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Friday 30 March 2018

Autism and Aspartic Acid - N-acetylaspartate (NAA), AGC1, CREB and EAAT1/2



Aspartic acid is a metabolite of the sweetener Aspartame; not a surprise when you taste it

Today’s post is about the amino acid aspartic acid, which our reader Tyler has been supplementing. The short version of this post would answer the question should we all follow Tyler and supplement L-Aspartic acid? I guess the answer is that it is well worth a trial.
There is a great deal of complex science showing that aspartic acid is dysfunctional in autism, but we cannot prove that supplemental aspartic acid corrects these dysfunctions. Supplemental aspartic acid will have other effects and it may be these that are beneficial in Tyler’s case and possibly yours.
Correcting the core dysfunctions relating to Aspartic acid would be very helpful, if it were possible.  You could write an entire book just about the contents of today’s post, so for most people understanding all of it will take quite some time.

You can read Tyler’s many comments on this subject by entering the following into google.

       aspartate "site:epiphanyasd.blogspot.com"

This gives you a list of those posts, where he left comments on aspartate.
Then press the “crtl” key and the “f” key and this will find the mentions of “aspartate” in that post and its comments

Here are some comments.





Malate-Aspartate Shuttle
I wonder if the reason supplemental aspartic acid may help some people comes from something called the Malate-Aspartate Shuttle.  Now this does get complicated, but is links together many things we already know:-
·        Disturbed calcium channel signaling and excess physical calcium in autistic brains

·        Abnormal myelination

·        Oxidative stress

·        Mitochondrial abnormalities

·        Microglial activation

·        Disturbed BDNF, VEGF, CRH etc.

There is also a very interesting potential protective genetic variation (SLC25A12) that appears to protect some siblings from developing autism.
It appears that the excessive level of (cytosolic) Ca2+ increases the expression of AGC1 (the mitochondrial aspartate/ glutamate carrier number 1) via CREB (cAMP response element-binding protein).
CREB is a cellular transcription factor that regulates numerous genes including c-fos, BDNF, tyrosine hydroxylase, numerous neuropeptides (somatostatin, enkephalin, VGF, corticotropin-releasing hormone CRH, etc.), and genes involved in the circadian clock (PER1, PER2). 

For instance, multiple stimuli induce the phosphorylation of the cyclic AMP-responsive element-binding protein (CREB) at Ser-133, which recruits the CREB-binding protein (CBP) to gene promoters. CBP catalyses the acetylation of histones, leading to changes in chromatin structure that facilitate the recruitment of RNA polymerase II and the activation of gene-transcription programs that promote synapse development12,15.
In addition to stimulating CREB-dependent gene transcription, neuronal activity-dependent calcium influx into neurons triggers the dephosphorylation of the transcription factor myocyte enhancer factor 2 (MEF2) by calcineurin, disrupting the association of MEF2 with histone deacetylases and leading to the recruitment of CBP and the stimulation of gene transcription26. 


Neuronal activity that functions through CREB, MEF2 and multiple other activity-regulated transcription factors — including SRF, Fos27 and NPAS428 — induces the transcription of the genes encoding proteins that function directly at synapses, including Bdnf, Arc and Ube3A12,15.
For example, activity-regulated MEF2 by activating Arc suppresses the number of excitatory synapses26,29, whereas CREB and NPAS4, by activating Bdnf transcription, control the number of inhibitory synapses that form on excitatory neurons28,30.

CREB is down regulated in Alzheimer’s.
CREB proteins are activated by phosphorylation from various kinases, including PKA.
A number of existing drugs can raise or lower PKA levels in the body. 
You would want to raise PKA in Alzheimer’s and some autism.
If AGC1 is over-activated this could be corrected using a PKA inhibitor, but this is easier said than done.  PKA does very many different things.
PDE4 inhibitors used to treat asthma and COPD (Ibudilast and Daxas) raise PKA.
PDE4 inhibitors also reduce microglial activation. See the reference to gliosis below.
As our reader Nat knows, Ca2+ ions are the source of numerous problems in autism.  The route problem seems to be too much calcium released from the stores within each cell (endoplasmic reticulum) and/or faulty voltage gated calcium channels allowing Ca2+ to enter from outside the cell.
The malate-aspartate shuttle “functions to move reducing equivalents into the mitochondrial matrix in the form of malate, whereas the major mitochondrial output through this complex is aspartate”. Specifically, AGC1 moves cytoplasmic glutamate into mitochondria, while moving mitochondrially synthesized aspartate out. Without AGC1, brain mitochondrial glutamate import and aspartate export are crippled. Studies with AGC1 knockout mice showed a dramatic drop in brain aspartate levels, with a concomitant reduction in NAA (N-acetylaspartate) synthesis.
AGC1 activation increases mitochondrial metabolism and oxidative stress
Reduction in N-acetylaspartate (NAA) synthesis causes hypomyelination.
N-acetylaspartate (NAA) is synthesized from acetyl CoA and aspartate. NAA is very important in the brain; too little NAA causes reduced myelination (hypomyelination) which is a feature of autism. Studies show that NAA is reduced in the brains of young people with autism, but interestingly not in older people with autism.
In autism we expect to see activated AGC1 and reduced NAA
It does look like NAA is something you do not want to be deficient in, but you also do not want too much. NAA is synthesized from acetyl CoA and aspartate and without going into details you might just say, why not just add some extra aspartate, which you can buy as an OTC powder. 
There is a very more complex explanation, which comes back to aberrant calcium signaling being the demon of most autism. That would suggest:-
“Pharmacological treatments able to modulate extracellular Ca2+ entry, intracellular Ca2+ release from the endoplasmic reticulum or putative upstream immune mechanisms affecting either or both the pathways are, at least in principle, already available”

Overexpression of mitochondrial aspartate/glutamate carrier AGC1/ aralar1 (annoyingly, some research use the term  Aralar1 while others use AGC1) encoded by encoded by the SLC25A12, caused by excess calcium Ca2+ either from intracellular release from stores in the endoplasmic reticulum or extracellular Ca2+. Recall the post about Gargus’ theory that a key nexus in autism is the IPR3 receptor,  in the Endoplasmic Reticulum within each cell. He believes this is core defect in much autism.

Canavan disease is a rare condition caused by far too much NAA, a genetic error prevents the normal breakdown of NAA and this disrupts myelination leading to death in childhood.

Also part of the malate-aspartate shuttle we have GLutamate ASpartate Transporter (GLAST) or Excitatory Amino Acid Transporter 1 (EAAT1). We know that both glutamate transporters EAAT1 and EAAT2 are over-expressed in the cerebellum of post-mortem tissue from autism patients.  Because EAAT expression is controlled in part by the extracellular concentration of glutamate, it is possible that the EAAT overexpression is due to the increased glutamate concentration seen in plasma and spectroscopic studies.

Glutamate & Glutamine
Since I have mentioned glutamate, note that numerous studies show high levels of glutamate but low levels of its precursor glutamine in autism. Gliosis is one possible explanation. This connects with those activated microglia which are another recurring feature of autism:-


“An increase in gliosis, which is characterized by enhanced activation of astrocytes and microglia, has been observed in the brains of individuals with autism. Interestingly, Ortinski et al. have reported that activated astrocytes downregulate the expression of glutamine synthetase, whereby glutamate is converted into glutamine, which in turn results in reduced glutamine coupled with elevated glutamate. In addition, glutaminase, another enzyme related to glutamate/glutamine metabolism via its conversion of glutamine into glutamate, has been shown to be upregulated in activated microglia. Thus, it is tempting to assume that the process of gliosis generation may be related to the etiology of autism, as mediated by activated astrocytes and/or activated microglia, which may disturb the regulation of certain types of enzymes and thereby alter the metabolism of glutamate/glutamine. Taken together with previous findings, our results demonstrating glutamate/glutamine abnormalities in the plasma of individuals with autism may be indicative of a gliosis process in the autistic brain.

Amino Acids and the BBB
Amino acids are present inside the brain but some do not cross easily across the BBB. If they do not cross the BBB then taking a supplement is not going to help much.
This point is quite relevant because one study showed that arginine deprivation plays a key role in Alzheimer’s, but the mass media pointed out that taking arginine supplements is not going to help. Had they read this blog they would have known that if you want to increase arginine in the brain, you want to take citrulline. So citrulline for Alzheimer’s?  well worth investigating.  
The amino acid transporters that control the level in the brain are themselves disturbed in autism. So it may be that the level of many amino acids in autistic brains is disturbed, regardless of diet and what the blood/urine levels indicate.

Amino acid gradients between brain and plasma





Amino acid concentrations in plasma and brain. The plasma and CSF concentrations were grouped and the CSF-to-plasma ratio expressed as percent of the plasma. CSF concentrations are assumed to approximate brain ECF (54,102). With the exception of glutamine, the concentrations of all AAs in the ECF are much lower than the concentrations of AAs in plasma.


The concentrations of all naturally occurring AAs in the cerebral spinal fluid (CSF) (presumably similar to the extracellular fluid (ECF) of the brain), with the exception of glutamine, are 10% or less than the plasma concentrations (Fig. 4) (54). This situation cannot be explained by the consumption of AAs by brain because the arteriovenous differences across brain of most AAs are imperceptible (55–57), as are the arteriovenous differences of ammonia (NH4+), a byproduct of AA catabolism (58). These observations indicate that AAs leave the brain against a concentration gradient. From this it may be concluded that active (e.g., Na+-dependent) systems on the abluminal membrane have an important role in maintaining both homeostasis of brain AA content as well as the lower concentration in the extracellular fluid. Based on similar observations Bradbury wrote “there is a strong indirect argument in favor of the hypothesis that most AA must be moved against a concentration gradient from interstitial fluid to blood” (34).



The present view of the BBB is that cerebral endothelial cells participate actively in regulating the composition of brain extracellular fluid and the AA content of the brain. The luminal and abluminal membranes work in a complementary fashion with the Na+-dependent transport of AAs occurring at the abluminal membrane, and with facilitative transport at the luminal membrane, or, in the case of LNAAs, at both membranes (97).

Although the BBB determines the availability and therefore the brain content of essential AAs, astrocytes and neurons participate in maintaining the extracellular concentrations. Astrocytes and neurons have Na+-dependent transport systems capable of transporting NAAs and acidic AAs (98–100). These systems are actively involved in regulating AA concentrations in ECF and are especially important in the maintenance of low concentrations of neurotransmitter AAs such as glutamate, aspartate, and glycine. On the other hand, it now seems clear that the BBB also participates in the active regulation of brain ECF composition, and the abluminal membrane is especially important in this role.

Characteristics of L-citrulline transport through blood-brain barrier in the brain capillary endothelial cellline (TR-BBB cells)


Background

L-Citrulline is a neutral amino acid and a major precursor of L-arginine in the nitric oxide (NO) cycle. Recently it has been reported that L-citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, L-citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of L-citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB. 

Conclusions

Our results suggest that transport of L-citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.
Our results demonstrated that L-citrulline transport might be mainly mediated by LAT1 in TR-BBB cells. Understanding the transport characteristics of L-citrulline to the brain through BBB might contribute to the transport strategy for L-citrulline as a potential therapeutic agent for cerebrovascular diseases such as brain ischemia. 



Disturbed amino acid transporters and levels in blood/urine      

Since we know that the amino acid transporters that carry amino acids across the blood brain barrier are disturbed in autism what is the relevance of blood/urine tests and standard reference levels? Unless you lest amino acid levels in spinal fluid (i.e. within the central nervous system) do lab tests really help? You certainly need to be aware of their limitations.
So as you can see even the summary is highly complicated.
Very many things associated with aspartate are messed up in autism and Tyler finds his son benefits from L-aspartate supplementation.
We saw in an earlier post that NT girls had three times the level of excreted aspartic acid than NT boys and that people with autism had very low levels of excreted aspartic acid.
If you could fix the underlying problem with elevated Ca2+ as proposed by Gargus, you would solve the AGC1/NAA problem. It may be that by supplementing L-aspartate you do have an effect on the  malate-aspartate shuttle, even though there is no solid understanding of exactly how this occurs.







IP3R controls the release of calcium from the ER (Endoplasmic Reticulum inside each cell). In the brain, calcium is used to communicate information within and between neurons, and it activates a host of other cell functions, including ones regulating learning and memory, neuronal excitability and neurotransmitter release – areas known to be dysfunctional in ASD. It also causes the release of Protein Kinase C which then acts to change the function of numerous proteins (via phosphorylation) and trigger a series of signaling cascades.

Rapid progress in our understanding of macrostructural abnormalities in autism spectrum disorders (ASD) has occurred in recent years. However, the relationship between the integrity of neural tissue and neural function has not been previously investigated. Single-voxel proton magnetic resonance spectroscopy and functional magnetic resonance imaging of an executive functioning task was obtained in 13 high functioning adolescents and adults with ASD and 13 age-matched controls. The ASD group showed significant reductions in N-acetyl aspartate (NAA) in all brain regions combined and a specific reduction in left frontal cortex compared to controls. Regression analyses revealed a significant group interaction effect between frontal and cerebellar NAA. In addition, a significant positive semi-partial correlation between left frontal lobe NAA and frontal lobe functional activation was found in the ASD group. These findings suggest that widespread neuronal dysfunction is present in high functioning individuals with ASD. Hypothesized developmental links between frontal and cerebellar vermis neural abnormalities were supported, in that impaired neuronal functioning in the vermis was associated with impaired neuronal functioning in the frontal lobes in the ASD group. Furthermore, this study provided the first direct evidence of the relationship between abnormal functional activation in prefrontal cortex and neuronal dysfunction in ASD.
This study extended prior work in this area by establishing that neural abnormalities, which have been identified using 1H-MRS predominantly in younger and likely lower functioning autistic individuals), may also be present in a higher functioning broadly inclusive ASD sample. None of the individuals in our sample had IQs in the mentally retarded range and the sample included individuals with diagnoses in the less severe end of the autism spectrum; approximately 43% of the sample was diagnosed as PDD-NOS or Asperger’s disorder. 
Recent postmortem studies have identified neuroinflammation as a compelling potential pathological mechanism for reduced levels of NAA in individuals with ASD (Laurence and Fatemi, 2005; Vargas et al., 2005). Vargas and colleagues documented microglial activation in postmortem middle frontal gyrus, anterior cingulate, and cerebellar tissue, and in cerebral spinal fluid (CSF) of children and adults with autism. Laurence and Fatemi found increased glial fibrillary acidic protein in area 9, area 40 and the cerebellum in autism. Ongoing neuroinflammatory processes may produce alterations in brain tissue that would result in reduced NAA (secondary to cell loss or damage), as was observed in the current study. 
We found that high functioning individuals with ASD have reduced levels of NAA compared to age-matched controls. The most consistent area of abnormality was observed in the left middle frontal gyrus. In addition, the relationship between level of NAA in the frontal lobes and NAA in the cerebellar vermis differed between groups. These findings of biochemical alterations in ASD may reflect early brain growth dysregulation and ongoing neuroinflammatory processes. Reduced levels of NAA in ASD were also directly related to neurofunctional abnormalities observed in the FMRI study of executive functioning. This study is the first to directly link functional activation to neuronal integrity in ASD, and provides direct evidence that primary neuronal dysfunction, in addition to hypothesized aberrant neural connectivity leads to neurofunctional impairment in high functioning individuals with autism spectrum disorders. 


Atypical trajectory of brain growth in autism spectrum disorders (ASDs) has been recognized as a potential etiology of an atypical course of behavioral development. Numerous neuroimaging studies have focused on childhood to investigate atypical age-related change of brain structure and function, because it is a period of neuron and synapse maturation. Recent studies, however, have shown that the atypical age-related structural change of autistic brain expands beyond childhood and constitutes neural underpinnings for lifelong difficulty to behavioral adaptation. Thus, we examined effects of aging on neurochemical aspects of brain maturation using 3-T proton magnetic resonance spectroscopy (1H-MRS) with single voxel in the medial prefrontal cortex (PFC) in 24 adult men with non-medicated high-functioning ASDs and 25 age-, IQ- and parental-socioeconomic-background-matched men with typical development (TD). Multivariate analyses of covariance demonstrated significantly high N-acetylaspartate (NAA) level in the ASD subjects compared with the TD subjects (F=4.83, P=0.033). The low NAA level showed a significant positive correlation with advanced age in the TD group (r=−0.618, P=0.001), but was not evident among the ASD individuals (r=0.258, P=0.223). Fisher's r-to-z transformation showed a significant difference in the correlations between the ASD and TD groups (Z=−3.23, P=0.001), which indicated that the age–NAA relationship was significantly specific to people with TD. The current 1H-MRS study provided new evidence that atypical age-related change of neurochemical aspects of brain maturation in ASD individuals expands beyond childhood and persists during adulthood.



In conclusion, the present findings demonstrated an absence of typical age-related medial prefrontal NAA decrement in ASD individuals during adulthood. Such an atypical relationship between age and NAA levels might contribute to a significant NAA increase in the ASD subjects compared with the TD adults. Although future studies should examine potential localization of atypical age-related NAA change and longitudinal course of autistic NAA abnormality, the current study has provided a new suggestion with regard to a role of atypical age-related NAA changes in the pathophysiology of ASD.


An interesting way to increase NAA:-

Supplement 'boosts' brain power
"They were asked to be more active and cut down on unhealthy snacks and fizzy drinks. 

At the same time, they were given two capsules a day of the VegEPA supplement, which contains an omega-3 fatty acid called EPA.

Tests done at the end of the three-month study found the children showed an increase in reading age of well over a year, their handwriting became neater and more accurate and they paid more attention in class.

Brain scans which identified a chemical called N-Acetylaspartate (NAA) which is linked to the growth of nerve fibres in the brain also showed dramatic changes, said Professor Puri.

Although the children were encouraged to change their diet, there was no evidence they did this to any great extent, suggesting the improvements in the children were a result of the supplement.


Brain growth 

"In three months you might expect to see a small NAA increase.

"But we saw as much growth as you would normally see in three years.

"It was as if these were the brains of children three years older. It means you have more connections and greater density of nerve cells, in the same way a tree grows more branches."

The boys in the study showed the most improvement, he added.

Omega-3 fatty acids are found naturally in oily fish such as mackerel, salmon, herring and tuna or seeds such as flax, pumpkin and hemp.

A systematic review of fish oil supplements in children published by the Food Standards Agency last year found there were too many inconsistencies in current evidence to come to any conclusion.

Professor Puri said he believed that it was EPA specifically which conferred the benefits which was why studies of fish oil supplements which also contain a fatty acid called DHA showed confusing results.

He is now planning to carry out a larger placebo-controlled study.

Professor Robert Grimble, professor of nutrition at the University of Southampton said it was vital that larger studies were done to clarify the issue.

"My view is we can't come to any clear conclusion until a proper trial is done.

"These small bits of weak data just confuse the public. The FSA looked at this very carefully and I wouldn't contradict that until we have more evidence." 



N-Acetylaspartate (NAA) is employed as a non-invasive marker for neuronal health using proton magnetic resonance spectroscopy (MRS). This utility is afforded by the fact that NAA is one of the most concentrated brain metabolites and that it produces the largest peak in MRS scans of the healthy human brain. NAA levels in the brain are reduced proportionately to the degree of tissue damage after traumatic brain injury (TBI) and the reductions parallel the reductions in ATP levels. Because NAA is the most concentrated acetylated metabolite in the brain, we have hypothesized that NAA acts in part as an extensive reservoir of acetate for acetyl coenzyme A synthesis. Therefore, the loss of NAA after TBI impairs acetyl coenzyme A dependent functions including energy derivation, lipid synthesis, and protein acetylation reactions in distinct ways in different cell populations. The enzymes involved in synthesizing and metabolizing NAA are predominantly expressed in neurons and oligodendrocytes, respectively, and therefore some proportion of NAA must be transferred between cell types before the acetate can be liberated, converted to acetyl coenzyme A and utilized. Studies have indicated that glucose metabolism in neurons is reduced, but that acetate metabolism in astrocytes is increased following TBI, possibly reflecting an increased role for non-glucose energy sources in response to injury. NAA can provide additional acetate for intercellular metabolite trafficking to maintain acetyl CoA levels after injury. Here we explore changes in NAA, acetate, and acetyl coenzyme A metabolism in response to brain injury
N-acetylaspartate (NAA) is one of the most abundant brain metabolites and is highly concentrated in neurons, but it remains to be determined why neurons synthesize so much of this particular acetylated amino acid. Early research implicated NAA in lipid synthesis in the brain, especially during postnatal myelination.
NAA is synthesized from acetyl CoA and aspartate, and because of the exceptionally high concentration in the human brain (~10 mM) some proportion of acetyl CoA must be utilized to maintain NAA levels 
The primary mitochondrial aspartate-glutamate carrier expressed in brain, heart, skeletal muscle, and several other tissues is known as aralar1 (Del Arco et al., 2002), which is part of a larger complex that comprises the so-called mitochondrial malate-aspartate shuttle. The malate-aspartate shuttle functions to move reducing equivalents into the mitochondrial matrix in the form of malate, whereas the major mitochondrial output through this complex is aspartate. Specifically, aralar1 moves cytoplasmic glutamate into mitochondria, while moving mitochondrially synthesized aspartate out. Without aralar1, brain mitochondrial glutamate import and aspartate export are crippled. Studies with aralar1 knockout mice showed a dramatic drop in brain aspartate levels, with a concomitant reduction in NAA synthesis
The connection between a lack of aralar1 expression and dramatically reduced NAA synthesis has at least two potential explanations. First, as suggested by Jalil et al. (2005) it could be due to the lack of mitochondrial aspartate output, which in turn would limit substrate availability for microsomal Asp-NAT to synthesize NAA. The other possible explanation is that the lack of glutamate uptake into neuronal mitochondria prevents intramitochondrial aspartate synthesis via the aspartate aminotransferase reaction which converts glutamate and oxaloacetate into α-ketoglutarate and aspartate. In this case the lack of intramitochondrial aspartate synthesis would be the limiting factor in NAA synthesis, leading to decreases in NAA levels. It is also possible that both of these mechanisms are responsible for the large drop in NAA levels observed in aralar1-deficient mice. 
One of the more interesting outcomes of aralar1 deficiency in addition to the large decrease in brain NAA levels is hypomyelination (Jalil et al., 2005; Wibom et al., 2009). The hypomyelination is hypothesized to result from the lack of availability of NAA and this conclusion is supported by the fact that galactocerebrosides, one of the myelin lipid classes that are reduced in Canavan disease 

The more NAA the more creative? 

A broadly accepted definition of creativity refers to the production of something both novel and useful within a given social context. Studies of patients with neurological and psychiatric disorders and neuroimaging studies of healthy controls have each drawn attention to frontal and temporal lobe contributions to creativity. Based on previous magnetic resonance (MR) spectroscopy studies demonstrating relationships between cognitive ability and concentrations of N-acetyl-aspartate (NAA), a common neurometabolite, we hypothesized that NAA assessed in gray and white matter (from a supraventricular slab) would relate to laboratory measures of creativity. MR imaging and divergent thinking measures were obtained in a cohort of 56 healthy controls. Independent judges ranked the creative products of each participant, from which a “Composite Creativity Index” (CCI) was created. Different patterns of correlations between NAA and CCI were found in higher verbal ability versus lower verbal ability participants, providing neurobiological support for a critical “threshold” regarding the relationship between intelligence and creativity. To our knowledge, this is the first report assessing the relationship between brain chemistry and creative cognition, as measured with divergent thinking, in a cohort comprised exclusively of normal, healthy participants.
Exactly how does variation in NAA concentration affect brain activity in normals? A rapidly growing literature links NAA to intelligence, working memory, attention, and memory both in health and disease (Ross and Sachdev, 2004), but the mechanisms underlying these relationships remain elusive. In the adult brain, there is substantial evidence that NAA is a marker of mitochondrial functioning, is involved in myelin lipid turnover, participates in axonglial signaling, and may be involved in brain nitrogen balance (Moffett et al., 2007). Substantial basic research has yet to unravel the complex role that NAA plays in higher cognitive functioning, including creativity, beyond mere correlation.

NAA is synthesized from acetyl CoA and aspartate, so you would not want to be short of either.





From your high school biology you may recall a process called aerobic respiration which is how you mitochondria convert Glucose (fuel) into ATP (energy).

As you can see one step in this process is the production of Acetyl CoA.

If you are breathing, you must be making Acetyl CoA.  So perhaps a little extra aspartic acid might help produce more NAA.

In extreme cases a lack of the mitochondrial carrier protein Aralar1 will causes very low levels of NAA. 

Canavan disease is a rare condition caused by too much NAA, a genetic error prevents the normal breakdown of NAA and this disrupts myelination leading to death in childhood.

Even after all that, we do not know for sure why supplementing Aspartate works for Tyler. Making more NAA would be a nice explanation but Aspartate does stimulate NMDA receptors.  


Autism is a severe developmental disorder, whose pathogenetic underpinnings are still largely unknown. Temporocortical gray matter from six matched patient-control pairs was used to perform post-mortem biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC), which participates in the aspartate/malate reduced nicotinamide adenine dinucleotide shuttle and is physiologically activated by calcium Ca2+. AGC transport rates were significantly higher in tissue homogenates from all six patients, including those with no history of seizures and with normal electroencephalograms prior to death. This increase was consistently blunted by the Ca2+ chelator ethylene glycol tetraacetic acid; neocortical Ca2+ levels were significantly higher in all six patients; no difference in AGC transport rates was found in isolated mitochondria from patients and controls following removal of the Ca2+-containing post mitochondrial supernatant. Expression of AGC1, the predominant AGC isoform in brain, and cytochrome c oxidase activity were both increased in autistic patients, indicating an activation of mitochondrial metabolism. Furthermore, oxidized mitochondrial proteins were markedly increased in four of the six patients. Variants of the AGC1-encoding SLC25A12 gene were neither correlated with AGC activation nor associated with autism-spectrum disorders in 309 simplex and 17 multiplex families, whereas some unaffected siblings may carry a protective gene variant. Therefore, excessive Ca2+ levels are responsible for boosting AGC activity, mitochondrial metabolism and, to a more variable degree, oxidative stress in autistic brains. AGC and altered Ca2+ homeostasis play a key interactive role in the cascade of signaling events leading to autism: their modulation could provide new preventive and therapeutic strategies.

Evidence linking altered energy metabolism to autistic disorder has been available for some time, including peripheral markers, such as increased plasma lactate levels, and rare instances of association between respiratory chain disorders and autism.  Interest in assessing the role of mitochondria

in this disorder has been revitalized by the association between autism and variants of the SLC25A12 gene, which encodes the predominant isoform of the mitochondrial aspartate (asp)/glutamate (glu) carrier (AGC) in brain.15,16 AGC belongs to a family of integral proteins that catalyze the transport of metabolites and cofactors across the inner mitochondrial membrane.17 In particular, AGC is important in energy metabolism

by transporting glutamate into mitochondria in exchange for matrix aspartate, a key regulatory step in the malate/aspartate reduced nicotinamide adenine dinucleotide (NADH) shuttle.18,19 Its two

isoforms, AGC1 and AGC2, also named aralar(1) and citrin, are encoded by the SLC25A12 and SLC25A13 genes, located on human chromosomes 2q24 and 7q21.3, respectively.19 AGC1 and AGC2 expression overlaps during early prenatal life, but diverges beginning in late gestation and into adulthood, with AGC1 predominantly expressed in the brain, heart and skeletal muscle, whereas AGC2 is mainly expressed in liver and kidney.20,21 In the CNS, AGC1 is highly expressed in neurons, whereas glial cells express both isoforms at much lower levels.20,21 Importantly, AGC activity is regulated by intracellular calcium (Ca2+) through four ‘EF-hand’ domains22 located at its N-terminus, hanging into the intermembrane

space.18,19 As Ca2+ concentrations in the mitochondrial intermembrane space and cytosol are in equilibrium, cytosolic Ca2+ can rapidly activate AGC transport, thereby increasing the NADH/NAD ratio in the mitochondrial matrix and consequently boosting electron flow through the respiratory

chain and adenosine triphosphate (ATP) generation by oxidative phosphorylation.18,19,23 Through this mechanism, AGC1 is important in the transduction of small Ca2+ signals to neuronal mitochondria.19 An

excessive amplitude and/or duration of Ca2+ spikes leading to AGC activation can, however, contribute to the formation of reactive oxygen species (ROS) and to oxidative stress.24 Genetic and/or environmental

factors could thus interfere with neuronal ATP production and with oxidative stress by affecting the AGC1 carrier, either directly or through Ca2+ homeostasis.



===================

Results

AGC activity is boosted by excessive Ca2+ levels in autistic brains

AGC activity, normalized by CS activity to adjust for differences in absolute mitochondria tissue content, displays a prominent threefold increase in neocortical homogenates from nonsyndromic autistic patients compared to matched controls in all six pairs (Wilcoxon’s test: Z=

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chelation by EGTA reduces asp/glu exchange rates to a much larger extent in patients than in controls (2.1- vs 0.35-fold, respectively), reducing case–control differences to only 36.1% (Figure 1a, middle). No difference between patients and controls is anymore detectable upon reconstitution of protein extracts from isolated mitochondria (P=0.17; Figure 1a, right). These results strongly point toward excessive Ca2+ concentrations as most likely responsible for increased asp/glu exchange rates in the brains of autistic patients.



AGC activation increases mitochondrial metabolism and oxidative stress

By increasing the availability of reducing equivalents in the mitochondrial matrix through AGC, enhanced cytosolic Ca2+ can be predicted to steadily boost mitochondrial metabolism and oxidative phosphorylation.18,19,23 Indeed, transcript amounts of the mitochondrial phosphate carrier (PiC), AGC1, and AGC2 (expressed at much lower levels compared to AGC1), are all increased (Supplementary Figure S2). Also COX activity is elevated to a similar extent in all six autistic patients compared to their matched controls (P<0 .05="" 3a="" figure="" span=""> 

No evidence of SLC25A12 gene contributions to autism vulnerability

Sequencing of the AGC1-encoding SLC25A12 cDNA and genomic DNA in these same six case–control pairs does not detect any nonsynonymous coding mutation
Figure 2 Calcium levels measured directly in the post mitochondrial supernatant by fluorimetry. (a) Calcium concentrations are significantly higher in the neocortical tissue of autism-spectrum disorder (ASD) patients compared to matched controls 

A SLC25A12 gene variant may confer protection in unaffected siblings

Single-marker analyses point toward the possible existence of a protective SLC25A12 gene variant preferentially transmitted from heterozygous parents to unaffected siblings of autistic patients.

The allele frequency of this haplotype is estimated at approximately 23–26% (Table 2b), making it a relatively common variant among unaffected siblings.
This study reports increased asp/glu exchange rates and significantly higher Ca2+ concentrations in postmortem neocortical tissue specimens of six nonsyndromic autistic patients compared to age-, sex- and PMI-matched controls. Altogether, our results strongly support excessive Ca2+ levels as primarily responsible for the observed activation of asp/glu exchange rates, whereas genetic contributions appear neither widespread nor necessary, at least in our postmortem and genetic samples.
Most importantly, direct measurements of neocortical Ca2+ concentrations clearly demonstrate a significant elevation of neocortical Ca2+ levels in all autistic patients compared to controls (Figure 2a)
Increased AGC transport rates, COX activities and Ca2+ levels consistently recorded in all six neocortical specimens from ASD patients crossvalidate each other, confirming the reliability and biological significance of these findings
In summary, altered Ca2+ homeostasis is the only factor shared by all autistic cortical tissue samples (Figure 2a), able to boost AGC activity,18,19,23 and previously linked to the pathogenesis of autism per se.
The existence of altered Ca2+ signaling in autism has been suggested in recent years by several lines of research.42 Gain-of-function mutations in the L-type voltage-gated Ca2+ channel Cav1.2 (CACNA1C) cause Timothy syndrome, a multisystem disorder including mental retardation and autism.43 Similarly, mutations in the L-type voltage-gated Ca2+ channel Cav1.4 (CACNA1F) cause the incomplete form of X-linked congenital stationary night blindness (CSNB2): gain-of-function mutations cause CSNB2 frequently accompanied by cognitive impairment and either autism or epilepsy, whereas CSNB2 due to lossof-function mutations is not accompanied by these symptoms.44 All of these gain-of-function mutations prevent voltage-dependent channel inactivation leading to excessive Ca2+ influx. Also mutations indirectly yielding increased cytosolic Ca2+ levels or amplifying intracellular Ca2+ signaling by hampering Ca2+-activated negative feedback mechanisms have been found associated with autism.42,45 The bioelectrical instability resulting from these mutations nicely parallels the high prevalence of seizures and/or EEG abnormalities present among autistic individuals 
We are currently in the process of correlating AGC activity and levels of oxidative stress with markers of immune activation, measured in the same tissue specimens assessed in this study. 
The present results can potentially pave the path to targeted preventive and therapeutic strategies. One important example is represented by thimerosal, an ethyl-mercury compound used as a preservative in vaccines.59,60 Thimerosal has drawn attention following initial anecdotal reports by some parents linking vaccinations to behavioral regression and to the onset of autism in their child within a matter of days or few weeks. Thimerosal is a Ca2+-mobilizing agent, capable of releasing Ca2+ from intracellular stores and increasing Ca2+ entry.61 Despite its short half-life compared to inorganic mercury, it undergoes preferential accumulation in the CNS, affecting the microglia and producing strain-dependent neurotoxic effects in rodents.62,63 This strain dependency, in conjunction with the present data, suggests that thimerosal could contribute to produce an unbalanced Ca2+ homeostasis in genetically vulnerable individuals. Indeed, a postnatal exposure to thimerosal is not reconcilable with the prenatal onset of neurodevelopmental anomalies leading to autism. Also large retrospective epidemiological studies confirm that thimerosal neither causes autism, nor provides large-scale contributions to its pathogenesis.2,60 However, our results suggest that thimerosal could conceivably precipitate an abrupt onset in a subset of children who would have otherwise developed autistic symptoms more insidiously. At the same time, we cannot exclude that thimerosal and other Ca2+-mobilizing environmental factors could also push genetically vulnerable individuals along the autism-spectrum toward more severe forms of the disease. On the basis of the present study, the elimination of thimerosal from vaccines, undertaken in the United States and Canada, is a well-justified safety measure. 

Pharmacological treatments able to modulate extracellular Ca2+ entry, intracellular Ca2+ release from the endoplasmic reticulum or putative upstream immune mechanisms affecting either or both the pathways are, at least in principle, already available. However, caution should be exercised in translating the present findings into therapeutic interventions prior to at least one replication in an independent cohort of brain samples and to assessments of Ca2+ homeostasis in vivo. 
Pharmacologically reducing Ca2+ entry into cells or blunting the oxidative damage produced by AGC activation seemingly represent more amenable and less dangerous therapeutic strategies. It is nonetheless difficult to predict the actual efficacy of treatments initiated during childhood on pathogenetic mechanisms active since early prenatal development. In this regard, the identification and functional characterization of protective SLC25A12 gene variants, if existent, could provide additional critical information on the contribution of AGC activation and oxidative stress to autism pathogenesis.

=======================

This study reports increased asp/glu exchange rates and significantly higher Ca2+ concentrations in postmortem neocortical tissue specimens of six nonsyndromic autistic patients compared to age-, sex- and PMI-matched controls. Altogether, our results strongly support excessive Ca2+ levels as primarily responsible for the observed activation of asp/glu exchange rates, whereas genetic contributions appear neither widespread nor necessary, at least in our postmortem and genetic samples.

Our results point toward the possible existence of a protective SLC25A12 gene variant in a sizable group of unaffected siblings. This cannot be conclusively demonstrated with our sample size of 104 families including one or more unaffected sibling.

On the other hand, our results would provide further support for key contributions of Ca2+-triggered AGC1 activity to autism pathogenesis. Increased asp/glu exchange rates provide more reducing equivalents (that is, NADH) to the respiratory chain and could foster oxidative stress,24 which was previously found increased measuring peripheral markers in autism.55 Also overexpression of AGC1 in cell culture has been recently found associated with a biphasic response, characterized initially by enhanced neurite outgrowth, which subsequently slows down and ends in early cell death.56 This response is seemingly compatible with an initial overproduction of ATP paralleled by a progressive build up of oxidative stress leading to cell damage. Oxidative stress, in addition to lipid and protein oxidation,55 can also produce genomic instability and stimulate cell cycle progression, pathophysiological events likely to be important in autism pathogenesis9,57,58 In this regard, the interindividual variability in oxidative damage reported in our study is not at all surprising, as the balance between ROS production and antioxidant agents leaves ample room for genetic and environmental influences

The present results can potentially pave the path to targeted preventive and therapeutic strategies. One important example is represented by thimerosal, an ethyl-mercury compound used as a preservative in vaccines.59,60 Thimerosal has drawn attention following initial anecdotal reports by some parents linking vaccinations to behavioral regression and to the onset of autism in their child within a matter of days or few weeks. Thimerosal is a Ca2+-mobilizing agent, capable of releasing Ca2+ from intracellular stores and increasing Ca2+ entry.61 Despite its short half-life compared to inorganic mercury, it undergoes preferential accumulation in the CNS, affecting the microglia and producing strain-dependent neurotoxic effects in rodents.62,63 This strain dependency, in conjunction with the present data, suggests that thimerosal could contribute to produce an unbalanced Ca2+ homeostasis in genetically vulnerable individuals. Indeed, a postnatal exposure to thimerosal is not reconcilable with the prenatal onset of neurodevelopmental anomalies leading to autism.

Also large retrospective epidemiological studies confirm that thimerosal neither causes autism, nor provides large-scale contributions to its pathogenesis. 2,60 However, our results suggest that thimerosal could conceivably precipitate an abrupt onset in a subset of children who would have otherwise developed autistic symptoms more insidiously. At the same time, we cannot exclude that thimerosal and other Ca2+-mobilizing environmental factors could also push genetically vulnerable individuals along the autism-spectrum toward more severe forms of the disease. On the basis of the present study, the elimination of thimerosal from vaccines, undertaken in the United States and Canada, is a well-justified safety measure.

Pharmacological treatments able to modulate extracellular Ca2+ entry, intracellular Ca2+ release from the endoplasmic reticulum or putative upstream immune mechanisms affecting either or both the pathways are, at least in principle, already available. However, caution should be exercised in translating the present findings into therapeutic interventions prior to at least one replication in an independent cohort of brain samples and to assessments of Ca2+ homeostasis in vivo. In particular, our findings in no way support the use of Ca2+ chelation as a therapeutic approach in autism. Ca2+ chelation has not only been purported of benefit in few anecdotal reports and small-sized open trials, but also carries a substantial risk to produce hypocalcemia, resulting in recent deaths of autistic children.64,65 Pharmacologically reducing Ca2+ entry into cells or blunting the oxidative damage produced by AGC activation seemingly represent more amenable and less dangerous therapeutic strategies. It is nonetheless difficult to predict the actual efficacy of treatments initiated during childhood on pathogenetic mechanisms active since early prenatal development. In this regard, the identification and functional characterization of protective SLC25A12 gene variants, if existent, could provide additional critical information on the contribution of AGC activation and oxidative stress to autism pathogenesis.



Solute carrier family 1 (glial high-affinity glutamate transporter), member 3, also known as SLC1A3, is a protein that, in humans, is encoded by the SLC1A3 gene.[5] SLC1A3 is also often called the GLutamate ASpartate Transporter (GLAST) or Amino Acid Transporter 1 (EAAT1) Excitatory.

GLAST is predominantly expressed in the plasma membrane, allowing it to remove glutamate from the extracellular space.[6] It has also been localized in the inner mitochondrial membrane as part of the malate-aspartate shuttle.[7]



VERY thorough but rather complex

N-Acetylaspartate (NAA) is employed as a non-invasive marker for neuronal health using proton magnetic resonance spectroscopy (MRS). This utility is afforded by the fact that NAA is one of the most concentrated brain metabolites and that it produces the largest peak in MRS scans of the healthy human brain. NAA levels in the brain are reduced proportionately to the degree of tissue damage after traumatic brain injury (TBI) and the reductions parallel the reductions in ATP levels. Because NAA is the most concentrated acetylated metabolite in the brain, we have hypothesized that NAA acts in part as an extensive reservoir of acetate for acetyl coenzyme A synthesis. Therefore, the loss of NAA after TBI impairs acetyl coenzyme A dependent functions including energy derivation, lipid synthesis, and protein acetylation reactions in distinct ways in different cell populations. The enzymes involved in synthesizing and metabolizing NAA are predominantly expressed in neurons and oligodendrocytes, respectively, and therefore some proportion of NAA must be transferred between cell types before the acetate can be liberated, converted to acetyl coenzyme A and utilized. Studies have indicated that glucose metabolism in neurons is reduced, but that acetate metabolism in astrocytes is increased following TBI, possibly reflecting an increased role for non-glucose energy sources in response to injury. NAA can provide additional acetate for intercellular metabolite trafficking to maintain acetyl CoA levels after injury. Here we explore changes in NAA, acetate, and acetyl coenzyme A metabolism in response to brain injury.
N-acetylaspartate (NAA) is one of the most abundant brain metabolites and is highly concentrated in neurons, but it remains to be determined why neurons synthesize so much of this particular acetylated amino acid. Early research implicated NAA in lipid synthesis in the brain, especially during postnatal myelination.
Subsequently it was discovered that mutations in the gene for the enzyme that deacetylates NAA, known as aspartoacylase or ASPA, lead to the fatal neurodegenerative disorder known as Canavan disease.
One line of research has focused on the lack of catabolism leading to a toxic buildup of NAA in the brain as the primary etiological component. Another line of research has suggested that the lack of catabolism results in an acetate deficiency in oligodendrocytes during brain development that subsequently limits acetyl coenzyme A (acetyl CoA) availability during this critical period of myelination. There is experimental support for both mechanisms, and it is possible that both are operative.
The loss of NAA after TBI is paralleled by a loss of ATP, acetyl CoA, and other metabolites associated with energy metabolism (Vagnozzi et al., 2007) indicating a substantial impact on neuroenergetics. The connections between NAA and brain energy metabolism are not entirely clear
NAA is synthesized from acetyl CoA and aspartate, and because of the exceptionally high concentration in the human brain (~10 mM) some proportion of acetyl CoA must be utilized to maintain NAA levels, and that proportion may change with brain injury. Signoretti and colleagues have shown that severe brain injury results in a very rapid drop in NAA levels that is paralleled by a similar reduction in ATP levels, suggesting that NAA is utilized rapidly in response to injury

NAA Synthesis and the Mitochondrial Malate-Aspartate Shuttle

The malate-aspartate shuttle functions to move reducing equivalents into the mitochondrial matrix in the form of malate, whereas the major mitochondrial output through this complex is aspartate. Specifically, aralar1 moves cytoplasmic glutamate into mitochondria, while moving mitochondrially synthesized aspartate out. Without aralar1, brain mitochondrial glutamate import and aspartate export are crippled.

The connection between a lack of aralar1 expression and dramatically reduced NAA synthesis has at least two potential explanations. First, as suggested by Jalil et al. (2005) it could be due to the lack of mitochondrial aspartate output, which in turn would limit substrate availability for microsomal Asp-NAT to synthesize NAA. The other possible explanation is that the lack of glutamate uptake into neuronal mitochondria prevents intramitochondrial aspartate synthesis via the aspartate aminotransferase reaction which converts glutamate and oxaloacetate into α-ketoglutarate and aspartate. In this case the lack of intramitochondrial aspartate synthesis would be the limiting factor in NAA synthesis, leading to decreases in NAA levels. It is also possible that both of these mechanisms are responsible for the large drop in NAA levels observed in aralar1-deficient mice. One of the more interesting outcomes of aralar1 deficiency in addition to the large decrease in brain NAA levels is hypomyelination


NAA and Lipid Synthesis 


Neurons provide key metabolites to their ensheathing oligodendrocytes for the purposes of myelination, myelin maintenance, and myelin sheath repair, including choline, palmitate, acetate, phosphate, and ethanolamine (Ledeen, 1984). NAA is among the trophic neuronally derived metabolites that are transferred to oligodendrocytes for use in myelination and myelin repair.

The synthesis of NAA requires the utilization of existing acetyl CoA and therefore NAA synthesis consumes a portion of brain acetyl CoA stores. Therefore NAA may be acting as a storage and transport form of acetate in the CNS that can be used for subsequent de novo synthesis of acetyl CoA, especially in oligodendrocytes


Inhibit AGC1?


The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.




Abstract

Ca2+ signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca2+ entry in mitochondria via the Ca2+ uniporter followed by Ca2+ activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca2+ signals via a mechanism that does not require the entry of Ca2+ in mitochondria, a mechanism depending on the activity of Ca2+-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/Pi carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca2+ in the intermembrane space and function together with the Ca2+ uniporter in decoding the Ca2+ signal into a mitochondrial response.


www.uniprot.org/uniprot/Q12482
Calcium-dependent mitochondrial aspartate and glutamate carrier. Transport of glutamate in mitochondria is required for mitochondrial transamination reactions and ornithine synthesis. Plays also a role in malate-aspartate NADH shuttle, which is critical for growth on acetate and fatty acids.

The aspartate/glutamate carrier isoform 1 is an essential mitochondrial transporter that exchanges intramitochondrial aspartate and cytosolic glutamate across the inner mitochondrial membrane. It is expressed in brain, heart and muscle and is involved in important biological processes, including myelination. However, the signals that regulate the expression of this transporter are still largely unknown. In this study we first identify a CREB binding site within the aspartate/glutamate carrier gene promoter that acts as a strong enhancer element in neuronal SH-SY5Y cells. This element is regulated by active, phosphorylated CREB protein and by signal pathways that modify the activity of CREB itself and, most noticeably, by intracellular Ca2+ levels. Specifically, aspartate/glutamate carrier gene expression is induced via CREB by forskolin while it is inhibited by the PKA inhibitor, H89. Furthermore, the CREB-induced activation of gene expression is increased by thapsigargin, which enhances cytosolic Ca2+, while it is inhibited by BAPTA-AM that reduces cytosolic Ca2+ or by STO-609, which inhibits CaMK-IV phosphorylation. We further show that CREB-dependent regulation of aspartate/glutamate carrier gene expression occurs in neuronal cells in response to pathological (inflammation) and physiological (differentiation) conditions. Since this carrier is necessary for neuronal functions and is involved in myelinogenesis, our results highlight that targeting of CREB activity and Ca2+ might be therapeutically exploited to increase aspartate/glutamate carrier gene expression in neurodegenerative diseases.

Studies in animal models have highlighted the relevance of AGC1 in the physiology of neurons. AGC1 knockout mice showed a dramatic drop in brain aspartate levels, with a concomitant reduction in N-acetylaspartate (NAA) synthesis and hypomyelination (Jalil et al., 2005). The connection between lack of AGC1 and drop in NAA synthesis may due to the lack of mitochondrial aspartate output, which in turn would limit availability of NAA-derived acetate needed for lipid biosynthesis resulting in hypomyelination. Numerous studies have indeed demonstrated that acetate moieties of NAA are incorporated into brain lipids during the development of the central nervous system, hence strongly suggesting that AGC1 may be crucially involved in the myelination). In support of this conclusion, children harboring mutations of the SLC25A12 gene display severe developmental delay, epilepsy, hypotonia hallmarked by hypomyelination and decreased NAA in the brain (Wibom et al., 2009; Falk et al., 2014). The chromosomal region containing the gene encoding AGC1 has also been identified as a putative autism susceptibility locus (Ramoz et al., 2004; Turunen et al., 2008; Palmieri et al., 2010). In addition, interest in the involvement of mitochondria in neurodegenerative and neuroinflammamtory disorders, such as Parkinson's and Alzheimer's disease, and multiple sclerosis is emerging (Lin and Beal, 2006)

Despite the well-established role of NAA in myelin biosynthesis, it is still unknown in which subcellular compartment the biosynthesis occurs. Different studies have provided evidence that the aspartate-N-acetyltransferase (Asp-NAT), the enzyme that catalyzes the biosynthesis of NAA, is localized in the mitochondria (Patel and Clark, 1979; Madhavarao et al., 2003; Arun et al., 2009). However, other studies performed in primary neuronal cultures established that Asp-NAT is located in the endoplasmic reticulum as well (Wiame et al., 2009; Tahay et al., 2012). A colocalization was reported by other authors (Lu et al., 2004; Ariyannur et al., 2010).

3.1. AGC1 expression is downregulated by inflammatory cytokines

3.3. The decrease in AGC1 expression in neuroinflammation is most likely caused by the downregulation of CREB

3.4. Cytosolic Ca2+ level affects AGC1 gene expression via CREB
Because Ca2+ is a well-known CREB inducer (Sheng and Greenberg, 1990) and is also known to stimulate AGC1 activity (Palmieri et al., 2001; Lasorsa et al., 2003; Contreras et al., 2007), we investigated whether alterations in the pool of intracellular Ca2 affect AGC1 gene expression. 

Glutamate is the only amino acid extracted by healthy myocardium in net amounts, with uptake further increased during hypoxic or ischemic conditions. Glutamate supplementation provides cardioprotection from hypoxic and reperfusion injury through several metabolic pathways that depend upon adequate transport of glutamate into the mitochondria. Glutamate transport across the inner mitochondrial membrane is a key component of the malate/aspartate shuttle. Glutamate transport in the brain has been well characterized since the discovery of the excitatory amino acid transporter (EAAT) family. 


Increased expression of the glutamate transporters EAAT1 and EAAT2 in the cerebellum of post-mortem tissue from autism patients has also been reported. Because EAAT expression is controlled in part by the extracellular concentration of glutamate, it is possible that the EAAT overexpression is due to the increased glutamate concentration seen in plasma and spectroscopic studies, as reviewed above.







Fig. 2 The malate – aspartate shuttle (MAS) and A TP generation. MAS is the main pathway for the transfer of reducing equivalents in the form of NADH from the cytosol into the mitochondria. Cytoplasmic malate dehydrogenase reduces oxaloacetate to malate while oxidizing 


A reminder of what belongs where in a cell:-




Components of a typical animal cell:

1.      Nucleolus

2.      Nucleus

3.      Ribosome (little dots)

4.      Vesicle


6.      Golgi apparatus (or "Golgi body")

7.      Cytoskeleton

  1. Smooth endoplasmic reticulum 

9.      Mitochondrion

10.  Vacuole

11.  Cytosol (fluid that contains organelles, comprising the cytoplasm)

12.  Lysosome

13.  Centrosome




Conclusion

I hope some people made it to the end of this post. Since it was written on two computers, there may be some duplication.

It appears that most people with autism would benefit from less calcium in their brains, but this is more complex than it sounds. We need to block voltage gated calcium channels that are open, when they should be closed. We need to reduce IP3 to keep calcium locked up in the endoplasmic reticulum.

If we cannot reduce the level of Ca2+, we need to look at CREB which is mediating much of the damage. We can modify CREB via drugs that increase or decrease PKA. The only problem here is that for most people that would mean decreasing PKA, but PKA also does some other good things. At least in Alzheimer’s things do not conflict you want more CREB and more PKA.

The amino acid transporters AGC1, EAAT1 and EAAT2 are likely over activated in much autism. So we should expect lots of problems with glutamate, aspartate and indeed mitochondrial function. 

It looks like most young people with autism would benefit from more NAA. One study showed that the omega 3 oil EPA can increase NAA.  At least that is simple.

More NAA may well help correct impaired myelination.

Ideas that may potentially help in some cases of autism:-

·        L-aspartic acid / L-aspartate

·        High EPA fish oil to increase NAA

·        PDE4 inhibitors (Ibudilast or Daxas)

·        PKA inhibitors (not easy)

·        IP3R blocker (only caffeine seems currently viable and is suboptimal)

         Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate   (IP3) receptor

·        Verapamil in those with over-active (open) L-type calcium channels 



Does oral L-aspartic acid actually reach the brain? Just try some. It tastes odd, like a homemade sweet/sour flavoring gone wrong and does not dissolve in water.  It should be harmless in moderation.







Saturday 24 March 2018

Stopping Over Medication of People with a learning disability, autism or both (STOMP) & Start Effective Personalized treatment (STEP). Also STALICLA


Lake Geneva with its famous fountain (Jet d'eau)

As some regular readers are well aware, people with autism are often prescribed psychotropic/psychiatric drugs to make them more manageable; this is particularly true of those who live in residential care homes and for those living in America. When it comes to adults, as Tyler just pointed out in a comment, 1 in 6 adult American takes one or more psychotropic/psychiatric drugs.
These drugs do not treat the underlying dysfunctions in most autism, but do produce a more compliant/sedated individual. They often lead to weight gain and its associated medical problems; some of these drugs are addictive, some can cause irreversible side effects (e.g. tardive dyskinesia).
In England there is a campaign called STOMP to stop this overuse of these psychiatric drugs in people with autism, which can only be a good thing.

STEP
Hopefully one day there will be a campaign to treat the underlying biological dysfunctions within autism, which could be called STEP (Start Effective Personalized treatment); not all treatments need to be drugs. 

STALICLA
One reader of this blog has recently established her own start-up company, Stalicla, in Switzerland with the aim of treating individual sub-types of autism. I assume that her STP1, comprising two repurposed drugs, targeting her Phenotype1 subgroup, said to represent 15% of the total idiopathic ASD patient population, is the one she gives her son.

Good luck Lynn - you are going to need it. 

STOMP
Stopping over medication of people with a learning disability, autism or both (STOMP) is the campaign in England.
“Public Health England says that every day about 30,000 to 35,000 adults with a learning disability are taking psychotropic medicines, when they do not have the health conditions the medicines are for. Children and young people are also prescribed them.” 

Here is a case study example of an over-medicated young man with autism.
While I fully support the STOMP campaign, I guess many STOMP supporters would think my PolyPill and Lyne’s STP1 are complete folly. Hopefully Lynn’s clinical trials will be successful, not to forget many people hope Dr Ben-Ari’s phase 3 bumetanide trial will be successful. Kritika and many others are waiting for results on CM-AT.
So it looks too early to launch my STEP campaign (Start Effective Personalized treatment for people with autism and/or ID and/or epilepsy, not forgetting Down syndrome, Pitt Hopkins, SATB2, Sotos, Turner, Fragile X and all the others).

Conclusion
This blog is all about using drugs more intelligently, not to sedate people with autism but rather to do the opposite, to improve their cognitive function so that they are more able to be self-supporting.
The inappropriate use of potent psychotropic/psychiatric drugs helps to give all drugs a bad name.
Fortunately Lynn chose her company name beginning with ST, which fits nicely with stepping and stomping; I get to squeeze two posts into one and use a nice photo of Lake Geneva and not a photo of someone’s foot.





Wednesday 21 March 2018

Amino Acids Disturbed in Autism? Very Likely


Amino acids in your body are the building blocks for making proteins. There are essential Amino Acids that you must obtain from diet and semi-essential amino acids that in young children are body is still not able to produce and non-essential amino acids that your body can produce. 

There was an earlier post on Amino Acids:- Amino Acids in Autism 



There are also sub-groups, Branch Chained Amino Acids (BCAAs) and aromatic.  The branched chained amino acids (BCAAs) compete with the aromatic amino acids for entry into the brain. Therefore, altering BCAA levels can affect the levels of the neurotransmitters serotonin, dopamine, epinephrine and norepinephrine in the brain. 

Alanine
Arginine (essential)
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine (essential) Aromatic
Isoleucine (essential) BCAA
Leucine (essential) BCAA
Lysine (essential)
Methionine (essential)
Phenylalanine (essential)  Aromatic
Proline
Serine
Threonine (essential)
Tryptophan (essential) Aromatic
Tyrosine  Aromatic
Valine
(essential)  BCAA 

Taurine is not an amino acid, but often gets treated as if it was one.

Arginine and its derivatives appear to play a critical role in some types of cognitive impairment including Alzheimer's and other forms of dementia.

I am writing new posts about certain individual amino acids that look interesting. Aspartic acid is next and the post looks like getting rather complex, so I decided to highlight an interesting very simplistic study that I stumbled across, that would otherwise get lost in other complex papers.  
I call it simplistic because it compares a control (NT) group with two groups of children with autism, one group has no intervention and the other group is made up of kids with some intervention, of any kind, under the umbrella of digestive/dietary/enzyme/antifungal. The control group was siblings of the children with autism. So three groups in total.
Nonetheless, this Disney science does show something quite surprising. In most cases it looks like any intervention produced results much closer to the reference range than no intervention. The study did not measure whether the intervention had any effect on the severity of autism, or compare the different interventions.
Since I am currently researching Aspartic acid I was drawn to the fact that Aspartic acid was nearly three times higher for the female controls compared with the males.  This I find notable, given the sex difference in autism, which is the biggest clue nature has left us. 


A total of 63 subjects were recruited, consisting of 34 autistic children with 31 males and three females aged 5–15 years (mean ± SD, 6.9 ± 2.5 years), and 29 controls with 13 males and 16 females also covering a range of 5–15 years (mean ± SD, 8.9 ± 3.3 years). The controls were derived primarily from siblings of the autistic group, where the sex ratio was more reflective of the general population compared to the sex balance of the autistic population which is recognised as approximately 4:1 (male:female).15 Measures were taken to account for the uneven sex-distribution in the interpretation of the results.


Twenty-two of the autistic children were receiving therapeutic treatments related to digestive function and nutritional uptake. These treatments included antifungal medication, to treat confirmed or suspected Candida infection of the digestive tract, probiotics for maintenance of gut microflora, dietary intervention (gluten- and/or casein-free diet), nutritional supplements, or the hormone secretin; this has been shown to be responsible for regulating pH of the duodenum and is, therefore, pertinent to the functioning of digestive enzymes. On this basis, the autism group was further subdivided into two groups for evaluation of urinary metabolites: treated autistic patients (n = 22; range, 5–15 years; mean, 6.9 ± 2.4 years; 91% male) and untreated autistic patients (n = 12; range, 5–12 years; mean, 7.0 ± 2.5 years; 91% male). To account for any sex-associated differences in urinary output, resulting from the disparity in the sex matching of the autistic and control groups, the control population was first subdivided on the basis of gender.

Two compounds emerged as significantly different on a gender basis for the control group: (i) glucose excretion was of a significantly higher mean concentration for the female control group compared with the males (P <0.05); and (ii) aspartic acid was nearly three times higher for the female controls compared with the males (P < 0.05). No other urinary metabolite concentrations proved to be significantly different between the female and male controls. On this basis, the data from the entire control group were compared with the treated and untreated autistic cohorts.




Plasma amino acids vs urinary amino acids 

There are numerous other studies and they do tend to use a blood test rather than a urine sample. Below is a relatively recent study from Egypt. 


Methods
Twenty autistic children were compared to twenty healthy age and sex matched normal children serving as control, where serum amino acids, urea, ammonia and protein electrophoresis were estimated.

Results
As regards essential amino acid levels, autistic children had significant lower plasma levels of leucine, isoleucine, phenylalanine, methionine and cystine than controls (P < 0.05),while there was no statistical difference in the level of tryptophan, valine, threonine, arginine, lysine and histidine (P > 0.05). In non-essential amino acid levels, phosphoserine was significantly raised in autistic children than in controls (P < 0.05). Autistic children had lower level of hydroxyproline, serine and tyrosine than controls (P < 0.05). On the other hand there was no significant difference in levels of taurin, asparagine, alanine, citrulline, GABA, glycine, glutamic acid, and ornithine (P > 0.05).

There was no significant difference between cases and controls as regards the levels of urea, ammonia, total proteins, albumin and globulins (alpha 1, alpha 2, beta and gamma) (P > 0.05).

Conclusions
Autistic children had lower levels of some plasma amino acids except for glycine and glutamic acids and phosphoserine were increased with normal serum levels of urea, ammonia, total proteins, albumin and globulins (alpha 1, alpha 2, beta and gamma). 
In conclusion, autistic children may have dysregulated amino acids metabolism as all amino acids except for glutamic acid, phosphoserine and glycine are decreased in patients than in control; the raised glutamic acid may suggest involvement of an altered glutamate transporter and is consistent with a biochemical basis for autistic disorders. Also, the lower amounts of essential amino acids are correlated with more severe autism.


Conclusion

The fair conclusion is that both excreted and plasma amino acids look to be disturbed in autism. Does this tell you anything actionable? Should you become obsessed by trying to reach the reference ranges?
The answer depends on who you ask and I guess who is paying.

The derivatives of some amino acids may indeed be disturbed as a protective mechanism in which the body is adapting to specific effects of that person's autism. This was suggested in the research as one explanation of why agmatine, a derivative of arginine, is elevated in schizophrenia.  In other words elevated agmatine is a good thing in that person, it may well be a biomarker for schizophrenia, but possibly a "good" biomarker. 
If normalizing amino acids does improve autism, then expect the CM-AT treatment from Curemark to pass its phase 3 trials and become an FDA approved therapy for autism. Interestingly when I looked into the Curemark patents a long time ago, secretin appeared and it made another appearance earlier in this post. Secretin is now viewed as a false hope for autism treatment and those who still use it are seen as quacks. In trials secretin was shown not to help most people with autism, but regular readers will know that this is different from saying nobody responds. 
Irrespective of what finally happens to CM-AT, it looks like individual amino acids do have a place in some personalized autism therapies. This may, in some cases, be irrespective of the reference ranges, in other words in some cases an abnormally high level of one amino acid may be required to get a specific beneficial therapeutic effect.  Staying within the reference range is clearly the safer option.
Now I am back to my complicated post about Aspartate and N-acetylaspartate (NAA), which does look very relevant to autism.







Thursday 15 March 2018

PolyPill Updated with Agmatine




After several months of testing Agmatine, including stopping and then re-starting, it is time to add it to my PolyPill.
The idea to trial Agmatine came from our reader Tyler. It ticks all the boxes, it really does have a benefit; that benefit continues for at least several months. When you stop taking it, the benefit stops and when you restart you see the same benefit return. It is safe, inexpensive and widely available if you live anywhere outside the EU. Since it is relatively recent to the market as a supplement, it can no longer be sold in the EU until someone applies for it to be approved; long established supplements bypass this recent legislation.
There were earlier posts evaluating why it might help some types of autism and now there even is one study on an animal model of autism. 





I think the positive effect very likely comes from the vasodilatory effect produced by the increased eNOS, there is also increased BDNF.  This I believe is why it also effective in two models of dementia. Agmatine is also an NMDAR antagonist, like Memantine and this is the mode of action proposed by autism researchers in the animal model above.  My opinion is that at this "bodybuilder's" dose the mode of action is not NMDAR antagonism.




The effect of Agmatine?



The energetic bunny on the left is the one taking 0.7g a day of Agmatine.
In the case of Monty, aged 14 with ASD, Agmatine gives him boundless energy, which in his case is beneficial.
I think the effect will manifest itself slightly differently in different people. In animal models it improves cognitive function.  
If you have autism + ADHD, then it might not be helpful. When I tried it on myself it made me feel slightly nauseous. In Monty's big brother it made him feel "different", but not better or worse and certainly not more energetic.


Other People’s PolyPills
Other readers of this blog have developed their own science-based “Polypill” therapies, for their specific type of autism. What works for my son may not help your child, but other  things discussed in this blog just might help.
High doses of the immuno-modulating Biogaia Gastrus probiotic bacteria clearly help some people greatly; but others get a negative reaction.
Immunomodulation by antibiotic is used successfully by some, but has some drawbacks. 
PAK1 inhibition ticks the science boxes and if you can obtain a potent PAK1 inhibitor it helps some people.

Butyric acid (from sodium butyrate, Miyairi 588 bacteria, more fiber or even rancid butter) is an HDAC inhibitor and is also required for gut wall integrity and likely BBB integrity. It is widely used in animal feed and some humans respond well to it, but the effect is dose dependent. HDAC inhibition can work epigenetically to change the expression of hundreds of "autism" genes, as highlighted in recent research using a potent cancer drug HDAC inhibitor.
Numerous individual amino acids (glutamine, taurine, methionine, aspartic acid etc) seem to help in some people. These tend to be OTC bulk powders like Agmatine.

In a sub-group of people with autism it is clear that digestive issues, unusual gut bacteria or food sensitivity is a major problem that is treatable relatively easily.

People with regressive autism may have (had) mitochondrial disease and this has a very specific therapy to protect from further regression and to allow for remyelination and mitochondrial biogenesis in the short term and hope for neurogenesis in the long term.




Saturday 10 March 2018

A Thinking Person’s Brief Guide to Autism


I wrote this list because most people visit this blog and do not go any further into the details, either because they think this blog is nonsense, or it is just too complicated.

It certainly does sometimes get complicated, but complex subjects require detailed attention. There is little point in looking superficially.  Understanding autism science is not beyond most people’s ability, but it does take time. It will not cost you a penny. Then you can make up your own mind as what is a novel therapy and what is likely a quack therapy.


Some doctors do know best.  While your family doctor no doubt was taught autism is untreatable, some doctors who have a child with autism think differently and I do not mean MAPS/DAN doctors. Some mainstream doctors read the same research as me and successfully treat their child – an example of personalized medicine. In the ideal world they would all publish their experiences, but most keep it private.

Autism is not a biological medical diagnosis, it is just an observational diagnosis based on guidelines published in a book called the Diagnostic and Statistical Manual of Mental Disorders (DSM). These guidelines have changed substantially over time. We are currently on DSM 5; in 1987 it was DSM3 Revised. Most people diagnosed today with autism would not have been given that diagnosis under DSM 3. This is one key reason for the autism diagnosis “epidemic”, but there are other reasons.

A vast amount of scientific research already exists about autism. More is published every day and very helpful lay summaries are available for free on the internet on medical news websites and blogs like Paul Whiteley's Questioning Answers. There is also the Simon’s Foundation’s excellent website for non-scientists at www.spectrumnews.org.

There are hundreds of different known biological causes of autism.  Scientists have already created 267 different genetic mouse models of autism and 78 types of induced autism. 192 rescue models exist, where the scientist could reverse autism in the mouse.  If you can reverse it in a mouse, perhaps you can treat it in a child? Not crazy to at least try.
https://gene.sfari.org/database/animal-models/genetic-animal-models/

Some autism/MR/ID caused by rare errors of the metabolism is treatable today, by mainstream medicine. Clinicians in Vancouver maintain a database; they focus on Mental Retardation (MR) / Intellectual Disability (ID), but many of these conditions can also be diagnosed as autism.


Science and medicine are not the same. Medicine applies drugs that are shown effective in large clinical trials on people with the same biological disorder.  This is evidence-based medicine.  Where a disorder exists with multiple causes, medicine struggles to cope. Many illnesses have multiple causes, or unknown causes, for example dementia, depression, epilepsy, multiple sclerosis etc. and even conditions outside the brain like chronic prostatitis.

Medicine has many poorly effective drugs for neurological conditions. There are drugs for dementia, ALS (motor neuron disease), Parkinson’s, Huntington’s, Schizophrenia, Bipolar etc., but they are not curative - they are better than nothing, but sometimes not by much. Drugs for autism should be seen in this perspective; generally they will be partially effective and only in specific people with the same particular biological dysfunction. Drugs for ALS, Alzheimer’s etc. currently just treat some features of the disease, they do not address the cause; some of these features are shared by others brain disorders. So using an ALS drug to treat autism or schizophrenia is not a crazy idea, if the same biological features are present.

There are numerous well-researched biological features present in some autism. Some of these same features are present in unrelated, better-researched, medical conditions where often they are treated. For example:- oxidative stress, elevated pro-inflammatory cytokines and reduced anti-inflammatory cytokines, activated microglia, disturbed growth factors (BDNF, NGF, IGF-1, VEGF etc.), numerous ion channel and transporter dysfunctions (NKCC1, KCC2, Cav1.2 etc.), NMDA hypofunction or hyperfunction, reduced estrogen and ERβ, excitatory-inhibitory imbalance, glutamate excitotoxicity, impaired autophagy, unusual myelination etc. 

Autistic brains are not just “wired up differently”. Brains are not computers and while some things are fixed as in a computer, much inside your brain is changing all time and so can potentially be modified.  You can induce even “autism” in a perfectly normal brain (with propionic acid) and then reverse it.  A slightly better analogy might be an old out-of-tune piano; tuning the strings (wires) is a complex process, but results in much improved function. Autistic brains can be tuned to improve their function and the person’s wellbeing.

Vaccines can trigger mitochondrial disease in children, which will appear as autism. This was proved by Dr Jon Poling a US neurologist trained at Johns Hopkins, who won $1.5 million in compensation for his daughter. Here interviewed on CNN:-


Vaccines caused one little girl's autism, but that does not mean they caused it in your child. How widespread this problem might be is something Dr Poling does comment on, but it is not something anybody would research.  Johns Hopkins is home to some of the best autism and mitochondrial disease researchers and clinicians. They even developed a cheap therapy to minimize the risk of possible damage from vaccinations to their young patients who already have mitochondrial disease. 

Autism does not kill you?
Vaccines save millions of lives and even Dr Poling does not suggest avoiding them. People with severe autism do have a life expectancy of less than 40 years mainly due to death from seizures, drowning and other accidents. People with the Asperger's type of milder autism have a nine times elevated risk of suicide and a disturbingly a high proportion of the tiny number of mass killers have an Asperger's/autism diagnosis (the other large group have suffered a previous head injury). So autism clearly can have troubling consequences.  Teaching anyone to shoot a gun who has a neurological disorder (autism, bipolar, schizophrenia, even ADHD, Intermittent Explosive Disorder etc), severe enough to get a medical diagnosis, is asking for trouble.

Does Autism need to be treated?
Some of the people with the newly included mild autism protest about the idea of treating autism. A significant minority of deaf people think their children born without hearing should not receive cochlear implants and so remain with them in silence, but most hearing people would likely want their child disabled by hearing loss to be treated. Medically diagnosed autism is currently based on guidelines from a book of mental disorders (DSM5). Most people probably think disorders in a psychiatrist’s manual should be treated, even if the very people with those disorders think they are just fine - they do after all have a disorder potentially clouding their judgement. If their autism is so mild and not troubling at all, then it does not warrant a medical diagnosis. You can be different without needing a medical diagnosis to support it.

Many observational diagnoses like Autism, ADHD, Bipolar and Schizophrenia are overlapping biologically
. Genetic studies have shown people can have biological elements of more than one diagnosis. So you can have autism with a little Bipolar and a touch of ADHD.  This means that some therapies trialed in Schizophrenia may show a positive effect in some autism, for example.

Clinical trials in Autism have all ultimately failed. All of the many clinical trials in autism to date have been viewed as failures and part of the reason is that there is more than one autism. There is no easy way to tell which person has which type, so trials include many people who are bound not to be responders.

People treating autism are all quacks? There undoubtedly are many people making money out of the families affected by autism and where money is involved, you will always find quack therapies of one kind or another.

Personalized medicine is one way forward. In cancer care personalized medicine is already being used, where drugs are tailored to exactly what is wrong in a specific person.  With hundreds of different variants of autism personalized medicine would be the ideal solution, but it is many decades away for most people.

Autism clusters and nexus where multiple dysfunctions converge.  The realistic way to treat autism in the 2020s will be to group people into similar clusters, not perfect matches, and treat by cluster.  This is possible because it seems that multiple different dysfunctions converge at a manageable number of so-called nexus. This means that rather than several hundred unique therapies you have just a few tens of therapies. Each person is then matched with their Polypill of a handful of those few tens of therapies.

Autism is a condition of opposites. In autism very often both extremes of the same condition exist, making the average meaningless. On a simple level the research finds many big heads, but also a fair number of tiny heads. There is very high cholesterol and very low cholesterol. There are very high levels of a particular growth factor and then some with virtually none.   So do not expect what works for someone else’s autism, to necessarily work for your child’s autism.

Many drugs have multiple modes of action. Lay people assume that drugs do just one thing, be it lower blood pressure, lower cholesterol or act as a diuretic.  This is not the case; most common drugs have multiple effects. For example many antibiotics have completely unrelated anti-inflammatory effects. A diuretic that affects one ion channel in your kidneys can also affect a related one in your brain, so yes a diuretic can treat some people’s autism, it is not crazy.

Many existing drugs can be repurposed. Many drugs already exist that can potentially be repurposed to treat neurological conditions. Big drug firms like to develop new drugs because their patents allow them to charge very high prices. Existing drugs are often now cheap generics, with well-known safety profiles, that can potentially be used off-label in personalized medicine, based on intelligent use of the vast wealth of autism research.

Off-label prescribing used to be widespread; it is when a drug is used for an application other than that for which it has been officially approved. For example, the diuretic Spironolactone is widely used off-label to treat acne in females.  Modern doctors are less willing to prescribe off-label either because they insist on strictly following the rules, or do not want to take a risk of being blamed for an adverse reaction.  Treating autism will inevitably require off-label prescribing.

Personalized therapy for the few? Hopefully the mainstream doctors who currently successfully treat their own children will persuade more of their peers to try and treat others. Until then, or until a drug like Bumetanide finally passes its stage 3 clinical trials for autism, I do not think much will change.  A small number of parents do read the research and persuade their physician to help them and others evidently self-treat. For those who are truly motivated there are options, which is what really matters.





Monday 5 March 2018

Autism and Non-Antibiotic Properties of Common Beta-lactam Antibiotics


If you are looking for personalized medicine, you or your doctor need to be a good detective. Not to mention you need some clues.
If you are treating a condition like autism and certain things cause a marked change in the severity of the condition, these are pretty good places to start.
In the case of our reader in Delhi, it is Beta-lactam antibiotics (penicillin, amoxicillin etc), that consistently seem to improve her son’s autism. Improvement during treatment with antibiotics is reported quite often in autism, but with all kinds of different antibiotic.  Nothing is simple.
For non-medical readers, there are several categories of antibiotics; common types including:-
·        Beta-lactams (e.g. Penicillins)

·        Macrolides (e.g. Erythromycin, Azithromycin)

·        Fluoroquinolones (e.g. Ciprofloxacin) 

·        Tetracyclines (e.g. Minocycline) 

Macrolides have already had a dedicated post about their immunomodulatory effects, which did also cover some history about Poland from Monty's homework.

Macrolide Antibiotics for Some Autism? Or better still, Azithromycin analogue CSY0073, or just Nystatin?



Beta Lactam Antibiotics
In earlier posts we came across something called glutamate transporter GLT1 (also known as EAAT2).
Glutamate is the major excitatory neurotransmitter, and is inactivated by uptake via GLT-1 (EAAT2) and GLAST (EAAT1) transporters.
Many people given the observational diagnosis of autism appear to have an underlying imbalance between excitatory and inhibitory neurotransmitters (E/I imbalance). By correcting the specific type of E/I imbalance, even profound symptoms of autism including MR/ID and epilepsy can be moderated. If you have autism and/or epilepsy tuning your E/I imbalance is likely the most important step you can take.
Some drugs increase the expression of GLT-1 and so reduce the amount of glutamate. Macrolide antibiotics are one of these drugs.
So if a person has too much glutamate and this causes/contributes to their E/I imbalance then improved behaviour while taking penicillin antibiotics, who have a simple explanation.
Since you would not want to take penicillin forever you would then look for a non antibiotic drug that also increases the uptake of Glutamate. Once such drug, Riluzole, does exist and has already been trialed on children with OCD. 
But beta-lactams have other effects, so it is not certain that GLT-1 accounts for the beneficial effect sometimes found in autism. Fortunately some researchers have assembled most previous research into a single review paper. This paper does not mention autism and does miss some things out.


There are seven categories:-
·        Antibiotic

·        Epileptogenic

·        Neuroprotective

·        Analgesic

·        Immunomodulatory

·        Anxiolytic

·        Antineoplastic



Antibiotic Effect
We all know something about bacteria. If you have a bacterial infection like an ear infection your doctor might prescribe you an antibiotic.

As well as inflaming your ear, the bacteria may well affect gene expression. We saw in a previous post that bacteria and viruses change the expression of many genes, but the study of this is in its infancy. In autism we know that many genes are miss-expressed, but this varies from person to person. So a bacteria or virus has the potential to make autism worse (e.g. PANS and PANDAS), but also better. Bacteria are not always bad.
A person whose autism responds to an antibiotic might have bacteria that are worsening his autism. This is simplest of explanation of all.

The question then is where is the bacteria? If it is an intestinal bacterium this could be proven by using an antibiotic that only works there, like Vancomycin.

Epileptogenic effects
In this review they concluded the effects relate to GABA and here we are talking about negative effects. 

penicillin is a potent epileptogenic agent = it is capable of causing an epileptic attack

“This could mean that penicillin is a competitive GABA specific antagonist, which would further explain its epileptogenic properties.”

The paper omits to point out that in some people beta-lactams protect from epileptic seizures. The effect on Glutamate is likely at least sometimes what stops seizures.


The really clever thing in the above case report is that appears that the effect on glutamate may be by an epigenetic mechanism (via GLT1), since the effect is long lasting. Read later in this post about the epigenetic effects of beta-lactams.

Neuroprotective properties
“These results suggest that the neuroprotective effect induced by beta-lactam antibiotics is due to their capacity to stimulate GLT1 expression and thus regulate the concentration of glutamate in the synaptic cleft. GLT1 is a glutamate transporter inducing its reuptake by astrocytes preventing excessive glutamate concentration in the synaptic cleft
It was subsequently shown that the neuroprotective effect of BLMs was due not only to glutamate down regulation, but also to a diminished glutamate-induced intracellular Ca2+ concentration and an increased uptake of glutamate
Another probable mechanism of neuroprotection induced by BLMs is down-regulation of oxidative stress and modulation of apoptotic pathways shown in rat spinal cord when CFX was administered for 7 days prior to induction of constrictive neuropathy. This effect was apparently mediated by both a reduction in proapoptotic proteins Bax, and an increment in the antiapoptotic protein Bcl2.
CFX (Ceftiaxone) may induce neuroprotection by other mechanisms besides GLT1 overexpression. Yamada and Jinno [51] reported that the antibiotic reversed axotomy-induced up regulation of GFAP, a neuronal damage marker, and increased neuronal survival; apparently not only through glutamatergic regulation, but also by direct reduction of glial hypereactivity. Supplementary to this is the finding of an attenuation of microglial activation induced IL-1 expression in an ischemic injury model when CFX was administered as a pre-treatment [52]. This result may indicate a direct action on glial cells since partial reduction of astrocytes and microglia was observed.”

Analgesic (pain killing) Properties
“Interestingly, despite the widespread clinical use of BLMs (beta-lactams), some of their known non-antibiotic effects have been either disregarded or misinterpreted as resulting from bacterial microbiome regulation. For example, Caperton, Heim-Duthoy [54] hypothesized that chronic inflammatory arthritis could have a bacterial component and that therefore the clinical course of a patient could be affected by administration of CFX (Ceftriaxone).
Both the anti-inflammatory and neuromodulating effects exerted by BLMs either peripherally or centrally may be related to their analgesic properties in some pathologies that are difficult to treat such as the complex regional pain syndrome [65] or to the analgesic effect of a single preoperative dose of CFX in a clinical protocol [66].

Immunomodulatory Properties 
Not many people seem to have read this paper. They did not flesh out immunomodulation, so I draw on a different paper. People who write about immunomodulation usually say that beta-lactams do not have this effect, but that appears to be incorrect. 

Recent work has suggested that beta-lactam antibiotics might directly affect eukaryotic cellular functions. Here, we studied the effects of commonly used beta-lactam antibiotics on rodent and human T cells in vitro and in vivo on T-cell–mediated experimental autoimmune diseases. We now report that experimental autoimmune encephalomyelitis and adjuvant arthritis were significantly more severe in rats treated with cefuroxime and other beta-lactams. T cells appeared to mediate the effect: an anti-myelin basic protein T-cell line treated with cefuroxime or penicillin was more encephalitogenic in adoptive transfer experiments. The beta-lactam ampicillin, in contrast to cefuroxime and penicillin, did not enhance encephalomyelitis, but did inhibit the autoimmune diabetes developing spontaneously in non-obese diabetic mice. Gene expression analysis of human peripheral blood T cells showed that numerous genes associated with T helper 2 (Th2) and T regulatory (Treg) differentiation were down-regulated in T cells stimulated in the presence of cefuroxime; these genes were up-regulated in the presence of ampicillin. The T-cell protein that covalently bound beta-lactam antibiotics was found to be albumin. Human and rodent T cells expressed albumin mRNA and protein, and penicillin-modified albumin was taken up by rat T cells, leading to enhanced encephalitogenicity. Thus, beta-lactam antibiotics in wide clinical use have marked effects on T-cell behavior; beta-lactam antibiotics can function as immunomodulators, apparently through covalent binding to albumin.

 Anxiolytic effects (reduce anxiety)
“CA (Clavulanic acid) has proven effective as an anxiolytic drug, since it was reported that this drug diminished anxiety-like conduct in both rodent and primate models”

Antineoplastic effects (preventing tumors)
“CFX (Ceftriaxone) elicit antitumor activity both in vitro and in vivo models”

Addiction
Addiction did not appear in the chart above, but it gets a mention in the text 
“When tested in an opiate dependence model, both CFX [72] and CA [73] inhibited both physical dependence and withdrawal symptoms. This could mean that the effect shown by CFX is not due to its particular molecular structure, but can be reproduced by other BLMs (several BLMs effects shown on Fig. 3)

Other effects
“CA (Clavulanic acid) has been shown to increase dopamine release”

Epigenetic Effects
These were not mentioned in the paper, but I do think epigenetics is a fundamental part of many diseases, including much autism.
The paper really explains why short term use of beta-lactams can stop a person with epilepsy having seizures for a long time.

Off-Target drug effects resulting in altered gene expression events with epigenetic and"Quasi-Epigenetic" origins.


This review synthesizes examples of pharmacological agents who have off-target effects of an epigenetic nature. We expand upon the paradigm of epigenetics to include "quasi-epigenetic" mechanisms. Quasi-epigenetics includes mechanisms of drugs acting upstream of epigenetic machinery or may themselves impact transcription factor regulation on a more global scale. We explore these avenues with four examples of conventional pharmaceuticals and their unintended, but not necessarily adverse, biological effects. The quasi-epigenetic drugs identified in this review include the use of beta-lactam antibiotics to alter glutamate receptor activity and the action of cyclosporine on multiple transcription factors. In addition, we report on more canonical epigenome changes associated with pharmacological agents such as lithium impacting autophagy of aberrant proteins, and opioid drugs whose chronic use increases the expression of genes associated with addictive phenotypes. By expanding our appreciation of transcriptomic regulation and the effects these drugs have on the epigenome, it is possible to enhance therapeutic applications by exploiting off-target effects and even repurposing established pharmaceuticals. That is, exploration of "pharmacoepigenetic" mechanisms can expand the breadth of the useful activity of a drug beyond the traditional drug targets such as receptors and enzymes.








DAO inhibition
As our reader Agnieszka pointed out in the comments section, one commonly prescribed beta-lactam antibiotic called Augmentin contains a second antibiotic, Clavulanic acid, to boost its effectiveness; by chance is also a very potent DAO inhibitor. Diamine oxidase (DAO), also known as histaminase, is an enzyme in your body that is used to inactivate histamine. Histamine is found in food that you eat as well as being produced in your body and released by your mast cells during an allergic reaction.

DAO neutralizes the histamine in food so it does not enter your bloodstream.
So this particular antibiotic should be avoided by those people who are histamine intolerant and so do not produce enough DAO. This is about 1% of the general population, but might be more common in those with autism although there is no data on this subject.

Some people believe that ADHD is associated with a reduced level of DAO.
Indeed there is a patent to treat ADHD with a combination of DAO and caffeine.



[0087] DAO can also be mixed with caffeine, strengthening the role of prevention and treatment of attention deficit hyperactivity disorder. Thus, also disclosed herein compositions comprising DAO and caffeine. 
[0088] Caffeine, a xanthine alkaloid group having stimulating properties for the treatment of attention deficit hyperactivity disorder. 
[0089] DAO content of the present invention per unit dose 0 · l-50mg, preferably 2-20mg. 
[0090] The present invention is caffeine content per unit dose 1-lOOmg, preferably 5-50mg. 
[0091] for the prevention and treatment of attention deficit hyperactivity disorder DAO or compositions comprising DAO may be before a meal or postprandial meal administration.
[0092] The use of DAO of the invention or compositions comprising DAO directly affect blood histamine levels, thus affecting the symptoms of attention deficit cumulative histamine levels induced hyperactivity disorder.

You can actually buy DAO supplements and of course caffeine.
Perhaps people consuming DAO inhibitors long term, such as NAC and Verapamil, and have chronic allergies or mast cell disorders might benefit from extra DAO. 




Most DAO is actually in your digestive tract, where the dietary histamine is.

You can measure DAO levels in your blood.

We can conclude that determination of DAO activity in serum is a useful diagnostic tool, together with detailed history to differentiate between food allergy and histamine intolerance.
We found that DAO activity was significantly lower in patients than in healthy control subjects.

Conclusion
I think there is plenty of food for thought here for parents of children whose autism and/or epilepsy improves when taking a beta-lactam antibiotic.  Hopefully some people will figure out which effect is the beneficial one and find something else to replicate it.

There is a lot previously written in this blog about upregulating GLT1, other than by a beta-lactam. My favoured option was Riluzole, but Bromocriptine will also do this, among its other actions. Riluzole is a drug for ALS, that has been trialed in children with OCD, without side effects.    

People technically without histamine intolerance (normal levels of DAO) who incidentally take large amounts of DAO inhibitors, may end up exacerbating an existing mast cell related problem. One potential solution for that small group might be taking an OTC DAO supplement.