but not obsessed by it.

France generally has quite an old fashioned view of autism, you will not find much in the way of ABA in France, and the State is certainly not going to be the one paying for it.




Millan nicely summarizes the implications:-

Neurodevelopmental Disorders (NDDs) are accompanied by aberrant "epigenetic" regulation of processes critical for normal and orderly development of the brain. Epigenetics refers to potentially-heritable (by mitosis and/or meiosis) mechanisms controlling gene expression without changes in DNA sequence. In certain NDDs, prototypical epigenetic processes of DNA methylation and covalent histone marking are impacted. Conversely, others involve anomalies in chromatin-modelling, mRNA splicing/editing, mRNA translation, ribosome biogenesis and/or the regulatory actions of small nucleolar RNAs and micro-RNAs. Since epigenetic mechanisms are modifiable, this raises the hope of novel therapy …


In the next post on epigenetics we will look at the research that is specific to  neurodevelopmental disorders.  It is interesting, but does not really have any obvious therapeutic implications.  One point I will highlight in this current post is the following:-

ASD is not associated with systemic differences in global DNA methylation



What this means is that, as far as one key type of epigenetics is concerned, autism is not characterized by too many or too few epigenetic tags; the problem is that they are not all in the right place.  Many alternative therapies in autism are rather simplistic.  It is not a case of too much methylation, or too little.

In the twin study the ASD Twin and his unaffected sibling has almost the same amount of total DNA methylation.


































Tuesday 3 February 2015

Autism & Schizophrenia - Histamine degradation via HMT (requiring SAMe) and via DAO

Today’s post is a little complicated because it links together various issues ranging from food allergies to severe headaches, brain inflammation to arthritis.

The common link here is histamine, which has been covered at length on this blog.  You may recall that the H1 histamine receptor is the one associated with hay fever, H2 is expressed in the intestines and is involved in regulating acidity levels, H3 is mainly found in the central nervous system (CNS).

The Histamine H4 receptor has been shown to be involved in mediating eosinophil shape change and mast cell chemotaxis.

Here is the full paper, for those interested in mast cells:-


In addition to all these receptors, histamine causes an increase in the pro-inflammatory cytokine IL-6.  IL-6 is elevated in autism and many other inflammatory conditions ranging from arthritis to traumatic brain injury (TBI). 

One of interesting interventions in this post is SAMe (S-Adenosyl methionine )and its precursor L-methionine.  We will see why a deficit of SAMe causes a problem when the body tries to degrade/deactivate histamine.

We will also see in a later post that the level of SAMe in the body modulates the release anti-inflammatory cytokines like IL-10 and IL-35.  Here is one link, for now.


5. Higher expression of IL-35 could be induced by higher hypomethylation status in tissues

Previous reports showed that epigenetic mechanisms, including methylation and demethylation, control T helper cell differentiation and cytokine generation [41]. As we discussed in our recent review [42], the ratio of cellular methylation donor S-adenosylmethionine (SAM) levels over S-adenosylhomocysteine (SAH) levels is an important metabolic indicator of cellular methylation status [43], [44]. A higher SAM/SAH ratio suggests a higher methylation status than normal (hypermethylation) whereas a lower SAM/SAH ratio indicates a lower methylation status than normal (hypomethylation).  A previous report showed that feeding rats with SAM, a methyl donor, inhibits the expression of TGF-βR1 and TGF-βR2 [45], suggesting that intracellular global methylation status regulates anti-inflammatory cytokine signaling.  … Cont/


Interestingly, I found that for decades SAMe  has been a mainstream drug therapy used in Italy to treat arthritis.
    

Histamine degradation

In mammals, histamine is metabolized by two major pathways: N(tau)-methylation via histamine N-methyltransferase (HMT) and oxidative deamination via diamine oxidase (DAO).

HMT and uses S-adenosyl-L-methionine (SAMe) as the methyl donor.  If SAMe is lacking HMT cannot degrade histamine.

In the brain, the neurotransmitter activity of histamine is controlled by N(tau)-methylation.  It is disputed whether diamine oxidase is found in the central nervous system.  Some sources say it is not, but other studies specifically measure DAO levels in the brain, finding them elevated in schizophrenia.

A common genetic polymorphism affects the activity levels of HMT in red blood cells.  This can be tested for.

People with low levels of DAO will not be able to degrade histamine in their body nor, it appears to me, in the brain.

People with low levels of SAMe will not be able to degrade histamine as they should, that has crossed the BBB (blood brain barrier).  Those same low levels of SAMe will have raised the inflammatory cytokines and reduced the anti-inflammatory cytokines.


Methionine metabolism


I am always very wary when I see charts like the one below.  Often they are used to justify all kinds of strange ideas.  So the following methionine description is just a cut and paste from Wikipedia.

If anything goes wrong in this metabolism, you might indeed expect strange things to happen.  The ratio of SAMe/SAH is measurable  and tends to be markedly low in people with ASD.  This why DAN doctors use vitamin B12 injections, other B vitamins and other exotic sounding “supplements”.

Metabolic biomarkers of increased oxidative stress and impairedmethylation capacity in children with autism




Methionine is an essential amino acid that must be provided by dietary intake of proteins or methyl donors (choline and betaine found in beef, eggs and some vegetables). Assimilated methionine is transformed in S-adenosyl methionine (SAM) which is a key metabolite for polyamine synthesis, e.g. spermidine, and cysteine formation (see the figure on the right). Methionine breakdown products are also recycled back into methionine by homocysteine remethylation and methylthioadenosine (MTA) conversion (see the figure on the right). Vitamins B6, B12, folic acid and choline are essential cofactors for these reactions. SAM is the substrate for methylation reactions catalyzed by DNA, RNA and protein methyltransferases.

The products of these reactions are methylated DNA, RNA or proteins and S-adenosylhomocysteine (SAH). SAH has a negative feedback on its own production as an inhibitor of methyltransferase enzymes. Therefore SAM:SAH ratio directly regulates cellular methylation, whereas levels of vitamins B6, B12, folic acid and choline regulates indirectly the methylation state via the methionine metabolism cycle.[44][45] A near ubiquitous feature of cancer is a maladaption of the methionine metabolic pathway in response to genetic or environmental conditions resulting in depletion of SAM and/or SAM-dependent methylation. Whether it is deficiency in enzymes such as methylthioadenosine phosphorylase, methionine-dependency of cancer cells, high levels of polyamine synthesis in cancer, or induction of cancer through a diet deprived of extrinsic methyl donors or enhanced in methylation inhibitors, tumor formation is strongly correlated with a decrease in levels of SAM in mice, rats and humans.[46][47]







Low levels of SAMe do seem to cause problems in some people and it is straightforward to increase it.  You can either give extra SAMe, which is expensive, or L-methionine, which is cheap.

Interestingly, L-methionine is used at Johns Hopkins to treat autism and apparently is particularly effective at increasing speech.

If L-methionine was effective it could be for reasons including:-

·        cellular methylation was dysfunction
·        histamine in the brain had been elevated
·        the level of pro/anti-inflammatory cytokines had been out of balance 

Here are some examples of the use of SAMe (methionine)




In its native form, SAMe is labile and degrades rapidly. However, several patents for stable salts of SAMe have been granted. Among them, toluenedisulfonate and 1,4-butanedisulfonate forms have been chosen for pharmaceutical development, and as a result, preclinical and clinical studies have been performed. Numerous studies over the past 2 decades have shown that SAMe is effective in the treatment of depression (46), osteoarthritis (78), and liver disease (911). Moreover, SAMe has a very favorable side-effect profile, comparable with that of placebos. Thus, SAMe offers considerable advantages as an alternative to standard medications.

Depression
Clinical studies performed as early as 1973 indicated that SAMe had antidepressant effects (38). Over the next 2 decades, the efficacy of SAMe in treating depressive disorders was confirmed in > 40 clinical trials. Several review articles that summarize these studies were published in 1988 (4), 1989 (5), 1994 (6), and 2000 (12). In a meta-analysis, Bressa (6) reviewed 25 controlled trials including a total of 791 patients. The outcome of this analysis showed that SAMe had a significantly greater response rate than did placebo and was comparable to tricyclic antidepressants. Brown et al (12) summarized the literature on the use of SAMe in depressive disorders up to the time of publication in 2000; they reported that SAMe had been studied in 16 open, uncontrolled trials (660 patients); 13 randomized, double-blind, placebo-controlled trials (537 patients); and 19 controlled trials comparing SAMe with other antidepressants (1134 patients). Significant antidepressant effects were observed in all 16 open trials. In 18 controlled trials, SAMe was as effective as was impramine, chlorimipramine, nomifensine, and minaprine. An important observation from these studies is that SAMe had far fewer side effects than did standard medications.
Neurologic disorders
Several studies indicate that a CNS methyl group deficiency may play a role in the etiology of Alzheimer disease (AD). Reduced SAMe concentrations were found in CSF (34) and in several different brain regions (51) of patients with AD. In addition, reduced phosphatidylcholine concentrations were found in postmortem brain tissue from AD patients (52), and significant changes in brain phospholipids that are dependent on SAMe metabolism were detected in vivo with 31p magnetic resonance spectroscopy in the early stages of AD (53). Deficiencies of folate and vitamin B-12 are common in the elderly (39, 40) and can lead to decreased CNS SAMe concentrations. Several studies indicate that elevated blood homocysteine concentrations, considered to be a marker for folate deficiency, vitamin B-12 deficiency, and impaired methylation, may be a risk factor for AD (5456). It is therefore important to note that preliminary studies using either SAMe (57) or alternative methyl group donors [such as betaine (58) or folate and vitamin B-12 (59, 60)] can improve measures of cognitive function. These treatments may be able to restore methyl group metabolism and normalize blood homocysteine concentrations. Reduced SAMe concentrations in CSF were also reported in patients with subacute combined degeneration of the spinal cord resulting from folate or vitamin B-12 deficiency (39) and in children with inborn errors of the methyl-transfer pathway who had demyelination (61). In these cases, treatment with methyl-group donors such as SAMe, methyltetrahydrofolate, betaine, and methionine was associated with remyelination and a clinical response (61).

Lancet. 1991 Dec 21-28;338(8782-8783):1550-4.

Association of demyelination with deficiency of cerebrospinal-fluid S-adenosylmethionine in inborn errors of methyl-transfer pathway.

We have shown that demyelination is associated with cerebrospinal-fluid S-adenosylmethionine deficiency and that restoration of S-adenosylmethionine is associated with remyelination.


Remyelination is also interesting.  Damage to the critical myelin layer has been suggested to occur with mitochondrial disease.  Most young people with autism show signs of mitochondrial disease (based on post mortem samples) but not old people with autism.

Demyelination is the loss of the myelin sheath insulating the nerves, and is the hallmark of some neurodegenerative autoimmune diseases, including multiple sclerosis.


Liver disease
The potential benefit of SAMe in treating liver disease stems from several important aspects of SAMe metabolism. In mammals, as much as 80% of the methionine in the liver is converted into SAMe (23). Hepatic glutathione, which is dependent on methionine and SAMe metabolism, is one of the principal antioxidants involved in hepatic detoxification. Studies have shown that abnormal SAMe synthesis is associated with chronic liver disease, regardless of its etiology. Early studies indicated that patients with liver disease are unable to metabolize methionine, resulting in elevated blood concentrations (67). Subsequent studies in patients with liver disease showed that the defect resulted from decreased activity of a liver-specific isoenzyme, MAT I/III; this defect effectively blocks the conversion of methionine to SAMe (68). Several well-designed experimental studies indicated that MAT I/III is regulated by cellular concentrations of both nitric oxide and glutathione. Thus, increased nitric oxide concentrations and decreased glutathione concentrations were shown to inhibit MAT I/III via mechanisms involving increased S-nitrosylation and free radical damage to the enzyme protein (69, 70). Experimental studies and clinical trials showed that parenteral and oral SAMe administration can increase glutathione concentrations in red blood cells (71) and in hepatic tissue (72, 73) and can effectively replenish depleted glutathione pools in patients with liver disease. The literature on the clinical potential of SAMe in the treatment of liver disease (including cholestasis, hepatitis, and cirrhosis) has been the subject of several review articles (911, 74, 75).
  
Osteoarthritis
The potential benefit of SAMe in treating osteoarthritis was discovered when patients enrolled in clinical trials of SAMe for depression reported marked improvement in their osteoarthritis symptoms (76). Nine clinical trials in Europe (77) and 1 in the United States (7) with a total of > 22 000 participants have confirmed the therapeutic activity of SAMe against osteoarthritis. SAMe has effects similar to those of the nonsteroidal anti-inflammatory drugs, but its tolerability is higher.
  

Back to DAO

I think we have established the one mechanism for histamine degradation has useful pointers for those interested in autism; now it is time to look at the other one.

D-amino acid oxidase (DAAO; also DAO, OXDA, DAMOX) is an enzyme. Its function is to oxidize D-amino acids to the corresponding imino acids, producing ammonia and hydrogen peroxide.

Recently, mammalian D-amino acid oxidase has been connected to the brain D-serine metabolism and to the regulation of the glutamatergic neurotransmission. In a postmortem study, the activity of DAAO was found to be two-fold higher in schizophrenia.
DAAO is a candidate susceptibility gene and may play a role in the glutamatergic mechanisms of schizophrenia.  Risperidone and sodium benzoate are inhibitors of DAAO.


Abstract

We review the role of two susceptibility genes; G72 and DAAO in glutamate neurotransmission and the aetiology of schizophrenia. The gene product of G72 is an activator of DAAO (D-amino acid oxidase), which is the only enzyme oxidising D-serine. D-serine is an important co-agonist for the NMDA glutamate receptor and plays a role in neuronal migration and cell death. Studies of D-serine revealed lower serum levels in schizophrenia patients as compared to healthy controls. Furthermore, administration of D-serine as add-on medication reduced the symptoms of schizophrenia. The underlying mechanism of the involvement of G72 and DAAO in schizophrenia is probably based on decreased levels of D-serine and decreased NMDA receptor functioning in patients. The involvement of this gene is therefore indirect support for the glutamate dysfunction hypothesis in schizophrenia.

Abstract
D-serine has been shown to be a major endogenous coagonist of the N-methyl D-aspartate (NMDA) type of glutamate receptors. Accumulating evidence suggests that NMDA receptor hypofunction contributes to the symptomatic features of schizophrenia. d-serine degradation can be mediated by the enzyme d-amino acid oxidase (DAAO). An involvement of d-serine in the etiology of schizophrenia is suggested by the association of the disease with single nucleotide polymorphisms in the DAAO and its regulator (G72). The present study aims to further elucidate whether the DAAO activity is altered in schizophrenia. Specific DAAO activity was measured in postmortem cortex samples of bipolar disorder, major depression and schizophrenia patients, and normal controls (n=15 per group). The mean DAAO activity was two-fold higher in the schizophrenia patients group compared with the control group. There was no correlation between DAAO activity and age, age of onset, duration of disease, pH of the tissue and tissue storage time and no effect of gender, cause of death and history of alcohol and substance abuse. The group of neuroleptics users (including bipolar disorder patients) showed significantly higher D-amino acid oxidase activity. However, there was no correlation between the cumulative life-time antipsychotic usage and D-amino acid oxidase levels. In mice, either chronic exposure to antipsychotics or acute administration of the NMDA receptor blocker MK-801, did not change d-amino acid oxidase activity. These findings provide indications that D-serine availability in the nervous system may be altered in schizophrenia because of increased D-amino acid degradation by DAAO.


Abstract
We examined the association of autism spectrum disorders (ASD) with polymorphisms in the DAO and DAOA genes. The sample comprised 57 children with ASD, 47 complete trios, and 83 healthy controls in Korea. Although the transmission disequilibrium test showed no association, a population-based case-control study showed significant associations between the rs3918346 and rs3825251 SNPs of the DAO gene and boys with ASD.


DAO as a target for the treatment of schizophrenia

As noted above, both D-serine and D-alanine show some effectiveness as add-on treatment in schizophrenia, in particular for the amelioration of negative and possibly cognitive symptoms. There are also comparable approaches and data regarding glycine augmentation. Since enzymes represent viable drug targets, DAO is receiving attention as a potential alternative therapeutic means to enhance NMDAR function in schizophrenia. The fact that DAO activity appears to be increased in schizophrenia provides another reason to propose that its inhibition might be beneficial. It is also intriguing that the original antipsychotic, chlorpromazine, was shown to be a DAO inhibitor in vitro over fifty years ago,2 confirmed recently and also found to apply to risperidone; whether these observations are relevant clinically are unknown, but they do provide a precedent for the potential therapeutic benefits of selective DAO inhibitors.
To date there have been no clinical trials of DAO inhibitors in schizophrenia, but several preclinical studies which, although findings remain preliminary, show that inactivation of DAO, either in ddY/DAO- mice or after pharmacological DAO inhibition in rats and mice, produces behavioural, electrophysiological and neurochemical effects suggestive of a pro-cognitive profile (Table 4). The Table includes the three DAO inhibitors for which functional data have been published thus far: AS057278,10 CBIO,201,203 and Compound 8.202 Several other small molecule DAO inhibitors have been patented but their behavioural effects have yet to be reported.62,204

Conclusions and future directions

DAO, as the enzyme which degrades the NMDAR co-agonist D-serine, has the potential to modulate NMDAR function and to contribute to NMDAR hypofunction in schizophrenia. Both genetic and biochemical data support an involvement of DAO in the disorder, however the processes involved are difficult to interpret. This is due to the many questions left unanswered concerning the neurobiology of DAO and its physiological roles. Notably there is still much that is unclear as to its localization and activity within the brain, and its spatial and functional relationships with its substrates. In addition, D-serine and thus DAO may have roles other than NMDAR modulation, whilst other DAO substrates, especially D-alanine, may also be relevant to any involvement of DAO in schizophrenia. Similarly, although recent preclinical data hint at potential therapeutic benefits of DAO inhibitors, extensive further study is required to establish their efficacy, tolerability, and mechanism.


Many drugs act as DAO inhibitors to a limited degree, even though this is not their intended mode of action.

We have heard about Sodium benzoate and Risperidone, but there are many others.


           

Results

Chloroquine and clavulanic acid showed greatest inhibition potential on diamine oxidase (> 90%). Cimetidine and verapamil showed inhibition of about 50%.
Moderate influence on DAO was caused by isoniazid and metamizole, acetyl cysteine and amitriptyline
(>20%). Diclofenac, metoclopramide, suxamethonium and thiamine have very low inhibition potential (<20%).  Interestingly cyclophosphamide and ibuprofen displayed no effect on DAO.

Conclusion

Since even levels of about 30% inhibition may be critical, most of the observed substances, can be designated as DAO inhibitors. Other drug components than active ingredients did not affect DAO activity or its interaction with a specific drug.


Note that cimetidine (Tagamet), a histamine H2-receptor antagonist drug used in promoting the healing of active stomach and duodenal ulcers.  Verapamil is in my “Polypill” and is a potent mast cell stabilizer.   Is this link back to histamine a coincidence?  I think not.









Conclusion

The experts are yet to conclude much, but it does seem that SAMe levels are low in autism and brain DAO levels are high schizophrenia (adult onset autism).  In Korea, DAO was shown to be dysfunction in autism.

It seems that, by coincidence, Risperidone happens to be an inhibitor of DAO and this indeed accounts for some its side effects.  Risperidone has actions at several 5-HT (serotonin) receptor subtypes, Dopamine receptors, Alpha α1/2 adrenergic receptors and even H1 histamine receptors.  Risperidone seems to be drug of last resort.

There are no selective DAO inhibitors currently in use.

We did see that two old drugs Tagamet and Verapamil are potent DAO inhibitors in vitro.

This suggest to me that while sodium benzoate has been trialed “successfully” in schizophrenia, perhaps it would be worth comparing the effect of Tagamet and Verapamil.

When it comes to autism/schizophrenia, it would seem that in some people one or more of the following might be helpful:-

·        Sodium benzoate, or cinnamon a precursor
·        Tagamet the H2 antihistamine, already used by some people with mastocytosis
 ·        Verapamil, the calcium channel blocker that actually does much more
·        SAMe, or L-methionine a precursor.