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Showing posts with label Verapamil. Show all posts
Showing posts with label Verapamil. Show all posts

Thursday, 24 May 2018

An Autism Case History - EpiphanyASD in a Pill





It is not quite that easy!


Initials:                        LT
Age:                           14 years old

Year
of Birth:              2003

Sex:                            Male

Date:                           24 May 2018

Diagnosis      
LT was diagnosed with autism in January 2007, at a multi-disciplinary assessment in London, at the age of 3 years 6 months.  At that time, LT was non-verbal but had some emerging vocalization. No tics, no seizures, no unusual physical features, no self-injury, no sleep disorder, no feeding disorder. Toilet trained. Very limited attention span. No imaginative play. Liked to jump.

IQ not tested.
No CARS (Childhood Autism Rating Scale) assessment.
TEACCH and PECS were recommended as therapy.
Further medical testing or referrals – none recommended (standard practice in the UK) 

LT has an older brother who is intelligent, multilingual and highly social.
Comorbidities
GI disease:                   None
Epilepsy:                       None
Asthma:                        Yes, mild asthma from early childhood
Allergy:                         Pollen
Sleep disorder:             None 

General Health          
Very healthy and almost never ill. When visiting his GP at the age of 14 the doctor commented how she had not seen him for three years, whereas she has seen his older brother twice a year.

Born via a planned caesarean section, without complications, APGAR score was 10.


Growth                      
Body is well proportioned, no obvious macro/microcephaly. No physical features of any syndromes/metabolic anomalies.

However, LT was initially on the 90th percentile for height and dropped to the 20th by the time he was 5 years old. He was a very muscular baby.  At the age of 10 his bone age (X ray of left hand) was estimated to be two years delayed.  IGF-I was normal, FT3 was slightly above the reference range.

At birth he fitted the research description of hyperactive pro-growth signaling pathways, even though there was no macrocephaly.

Regression at age 8              
Aged 8, a big regression took place with self-injurious behavior (SIB) and aggression to others. He would slam his head into walls, other people, car windows, punch himself etc, but he was still small enough to be physically controlled/restrained by larger adults. He could not be controlled by smaller/older adult family members.

This aggression could occur immediately on waking until finally falling asleep at night, it was not predictable.  At that time in the afternoons, LT had a male 1:1 assistant with experience from a school for severe autism and in the mornings a very firm-minded tall female 1:1 assistant. LT’s father imposed a policy of zero acceptance of any SIB, to avoid it becoming a permanent acquired behavior. SIB was physically blocked.
The regression was triggered by the departure of his long time full-time 1:1 female assistant. It was an emotional trauma.  Occasional visits from her just made the situation worse.  In response no drugs were used, just a consistent firm behavioral approach. Over a ten month period the situation slowly stabilized, but skills were lost and bad habits (SIB) were acquired.  LT subsequently did see his assistant again and sees her regularly to this day.
Throughout this time his classmates and teacher at school were remarkably understanding. He was never excluded from school. His assistant ensured nobody at school got hurt.
Since assistants will inevitably come and go, from the age of 8 LT has had two part-time assistants rather than one full time.  As and when subsequent assistants have left, he has not had any troubling emotional reaction. 

Summer-time raging and loss of cognitive function
Summertime raging with self injurious behavior and aggression to others developed from the age of 9.

Later it became clear that in addition there was a loss of cognitive function during the summer months. This became evident once it was possible to teach mental math, from aged 9 onwards.  For example, at the age of 11, simple verbal tasks like 7 x 8 = ?, that had previously been mastered, could not be answered in the summer months.

The raging and cognitive loss were ultimately treatable.

Winter-time raging

Summertime raging was resolved and then winter-time raging developed. This was traced back to the cytokines released to signal reabsorption of milk teeth roots (a proves that takes months) and the eruption of permanent teeth. It was not tooth ache, i.e. pain. LT has retarded bone age and apparently this applied to his teeth development as well.

He lost his later milk teeth always in the winter.

The winter time raging did not respond to his summertime therapy, but responded very well to a low dose of ibuprofen. Summertime raging does not respond to Ibuprofen 

PANS-like episode aged 13
At Christmas time, following a minor viral infection, LT developed acute onset profound verbal tics. LT does not have Tourette’s type autism and had never exhibited such behavior previously. The tics were treated as a PANS/PANDAS flare-up with 5 days of prednisone. Over a two week period the tics faded away and have never returned.

Intellectual disability 
IQ was never measured, LT’s ABA consultant said there was no point, but the very much more rigorous ABBLS was completed, see below. Evidently, prior to pharmacological treatment at the age on 9, there was a 5 year developmental delay.
With hindsight, IQ pre-treatment was probably in a similar range to Down Syndrome (DS) meaning less than 70.

At the age of 14, LT’s academic performance now puts him in the top half of his class of 12 year old neurotypical peers. His grades are mainly As, with maths and computing being particular strengths. 

Other testing:    No genetic testing, MRI or EEG.

Family History:          

LT has a 7 years younger, very distant cousin who is non-verbal with autistic disorder. They have shared great great great maternal grandparents. The cousin has parents who are both doctors and were high academic achievers as medical students.

The father’s family has a large number of Cambridge-educated doctors on both the grandmother's and grandfather's side; one gave his name to the scale still used to assess severity of Ulcerative Colitis and helped develop the first H2 anti-histamine drug. The father and uncle are engineering graduates from top universities. One distant cousin was a math’s protégé at Oxford University.  One distant cousin has bipolar. One uncle has type-1 diabetes.

The mother is an academic alpha female in a stressful creative profession. The maternal grandmother was a teacher and grandfather was an army Colonel.
The maternal grandmother and her children all had premature hair greying, which may be linked to Bcl-2 expression and Wnt signaling, both implicated in autism. Thickness and greying of hair share biological mechanisms, which overlap with those controlling development of dendritic spines. LT and his father have very dense hair, mother has thin hair.
Maternal grandparents both smoked and the grandfather has COPD (Chronic Obstructive Pulmonary Disease). Oxidative stress is a core feature of COPD, because anti-oxidant genes are silenced; these effects are known to be heritable via epigenetic tags. 
The family fits the high IQ  type of autism (some autism genes are linked to intelligence and some bipolar genes are linked to creativity, which helps explain why some actors/artists are bipolar) with oxidative stress raised during pregnancy, anti-oxidant response possibly weakened, no oxytocin surge during delivery and no microbiota transfer at birth (C-section delivery).  No pets at home during pregnancy (a good source immuno-stabilizing bacteria).  No obesity in the family.

Education
LT has attended the same mainstream international school, following the English curriculum, since the age of 3. Class sizes are very small, about 12 pupils. From the age of 4 he has had a 1:1 assistant eight hours a day, throughout the year.
LT commenced a parent-managed ABA (Applied Behavioral Analysis) inspired home program shortly after diagnosis.  Both parents attended a 2 day training program to learn the use of PECS (Picture Exchange Communication System).  PECS was applied and shortly thereafter LT became partly verbal at the age of 4, speaking single words.
1:1 assistants were recruited mainly from the local University and trained to apply ABA, with elements from Floortime and the Canadian Hanen Program. There was some supervision from US-trained Behavioral Consultants that would fly in for training. A large collection of specialist training material was acquired from the US. 
Extensive use was made of professional (i.e. expensive) special needs language teaching software (Laureate Learning) from the age of 4 until 8 years old.
Later, web-based reading software (Headsprout) was used and years later special maths teaching software (Math Wizz). Neither are made for special needs, but both are very compatible with an ABA approach.
LT spent an extra year in kindergarten and in primary/junior school was held back 2 years at the age of 9, following a request from the parents.
In primary school (English system) he went Year 1, Year 2, Year 3 (started bumetanide) then back to Year 2, then Year 3, Year 4, Year 5, Year 6 and currently attends Year 7 in secondary/high school
The equivalent in the US system would have been, he went K, 1st, 2nd, then 1st, 2nd 3rd, 4th etc.
From the age of 13, LT attended school full time, prior to that he attended only the morning and then went home after lunch to work 1:1 with his assistant for three hours.
During school holidays LT has a 1:1 home learning program.
LT learnt to read and write at home as result of the unrelenting efforts of his assistant. He started to learn maths from the age of 8, prior to that he could not master the basic concepts, or understand the relevant vocabulary.
From the age of 9, LT has been able to keep up with his new peer group at school, two years his junior.
At the age of 14, in a class with 12 year old neurotypical children, LT takes the same assessments as the rest of the class and his grades currently place him in the top half of the class. He is now particularly good at things like arithmetic, algebra, coordinates, spelling and has neat handwriting (very unusual in autism). He is still clearly autistic and his speech is limited to what he wants to say; there is no small talk.
LT started to learn the piano aged 8. He progressed from an extremely basic level and a desire to hit his teacher to his current level 4 of the popular Faber Music piano course (there are just 5 levels). When he plays in public people are very surprised, he does not play like someone with any cognitive impairment. His peers as school have asked “how can he play like that?” 

Motor Skills
Like many people with classic autism LT had problems with both fine and gross motor skills as an infant. After a great deal of 1:1 therapy, motor skills are now normal.
LT started to learn to ski at the age of 5 with a special needs instructor. Progress was initially slow, but 9 years and one broken collar bone later, LT can confidently ski on red slopes and deal with all the various types of lifts you encounter in the Alps.
Stamina improved considerably after starting to take Agmatine, which is evident at school where they are timed to run 2.5 km (1.5 miles) and when swimming.

Behavioral Treatment (age 3- 8)
From diagnosis aged three, until nine years old, therapy was exclusively based on behavioral interventions. Extensive use of ABA (Applied Behavioral Analysis) and VB (Verbal Behavior) with 40 hours a week with a 1:1 Assistant.
At the aged of 9, LT had mastered almost all the skills in the very extensive ABBLS (Assessment of Basic Language and Learning Skills) assessment. The language skills and other basic learner skills that are tracked by this tool are those that are acquired by most typically developing children by the time they reach four to five years of age. LT’s elder brother had acquired these while he was three years old.
LT’s skill acquisition to the age of eight was seen by the ABA consultants as nothing unusual in someone with classic autism. There was slow but continuous progress. 
All learning was taking place at home with school attended mainly for socialization.

Pharmacological Treatment (age 9 onwards)
In late 2012 a small clinical trial was published by Ben Ari and his clinical associate Lemmonier; it showed the benefit of the NKCC1/2 blocker bumetanide in autism. This paper was studied by LT’s father and contact has been maintained for several years with Dr Ben Ari, who originated and patented this therapy.
Bumetanide (1mg per day) was commenced just before Christmas December 2012, unknown to the school, or LT’s assistants.
On returning to school in January 2013 the Head Teacher summoned LT’s father and asked what had happened to LT. He was “so joyous” and “like a different child”.
At the suggestion of his original ABA consultant, LT’s father had been asking LT every school day for 5 years “what did you have for lunch at school today?”. The usual answer would be no answer, the wrong answer, but sometimes a brief correct answer. From now on LT would say precisely what he had eaten “peas, potatoes and chicken – cake for dessert”. The assistant was there to confirm what had really been eaten for lunch. 
LT’s 1:1 assistant at that time described the effect of bumetanide as making him “more present”. Since his assessment at the age of 3, it was always noted that LT had a very short attention span and would not be able to focus on the class teacher for more than a couple of minutes. LT was never hyperactive, quite the opposite. He was physically present but not mentally.
Later on it would be realized that the most potent effect of long term bumetanide use in strictly defined autism (SDA) is enhanced cognition, which leads to accelerated acquisition of new skills.  IQ has long been seen as the best predictor of more favorable outcomes in autism.  
Bumetanide use has continued for five years, with occasional pauses to confirm it still works.  Different doses were tested and currently the dose is 2mg once a day.
When stopping bumetanide for a week and returning to his web-based maths learning program, LT was unable to complete previously mastered tasks, no matter how many times he tried. Having recommenced bumetanide, the same maths problems were attempted a week later and could be solved. 
Blood potassium levels were checked regularly at the beginning, but were always high normal (5.0 mmol/L).  Bumetanide is taken with 250mg of K+ per 1mg of bumetanide. Diet is rich in potassium, with bananas and other fruit.
Dehydration, another potential problem, is entirely self-regulated with LT drinking more water. Total consumption is 2.5 to 3 liters per day.
Diuresis occurs mainly within one hour of taking bumetanide and has never caused a problem at home or school. LT takes his bumetanide at least an hour before leaving home for school.
Bumetanide’s suggested mode of action is lowering intracellular chloride via blocking NKCC1 cotransporters in the brain.  Bumetanide crosses the blood brain barrier very poorly and many researchers are dubious it can have any effect. Bumetanide is a partial solution.
A new drug is being developed by Dr Ben Ari that will cross the blood brain barrier more effectively than bumetanide and have less effect on NKCC2, so producing less diuresis.
An alternative strategy discussed in the literature is to improve the pharmacokinetics of bumetanide, by slowing its excretion via OAT3 (organic anion transporter 3) and thus increasing plasma concentration. There are many OAT3 inhibitors, the best known and most potent is probenecid, used to treat gout by increasing the excretion of uric acid. Some foods are OAT3 inhibitors. One readily available substance is chlorogenic acid (more precisely 1,3- and 1,5-dicaffeoylquinic acid) which is sold as a coffee-based weight loss supplement. Interestingly, coffee, but not caffeine, has been shown to reduce the risk of gout.
Little is known about exactly how bumetanide is transported/excreted across the blood brain barrier.
Bumetanide’s autism benefit appears to be from lowering intracellular chloride and hence making GABAA become more inhibitory. Excitatory-Inhibitory (E/I) imbalances are widely believed to be at the core of autism.  An E/I imbalance during so-called Critical Periods, will result in permanent changes to the developing brain, nonetheless it appears that correcting an E/I imbalance in later years can still be highly beneficial, though not curative. 
Another experimental therapy also makes GABAA become more inhibitory. This uses very low doses of clonazepam to modify the behavior of GABAA receptors that contain the α3 sub unit.  In LT the effective dose of clonazepam is just 0.03mg, which might be considered sub-clinical, but as predicted by Professor Catterall, it does have a beneficial effect (a bumetanide-like effect). It has no side effects and there is no tolerance develops at this tiny dose, after four years of use.
At the time low dose clonazepam was introduced, LT would go swimming at 5pm most days. He was not really interested to do much independently in the water, he was very passive. This passive behavior was notably changed once the effective clonazepam dose had been found. He became more like a typical child playing in a swimming pool. Instead of sitting on the steps he wanted/demanded interaction/play with the attending adult.  The effect was not as profound as that seen in the first months of bumetanide, but noticeable nonetheless.
After 4 years of bumetanide the effect was still there, but there was a desire to accelerate skill acquisition to keep up with neurotypical school peers.
A new strategy was adopted to further reduce intracellular chloride, this time using a method first documented in the 1850s, when potassium bromide (KBr) was used to treat epilepsy. Reading old case studies from Great Ormond Street Hospital in London it appeared to LT's father that some children with epilepsy, MR/ID and undiagnosed autism improved behaviorally and developed age-appropriate play when treated with KBr. Lack of age-appropriate play is a hallmark of autism.  Modern research shows that bromide ions compete with chloride ions to enter cells and the result is a lower intracellular concentration of Cl-. The limiting factor in the use of KBr is that it increases mucous secretions and so causes acne (and can make asthma worse), in a dose dependent fashion. At a low dose of 400mg per day there is a cognitive gain without significant spots. KBr is still used at high doses to treat pediatric epilepsy in Germany and Austria. Some leading US neurologists regret they cannot prescribe it; technically they could ask the FDA for permission on a patient by patient basis.

Another strategy to reduce intracellular chloride is to target chloride ions that enter neurons via the AE3 exchanger, this is possible using Acetazolamide (Diamox). This therapy does seem to work for some people, but was not tolerated by LT, it caused reflux.
KBr has a very long half-life and so it takes 4-5 weeks to reach the maximum effect. 
Bumetanide took about two weeks to lower chloride and show behavioral and cognitive improvements.
Low dose clonazepam takes three days, as was predicted by its half-life.
The cognitive loss in severe autism has parallels with that in Down Syndrome (DS). Bumetanide has been patented as a therapy for DS by Ben Ari, based on the results from mouse studies.
In mouse models of Down Syndrome both a negative allosteric modulator and a selective inverse agonist of α5 sub-unit of the GABAA receptor improve cognition. 
Mouse research has shown that poor learners have greater GABRA5 expression than good learners and that in mice GABRA5 expression can be normalized by eating cinnamon, or its metabolite sodium benzoate (NaB); this makes a poor learner become a good learner, at least in mice.
So it may be that increasing the effect of α3 sub-unit of the GABAA and reducing the effect of the α5 sub-unit of the GABAA can both improve cognition. For the moment the latter remains unproven. NaB is an approved food additive, E211. Ceylon cinnamon, which is safe for long term consumption, is metabolized to NaB. People who are histamine intolerant have to avoid DAO inhibitors such as cinnamon and NaB. 

Summertime raging and loss of cognitive gains
From the aged of 8 it became apparent that summer provoked behavioral deterioration. At this point there was no obvious allergy, but behavior improved when moving to the mountains in summer. At first, OTC mast cell stabilizers were investigated; some common H1 antihistamines are partial mast cell stabilizers. Rupatadine, azelastine, ketotifen, loratadine and cetirizine were all tried, as was the flavonoid quercetin.
Some of the above did indeed help reduce the summertime self injury, but not to a satisfactory level.
A final solution was found in a small dose of the Cav1.2 blocker, verapamil. 
When mast cells degranulate, one step requires activation of an L-type calcium channel. This is why most mast cell stabilizers are actually calcium channel blockers.
It should be noted that mutation in the CACNA1C gene, which encodes the Cav1.2 ion channel, leads to a severe kind of autism called Timothy Syndrome. Because Cav1.2 is widely expressed in the heart those affected have a very poor prognosis.
In addition, verapamil blocks the potassium ion channel Kv1.3.  Potassium channels, Kv1.3 and KCa3.1, have been suggested to control T-cell activation, proliferation, and cytokine production. Kv 1.3 is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases.  Research has shown that peptides from parasitic worms that suppress the body's immune response do so by blocking Kv1.3. A drug therapy based on these peptides is being developed.
Verapamil also upregulates autophagy, which is impaired in many neurological disorders, such as Huntington’s. Lack of autophagy has been linked to the synaptic pruning deficits found in autism.
Verapamil has a short half-life of about 3 hours. Only a small dose is required to prevent the onset of SIB and the preceding agitation (described by LT as “spray the fire in my head”).
From the age of 10, LT’s summertime raging has been treated with 40-80 mg of Verapamil split into 2-3 doses from May until late November.
On the occasions that he has missed his 1pm dose in the peak allergy period, he has repeatedly developed aggression and self-injury by 4 or 5pm.
When he has taken verapamil there has never been any aggression and or self-injury.
Once self-injury was removed as a concern, learning progressed during the long summer school holidays. It became clear that during summer cognition was reduced as if bumetanide was no longer working.
It has been shown that the expression KCC2, the cotransporter that allows Cl- to leave neurons is affected by inflammatory cytokines like IL-6. It therefore appears plausible that the histamine and IL-6 released directly and indirectly by mast cell degranulation was causing an increase in neuronal Cl- and thus undoing the good work being done by bumetanide. Inflammation also increases α5 GABAA receptor activity and can thus reduce cognitive function.
At this point, the bumetanide dose was raised from 1mg once a day to 2mg in the morning and on occasion 1mg in the late afternoon.
The combination of an increased dose of bumetanide and the use of verapamil, cetirizine and azelastine has produced a very favorable result (no SIB and minimal summertime cognitive decline). Perhaps of note is that cetirizine is an eosinophil stabilizer, which may also be helpful and not just for asthma.
OTC therapies that have a helpful effect in summer are L-histidine, curcumin and L. reuteri DSM 17938 (sold as Biogaia Protectis). The amino acid histidine is a precursor to histamine and it seems that the body’s feedback loops can be tricked into not degranulating mast cells by slightly increasing the level of circulating histidine. The immunomodulatory effects of L. reuteri DSM 17938 have been well studied; the effect however does not continue after prolonged use. Curcumin is a very widely studied natural substance that performs much better in vitro than in vivo, due to very poor bioavailability. Modified versions of curcumin have been developed and there is a marginal benefit. Histidine is extremely cheap and easy to administer. Modified curcumin and L. reuteri are quite expensive.
It is reported by others that at a higher dose verapamil is as effective as an H1 antihistamine in treating allergy. 

IPR3
It appears that aberrant calcium channel signaling is a key feature of much autism. Gargus has suggested that IP3R is a nexus for different dysfunctions that lead to autism. IP3R controls the release of calcium stored within cells (the endoplasmic reticulum).
Excessive calcium within cells is known to be damaging. L-type calcium channels that remain open will raise intracellular calcium and the same is true with IP3R. Caffeine can be used to inhibit calcium release via IP3R.
Gargus has not proposed an IP3R therapy.  


RORα

RORα is another proposed nexus where different dysfunctions  that lead to autism may converge. One potential RORα agonist is estradiol.  We know that in much autism there is elevated testosterone and reduced estradiol; we also know that estrogen receptor beta is under-expressed. Estradiol is known to be highly neuroprotective and may help protect females from developing autism. Females lacking in estradiol, for example in Turner Sydrome, may exhibit features of autism. A logical therapy would be to either use estrogens, or reduce testosterone (effectively the same thing). Ideally you would do this just in the brain; a brain selective pro-drug of estradiol, called DHED, actually exists. Less ideal therapies range from estradiol itself, to phytoestrogens or a high soy diet, to drugs reducing testosterone, like spironolactone; these will have effects beyond the brain.

Wintertime raging
Having solved summertime raging, wintertime raging appeared. As expected, verapamil had no effect.
Ultimately the likely trigger was traced back to the very slow loss of milk teeth and eruption of permanent teeth. Both reabsorption of roots and the eruption new teeth is signaled using pro-inflammatory cytokines.
Moderate use of Ibuprofen, as and when behavior began to deteriorate, resolved the problem. Ibuprofen has no effect on summertime raging.

PANS-like episode aged 13
PANS (Pediatric Acute-onset Neuropsychiatric Syndrome) and PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections) are infection-induced autoimmune conditions that disrupt a patient’s normal neurologic functioning, resulting in a sudden onset of Obsessive Compulsive Disorder (OCD) and/or tics and cognitive loss.
The import part is acute-onset; behavior changes overnight.
LT exhibits the classic traits of autism including stereotypy/stimming but never tics, which are a feature of Tourette’s-type autism.
Just before Christmas LT was recovering well from what presented as mild viral infection that had not warranted any medical intervention. He suddenly developed very loud verbal tics.
It is well known in PANS that delayed treatment severely affects prognosis. The sooner the patient is treated, the more complete recovery will be. Diagnosis is based on a very specific set of laboratory tests, only available in the US.
LT was treated from the third day of the tics as if he had PANS flare-up. He was treated with 40mg of prednisone for 5 days, requiring no taper.
Over a two week period the tics faded away. There have been no more tics.

Use of antioxidants
A recurring feature in autism research is oxidative stress. Two clinical trials have shown the benefit of the antioxidant NAC (N-acetylcysteine) in autism.
In LT the effect of NAC is the immediate disappearance of stereotypy and a type of anxiety. Without NAC, LT always wants to know what is happening next, to the point of obsession.
Oxidative stress has been shown to vary throughout the day and LT’s therapy is tailored to match it. Oxidative stress causes a cascade of further disruptions and causes many of the side effects of type-1 diabetes, for example.
LT takes 2,400 mg of NAC per day (a dose slightly lower than in the clinical trials). He has 600mg immediate release NAC at 7am, 600mg sustained release at 7am and then 600 mg sustained release at 1pm and 5pm. 
There have been no side effects after more than 4 years. 

Anti-inflammatory
Numerous studies (e.g. Ashwood) show elevated pro-inflammatory cytokines and reduced  anti-inflammatory cytokines as a feature of autism; but specific subgroups exist. Activated microglia is another feature of autism, which also suggests chronic inflammation.
Numerous anti-inflammatory strategies have been researched.
Atorvastatin has potent anti-inflammatory effects that are very well studied. It also affects the autism/cancer proteins RAS, PTEN and BCL2.
RASopathies are associated with MR/ID and indeed autism. Mutations in PTEN generally cause loss of function in PTEN and are associated with macrocephaly, enlarged corpus callosum, MR/ID and autism. Loss of function of PTEN is also found in some cancers, for example prostate cancer.
Because autism is polygenic and hundreds of genes are over/under expressed, it is not necessary to have a mutation to have misexpression. The mutation is just the extreme case (be it Cav1.2 or PTEN).
The effect of Atorvastatin is visible from the first dose and fades away the next day if therapy is stopped. The effect is very specific, it releases cognitive inhibition; it is as if the person with autism has the desire and capability to do something, but some barrier prevents him from doing it.
In broader severe autism, this is very important, Why does a child with autism who can verbalize never speak?
At the age of 9, LT was having piano lessons at home twice a week. He would practice the piano only if his assistant or father sat beside him. He never played independently.
After taking 10mg Atorvastatin for the first time, the next day LT went himself to his piano and started playing, without any prompting of any kind. He then began to practice on a daily basis.
As a child aged 3, LT had the habit of coming to the entry of the room with the television and watching from around the corner of the wall. He wanted to watch but could not enter the room. At the time it was thought he somehow just liked the visual sensation of peering around corners.
When he later moved to a multi-level house, LT would not come downstairs by himself; he would wait at the top of the stairs for someone to lead him down, every morning.  With atorvastatin not only did this behavior disappear, but it reappeared the day after Atorvastatin was withdrawn.
During one test withdrawal of the treatment, he got “stuck” in the kitchen and could not leave the room.

Sulforaphane Nrf2 and HDAC
In 2014, and again in 2017, Talalay/Zimmerman published research that sulforaphane from broccoli showed a benefit in autism. Sulforaphane is an HDAC inhibitor and thus has potential epigenetic properties, like some cancer drugs. Sulforaphane may also activate the Nrf2 redox “switch” and so be protective in conditions associated with oxidative stress.
LT’s father did contact the researchers and shortly after the first research was published LT started to take a broccoli sprout supplement. It did produce a very obvious effect and within 30 minutes; LT was laughing so much, be went to look at himself in the bathroom mirror. The more general effect was an unmissable increase in speech.
After three years of use the positive effect of sulforaphane/broccoli is no longer visible, even trying alternative brands.
In the 2017 clinical trial the authors found one responder retained the benefit of sulforaphane after the trial ended. They suggest an epigenetic switch may have been activated.  

Mitochondria and Microvasculature
A distinct type of autism has been characterized by Kelley at Johns Hopkins, Autism Secondary to Mitochondrial Disease (AMD). Kelley suggests that almost all regressive autism is caused by mitochondrial dysfunction and usually deficiency of the rate-limiting complex 1.
By stabilizing the mitochondria with antioxidants and then trying to stimulate more complex 1, a gradual improvement can occur.
Mitochondrial disease effectively starves the brain and body of energy (ATP), so lack of exercise endurance is exhibited in people with a genuine mitochondrial dysfunction.
One feature of autism is that growth factors (BNDF, IGF-1, NGF, VEGF etc) are disturbed, but the disturbance varies greatly by the type of autism.  Vascular endothelial growth factor (VEGF) in particular and its receptors are known to be disturbed and this has implications for microvasculature. Studies suggest that unstable, rather than reduced blood flow occurs in autistic brains.
In sports medicine, exercise endurance is a key target and it can be raised by improving the energy production from mitochondria and by improving the circulation of blood throughout the body by targeting eNOS (Endothelial Nitric Oxide Synthase) and NO (Nitric Oxide).
In Mild Cognitive Impairment (MCI) studies have shown the benefit of improved cerebral blood flow using cocoa flavanols to indirectly affect NO and hence improve memory.
Studies show that eNOS and NO can be safely increased by Agmatine and NO can be increased  using L-citrulline, which then produced more L-arginine. These supplements are widely used by sportsmen and women.
A small dose of Agmatine (1 g) has a near immediate substantial effect on LT, making him far more energetic.  It moved him from being rather passive physically, to being active. This has been very evident from his performance at school during physical activities, where it has been widely noted. At home LT started trampolining before breakfast and late in the evening.

Sensory Overload and Sensory Gating
An apparent over-sensitivity to sensory stimuli is a common observation in autism and is often the precursor to behavioral problems. In some younger children these can be trivial, but in more severe autism it can produce profound behavioral problems that never fade away.
Hypokalemic sensory overload and hypokalemic periodic paralysis are described in the literature. LT had sound sensitivity as a young child, in particular an inability to cope with the sound of crying. Tests were carried out to establish whether LT’s tolerance to the sound of crying improved after oral potassium. He consistently tolerated a high volume of a recording of this sound, when played 20 minutes after 250mg of potassium. Following ABA, he was purposefully exposed to this sound and taught to understand why people cry and modify his response, to the extent that his response changed to laughter, which again has to be modified towards empathy. 
Aged 10, LT developed a phobia to traveling in elevators/lifts. This was because the elevator he regularly used to visit his Grandparents was the old-fashioned type, with an internal sliding gate that you close by hand, which is extremely noisy.  He refused to use the elevator from that point on.  People with autism very easily form habits, or are allowed to form them, following the path of least resistance.  Elevators are a part of modern life and hard to avoid.
After a few weeks of this behavior, LT was given 500mg of potassium and half an hour later willingly entered the elevator and coped with the ride. The behavior has never recurred.
Sensory gating is another common issue in autism and schizophrenia, the individual is not able to filter out repetitive background sounds, like a clock ticking or the sound of a noisy eater. Sensory gating can be measured by looking at the P50 response on an EEG. α7 nicotinic acetylcholine receptor (α7 nAChR) agonists, like nicotine, can correct impaired P50 gating. A low dose of a PDE4 inhibitor is another suggested therapy
LT does exhibit was presents as impaired P50 gating. It is really only evident when his pharmacological therapy is halted for a few days. Then he finds all kinds of unavoidable noises very annoying, even the sound of a person sitting next to him eating. 

Typical Psychiatric Drugs
LT has never been treated with any of the usual antipsychotics, stimulants, anti-depressants, or anti-anxiety drugs sometimes prescribed in autism. His use of clonazepam is at a dose far below its standard clinical use.

Current status
In September 2017 LT moved to secondary/high school where some of the teachers recall how he used to be 10 years previously. Initially there was some trepidation and the view by some that a boy with classic autism should not be there. The school does have a boy with Asperger’s. However, LT surprised his new teachers, achieving grades placing him in the top half of his class. He is now extremely attentive in class, no attention deficit anymore, and has clearly not reached his intellectual limit. He has likely already far surpassed his intellectual limit, had he remained untreated.
As the end of the first year of high school approaches, LT continues to keep up academically with his peers. His agmatine-boosted physical performance has been maintained and he competes very well in long distance running and swimming.
LT is still intellectually far away from the trajectory followed by his older brother, but LT is keeping up academically with many of his classmates who are neurotypical, with average IQs.
A significant number of people diagnosed very young with autism do indeed make dramatic progress by the age of 6.  Zappella proposed his Dysmaturational Syndrome that he says applies to about 6% of early childhood autism, but they all have Tourette’s type autism (with tics).   There is an additional group without tics that also achieve what Fein calls Optimal Outcome, essentially they lose their autism diagnosis. In total it is 10-15% of cases that seem to “get better” all by themselves, regardless of intervention. As more diagnosis takes place even before 2 years of age and autism threshold grows ever wider, Optimal Outcome may become even more common.  
The definition of autism has been greatly watered down in recent years (DSM3 to DSM5). LT started with DSM3-type autism and by the age of 8 he still had it. DSM5 autism includes very much milder variants, some of which are trivial.
Each therapy used by LT has been found to be reversible based on careful withdrawal trials.



People with strictly defined autism (SDA) start to acquire skills with a delay compared to NT peers and thereafter acquire skills at a slower rate and hence fall ever further behind, making inclusion at school a delusion. The aim is to have similar skills to NT peers to make inclusion effective.
People with SDA often leave high school with an educational level of a 7 to 10 year old.

From the age of 12, LT ceased having any autism-specific learning curriculum; he just follows the curriculum of his mainstream school.  

Anecdotal Evidence
LT’s piano teacher exclusively teaches people with disabilities (mainly severe autism and a few with Asperger’s) and so has great experience of the disorder. She says while she has taught people who learnt to play as well as LT does today, this has never happened before with a child who started in his kind of condition at 8/9 years old.
The American ABA consultant (with Ph.D. and 20 years of experience) knowing LT from the age of 8, before he started bumetanide, told the family that of all her clients, LT is the one she sees the least but has improved the most and how strange that is. 


Current Therapy

The current therapy, called the Autism PolyPill, may be found in the link below.  

https://epiphanyasd.blogspot.com/p/polypill-for-autism.html

Autism is a highly heterogeneous condition, but there appear to be broad sub-types. At least some people with an autism diagnosis respond to each individual therapy in the PolyPill. Some people respond to almost the entire combination of therapies; other people respond to none.


Future Therapy

Some other interesting therapies remain to be investigated and it is clear that more improvement is possible because short term therapy with the flavones nobiletin and tangeretin produces a marked change in cognition and behaviour. The effect only lasts two or three days.  Tangeretin is a PPAR gamma agonist, among other properties. It reduces cholesterol when used long term, but its autism benefit is transient.  

The ketone Beta-Hydroxy Butyrate (BHB) also looks interesting; it has epigenetic properties amongst its other effects. 






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=

2.201, P<0 .05="" 1a="" a="" agc1="" ca="" figure="" increase="" instead="" left="" levels="" modest="" nonsignificant="" only="" protein="" s1="" show="" sup="" upplementary="" very="">2+
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.