Sunday, 26 February 2017

Secondary Monoamine Neurotransmitter Disorders in Autism – Treatment with 5-HTP and levodopa/carbidopa?

This post is about monoamine neurotransmitter disorders in Autism, that are usually a down-stream consequence of other miscellaneous dysfunctions, which makes them “secondary” dysfunctions.

There was a post on this blog way back in 2013 on catecholamines:

Classical monoamine is a broader term and encompasses:-

       ·          Classical Tryptamines:

Drugs used to increase or reduce the effect of monoamines are sometimes used to treat patients with psychiatric disorders, including depression, anxiety, and schizophrenia.

This blog does go on rather ad nauseam about histamine, so today it will skip over it.  It does not cause autism, but it certainly can make it much worse in some people.

Tryptophan is a precursor to the neurotransmitters serotonin and melatonin.  For years it has been known that odd things are going on in some people with autism regarding tryptophan, serotonin and indeed melatonin. This research does not really lead you anywhere.

Other than being converted to serotonin and melatonin, tryptophan has the potential to be converted in the gut into some very good things and some bad ones; this all depends on what bacteria are present. People lucky enough to have Clostridium sporogenes will produce a super potent, but apparently very safe, antioxidant called 3-Indolepropionic acid (IPA), which is seen as an Alzheimer’s  therapy.  To be effective you would need a constant supply of IPA, and that is exactly what you get from the right bacteria living in your gut.

Some people with autism have high levels of serotonin in their blood and so do their parent(s). It is known that in the brain many people with autism have low levels of serotonin.  Various mechanisms have been proposed to explain this using the body’s feedback loops, including mother to child.

Many people with autism take 5-HTP which is an  intermediate in the synthesis of both serotonin and melatonin from tryptophan.

Serotonin itself does not cross the blood brain barrier (BBB).

Too much serotonin in your brain has a negative effect and so taking too much 5-HTP supplement produces negative effects.

Many people take melatonin at small doses for sleep. At larger doses it has many other beneficial effects that range from resolving GI problems to reducing oxidative stress in mitochondria. 

Of the Catecholamines, it is dopamine that gets the most attention in neuro-psychiatric disorders and schizophrenia in particular.

There is a dopamine hypothesis for schizophrenia, but there is also a glutamate hypothesis of schizophrenia. 

If you read the research, it is actually ADHD that has the strongest connection to dopamine.  When you look closer still, you will see that even that connection is quite weak.

The conclusion is that ADHD, just like autism and schizophrenia is usually multigenic, meaning that numerous little things went awry, rather than one single dysfunction.

Tourette's syndrome and related tic disorders may be associated with either too much dopamine or overly sensitive dopamine receptors. 

It is fair to say that secondary monoamine neurotransmitter disorders can occur in autism, ADHD and indeed schizophrenia.

There is a long list of primary monoamine neurotransmitter disorders and much is known about them.

Monoamine Neurotransmitter Disorders  

I found an excellent paper that tells you pretty much all you could want to know about monoamine neurotransmitter disorders.  It also has nice graphics to explain what is going on.

Most people with autism are unlikely to have a primary disorder, but if they did, treating it should have a big impact on them.

BH4 =tetrahydrobiopterin. TH-D=tyrosine hydroxylase deficiency. AADC-D=aromatic L-amino acid decarboxylase deficiency. DTDS=dopamine transporter deficiency syndrome. PLP-DE=pyridoxal-phosphate-dependent epilepsy. P-DE=pyridoxine-dependent epilepsy. AD GTPCH-D=autosomal dominant GTP cyclohydrolase 1 deficiency. SR-D=sepiapterin reductase deficiency. AR GTPCH-D=autosomal recessive GTP cyclohydrolase 1 deficiency. PTPS-D=6-pyruvoyltetrahydropterin synthase deficiency. DHPR-D=dihydropteridine reductase deficiency. HIE=hypoxic ischaemic encephalopathy. PKAN=pantothenate kinase associated neurodegeneration. DNRD=dopa non-responsive dystonia. PKD=paroxysmal kinesogenic dyskinesia.

People with a secondary disorder would typically be identified by testing their spinal fluid for the metabolites of the monoamine.  So for serotonin you measure  5-HIAA (5-hydroxyindoleacetic acid) and for dopamine you measure  HVA (homovanillic acid).

Figure 2: The monoamine neurotransmitter biosynthesis pathway BH4 is synthesized in four enzymatic steps from GTP. BH4 is a necessary cofactor for TrpH and TH, the rate limiting enzymes in monoamine synthesis. Tryptophan is converted to 5-HTP by TrpH. Tyrosine is converted to L-dopa by TH. The conversion of 5-HTP to serotonin and of L-dopa to dopamine is catalyzed by AADC and its cofactor PLP.  When BH4 acts as a cofactor for TH and TrpH, it is converted to PCBD, which in turn is converted to BH4 (in the BH4 regeneration pathway) by a two-step process involving PCD and DHPR. After synthesis, uptake of monoamine neurotransmitters into the synaptic secretory vesicles requires the vesicular monoamine transporter VMAT (not shown).⁶ After synaptic transmission, serotonin and dopamine are metabolised through similar pathways, which involve MAO enzymes and COMT. Presynaptic reuptake of the monoamines is facilitated by DAT and SERT (not shown).⁷ Metabolic pathway of BH4 synthesis is shown in light blue, monoamine synthesis in light green, monoamine catabolism in dark blue, and BH4 regeneration in red. The biogenic amines are illustrated in light green circles and the cofactors (BH4 and PLP) are represented by light blue circles. Enzymes in the monoamine neurotransmitter pathway are underlined. GTPCH=GTP cyclohydrolase 1. H₂NP₃=dihydroneopterin triphosphate. PTPS=6-pyruvoyltetrahydropterin synthase. 6-PTP=6-pyruvoyltetrahydropterin. AR=aldose reductase. SP=sepiapterin. SR=sepiapterin reductase. BH4 =tetrahydrobiopterin. TrpH=tryptophan hydroxylase. TH=tyrosine hydroxylase. DHPR=dihydropteridine reductase. PCBD=tetrahydrobiopterin-α-carbinolamine. PCD=pterin-4αcarbinolamine dehydratase. qBH₂=(quinonoid) dihydrobiopterin. 5-HTP=5-hydroxytryptophan. L-dopa=levodihydroxyphenylalanine. COMT=catechol-O-methyltransferase. 3-OMD=3-ortho-methyldopa. VLA=vanillactic acid. AADC=aromatic L-amino acid decarboxylase. PLP=pyridoxal phosphate. DBH=dopamine β hydroxylase. PNMT=phenylethanolamine N-methyltransferase. MAO=monoamine oxidase. AD=aldehyde dehydrogenase. 3-MT=3-methoxytyramine. DOPAC=3,4-dihydroxyphenylacetic acid. 5-HIAA=5-hydroxyindoleacetic acid. HVA=homovanillic acid. MHPG=3-methoxy-4-hydroxylphenylglycol. VMA=vanillylmandelic acid.

The paper is very clear about what to:-

Secondary neurotransmitter disorders

Neurotransmitters abnormalities indicative of dopamine or serotonin depletion are becoming increasingly recognized as secondary phenomena in several neurological disorders. Concentrations of HVA and 5-HIAA in CSF in such patients are mostly within the range deemed abnormal for primary neurotransmitter disorders, but generally do not reach the lowest levels.

A secondary reduction in HVA is reported in perinatal asphyxia, disorders of folate metabolism, phenyl ketonuria, Lesch-Nyhan disease, mitochondrial disorders, epilepsy (and infantile spasms), opsoclonus-myoclonus, pontocerebellar hypoplasia, leukodystrophies, Rett’s syndrome, and some neuropsychiatric disorders.  Many patients who have no specific diagnosis but who present with neuromuscular or dystonic symptoms have low HVA concentrations in CSF, which suggests dopaminergic depletion. These patients also often present with dyskinesia, tremor, and eye-movement disorders similar to those seen in many of the primary monoamine neurotransmitter disorders. Cortical atrophy is associated with low levels of 5-HIAA in CSF. Low concentrations of HVA and 5-HIAA have been reported in patients with type 2 pontocerebellar hypoplasia and in a syndrome that involves spontaneous periodic hypothermia and hyperhidrosis.  Whether the latter syndrome is a primary or secondary neurotransmitter disorder is still unclear because the underlying cause is unknown. Patients with neonatal disease onset who have severe motor deficits and abnormalities on brain MRI seem particularly vulnerable to secondary reductions in HVA production. Such disruption of normal brain function is likely to impair biogenic monoamine synthesis, and the resultant neurotransmitter deficiencies in critical periods of neurodevelopment are thought to prevent development of certain brain functions. The possibility of treating such patients with levodopa, 5-hydroxytryptophan, or both should be considered, therefore, to improve brain maturation and neurological outcome.

When you look at autism specifically it is usually 5-HIAA and not HVA that is disturbed.  

Now for two papers by one of our reader Roger’s favourite researchers, Vincent Ramaekers. Ramaekers is one of the specialists for central folate deficiency and even better is a researcher/clinician who replies to my emails. 


Patients with autism spectrum disorder (ASD) may have low brain serotonin concentrations as reflected by the serotonin end-metabolite 5-hydroxyindolacetic acid (5HIAA) in cerebrospinal fluid (CSF).


We sequenced the candidate genes SLC6A4 (SERT), SLC29A4 (PMAT), and GCHFR (GFRP), followed by whole exome analysis.


The known heterozygous p.Gly56Ala mutation in the SLC6A4 gene was equally found in the ASD and control populations. Using a genetic candidate gene approach, we identified, in 8 patients of a cohort of 248 with ASD, a high prevalence (3.2%) of three novel heterozygous non-synonymous mutations within the SLC29A4 plasma membrane monoamine transporter (PMAT) gene, c.86A > G (p.Asp29Gly) in two patients, c.412G > A (p.Ala138Thr) in five patients, and c.978 T > G (p.Asp326Glu) in one patient. Genome analysis of unaffected parents confirmed that these PMAT mutations were not de novo but inherited mutations.

Expression of mutations PMAT-p.Ala138Thr and p.Asp326Glu in cellulae revealed significant reduced transport uptake activity towards a variety of substrates including serotonin, dopamine, and 1-methyl-4-phenylpyridinium (MPP+), while mutation p.Asp29Gly had reduced transport activity only towards MPP+. At least two ASD subjects with either the PMAT-Ala138Thr or the PMAT-Asp326Glu mutation with altered serotonin transport activity had, besides low 5HIAA in CSF, elevated serotonin levels in blood and platelets. Moreover, whole exome sequencing revealed additional alterations in these two ASD patients in mainly serotonin-homeostasis genes compared to their non-affected family members.


Our findings link mutations in SLC29A4 to the ASD population although not invariably to low brain serotonin. PMAT dysfunction is speculated to raise serotonin prenatally, exerting a negative feedback inhibition through serotonin receptors on development of serotonin networks and local serotonin synthesis. Exome sequencing of serotonin homeostasis genes in two families illustrated more insight in aberrant serotonin signaling in ASD.

In this context, we found that isolated low brain serotonin concentration, as reflected by the 5HIAA in the CSF, is associated with PDD-NOS and the functional (heterozygous) c.167G > C (p.G56A) mutation of the serotonin re-uptake transporter gene (SERT/SCL6A4) combined with a homozygous long (L/L) SERT gene-linked polymorphic promoter (5-HTTLPR) region [21]. Moreover, daily treatment with serotonin precursor 5-hydroxytryptophan and aromatic amino acid decarboxylase (AADC) inhibitor carbidopaled to clinical improvements and normalization of the 5HIAA levels in the CSF and urine, indicating that the brain serotonin turnover was normalized [22]. In an attempt to gain some insight into the brain serotonin physiology and underlying mechanisms of abnormal brain metabolism, we report in patients with ASD and low brain 5HIAA mutations in the serotonin transporter SCL29A4, an observation that may provide some bases for improving the application of various therapeutic tools.

Whole blood serotonin and platelet serotonin content are increased in about 25 to 30% of the ASD population and their first-degree relatives. Because the fetal blood–brain barrier during pregnancy is not yet fully formed, the fetal brain will be exposed to high serotonin levels, leading through a negative-feedback mechanism to a loss of serotonin neurons and a limited outgrowth of their terminals. This hypothesis has been confirmed by rat studies using the serotonin agonist 5-methoxytryptamine between gestational days 12 until postnatal day 20 [42].

Tryptophan hydroxylase (TPH; EC catalyzes the first rate-limiting step of serotonin biosynthesis by converting l-tryptophan to 5-hydroxytryptophan. Serotonin controls multiple vegetative functions and modulates sensory and alpha-motor neurons at the spinal level. We report on five boys with floppiness in infancy followed by motor delay, development of a hypotonic-ataxic syndrome, learning disability, and short attention span. Cerebrospinal fluid (CSF) analysis showed a 51 to 65% reduction of the serotonin end-metabolite 5-hydroxyindoleacetic acid (5HIAA) compared to age-matched median values. In one out of five patients a low CSF 5-methyltetrahydrofolate (MTHF) was present probably due to the common C677T heterozygous mutation of the methylenetetrahydrofolate reductase (MTHFR) gene. Baseline 24-h urinary excretion showed diminished 5HIAA values, not changing after a single oral load with l-tryptophan (50-70 mg/kg), but normalizing after 5-hydroxytryptophan administration (1 mg/kg). Treatment with 5-hydroxytryptophan (4-6 mg/kg) and carbidopa (0.5-1.0 mg/kg) resulted in clinical amelioration and normalization of 5HIAA levels in CSF and urine. In the patient with additional MTHFR heterozygosity, a heterozygous missense mutation within exon 6 (G529A) of the TPH gene caused an exchange of valine by isoleucine at codon 177 (V177I). This has been interpreted as a rare DNA variant because the pedigree analysis did not provide any genotype-phenotype correlation. In the other four patients the TPH gene analysis was normal. In conclusion, this new neurodevelopmental syndrome responsive to treatment with 5-hydroxytryptophan and carbidopa might result from an overall reduced capacity of serotonin production due to a TPH gene regulatory defect, unknown factors inactivating the TPH enzyme, or selective loss of serotonergic neurons.

Carbidopa is a drug given to people with Parkinson's disease in order to inhibit peripheral metabolism of levodopa. This property is significant in that it allows a greater proportion of peripheral levodopa to cross the blood–brain barrier for central nervous system effect.

L-DOPA/levodopa is the precursor to the neurotransmitters dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline) collectively known as catecholamines. Furthermore, L-DOPA itself mediates neurotrophic factor release by the brain and CNS. As a drug, it is used in the clinical treatment of Parkinson's disease.


Based upon the hypothesis that brain monoamine metabolism is disorganized in some children with an autistic disorder, we tried low dose levodopa therapy (0.5 mg/kg/day) proposed by Segawa, et al. We treated 20 patients with an autistic disorder diagnosed according to DSM-IV, and evaluated the effectiveness. A double blind cross over method was applied in this study because of the small number of patients. Drug effects were observed carefully by the psychologists and pediatric neurologists using an evaluation sheet consisting of twenty items. No significant effectiveness was observed in this study, although four cases (20%) showed some improvement. In conclusion, administration of low dose levodopa to autistic children resulted in no clear clinical improvements of autistic symptoms.

A team led by Wen-Hann Tan,  of the Genetics Program at Children’s, is completing a phase I clinical trial examining the safety and dosing of levodopa, a drug commonly used for Parkinson disease, in patients with Angelman syndrome. The results will inform a planned phase II treatment trial, to be conducted in collaboration with University of California San Francisco, University of California San Diego, Vanderbilt University, Baylor College of Medicine and Greenwood Genetic Center. [For more information on Angelman research and events, check out this Facebook page.]

Research suggests that levodopa may increase the activity of an important brain enzyme known as CaMKII, which is involved in learning and memory, and that may be decreased in Angelman syndrome. In a mouse model of Angelman syndrome, low activity of CaMKII is associated with neurologic defects. Levodopa reverses the chemical modification that underlies decreased CaMKII activity. When this same modification is reversed in mice by genetic means, they show improvement in neurologic deficits, and it’s hoped that levodopa can do the same in humans.

Parkinson's disease

We saw in an earlier post that people with Down Syndrome are prone to early onset Alzheimer’s. In the case of lack of dopamine the risk might be towards Parkinson's disease (PD). 

There was a recent post on PANS/PANDAS/Tourette’s which like PD results from dysfunction in the basal ganglia region of the brain.

The basal ganglia, a group of brain structures innervated by the dopaminergic system, are the most seriously affected brain areas in PD. The main pathological characteristic of PD is cell death in the substantia nigra, where greatly reduced activity of dopamine-secreting cells caused by cell death.

When a decision is made to perform a particular action, inhibition is reduced for the required motor system, thereby releasing it for activation. Dopamine acts to facilitate this release of inhibition, so high levels of dopamine function tend to promote motor activity, while low levels of dopamine function, such as occur in PD, demand greater exertions of effort for any given movement. Thus, the net effect of dopamine depletion is to produce hypokinesia, an overall reduction in motor output. Drugs that are used to treat PD, conversely, may produce excessive dopamine activity, allowing motor systems to be activated at inappropriate times and thereby producing dyskinesias.

The drugs used in PD only treat some of the symptoms and are not curative, but do offer effective ways to increase dopamine levels.

High rates of Parkinsonism in adults with autism? Or is it partly drug-induced Parkinsonism

There is a study suggesting high rates of Parkinsonism in adults with autism.  I think some of this is more likely to be drug-induced Parkinsonism, either caused by currently taken drugs, or those taken in earlier years, which is not mentioned in the study. 


While it is now recognized that autism spectrum disorder (ASD) is typically a life-long condition, there exist only a handful of systematic studies on middle-aged and older adults with this condition.

We first performed a structured examination of parkinsonian motor signs in a hypothesis-generating, pilot study (study I) of 19 adults with ASD over 49 years of age. Observing high rates of parkinsonism in those off atypical neuroleptics (2/12, 17 %) in comparison to published population rates for Parkinson’s disease and parkinsonism, we examined a second sample of 37 adults with ASD, over 39 years of age, using a structured neurological assessment for parkinsonism.
Twelve of the 37 subjects (32 %) met the diagnostic criteria for parkinsonism; however, of these, 29 subjects were on atypical neuroleptics, complicating interpretation of the findings. Two of eight (25 %) subjects not taking atypical neuroleptic medications met the criteria for parkinsonism. Combining subjects who were not currently taking atypical neuroleptic medications, across both studies, we conservatively classified 4/20 (20 %) with parkinsonism.
We find a high frequency of parkinsonism among ASD individuals older than 39 years. If high rates of parkinsonism and potentially Parkinson’s disease are confirmed in subsequent studies of ASD, this observation has important implications for understanding the neurobiology of autism and treatment of manifestations in older adults. Given the prevalence of autism in school-age children, the recognition of its life-long natural history, and the recognition of the aging of western societies, these findings also support the importance of further systematic study of other aspects of older adults with autism.

Drug induced Parkinsonism

Any drug that blocks the action of dopamine (referred to as a dopamine antagonist) is likely to cause parkinsonism. Drugs used to treat schizophrenia and other psychotic disorders such as behaviour disturbances in people with dementia, known as neuroleptic drugs, are possibly the major cause of drug-induced parkinsonism worldwide. Parkinsonism can occur from the use of any of the various classes of neuroleptics.
The atypical neuroleptics – clozapine (Clozaril) and quetiapine (Seroquel), and to a lesser extent olanzapine (Zyprexa) and risperidone (Risperdal) – appear to have a lower incidence of extrapyramidal side effects, including parkinsonism. These drugs are generally best avoided by people with Parkinson’s, although some may be used by specialists to treat symptoms such as hallucinations occurring with Parkinson’s.
For people with Parkinson’s, anti-sickness drugs such as domperidone (Motilium) or ondansetron (Zofran) are the drugs of choice for nausea and vomiting.
As well as neuroleptics, some other drugs can cause drug-induced parkinsonism. These include some medications for dizziness and nausea such as prochlorperazine (Stemetil); and metoclopromide (Maxalon), which is used to stop sickness and in the treatment of indigestion.
Calcium channel blocking drugs used to treat high blood pressure, abnormal heart rhythm, angina pectoris, panic attacks, manic depression and migraine may occasionally cause drug-induced parkinsonism. Calcium channel blocking drugs are, however, widely used to treat angina and high blood pressure, and it is important to note that most common agents in clinical use probably do not have this side effect. These drugs should never be stopped abruptly without discussion with your doctor.
A number of other agents have been reported to cause drug-induced parkinsonism, but clear proof of cause and effect is often lacking. Amiodarone, used to treat heart problems, causes tremor and some people have been reported to develop Parkinson’s-like symptoms. Sodium valproate, used to treat epilepsy, and lithium, used in depression, both commonly cause tremor which may be mistaken for Parkinson’s.

Dopamine Receptors vs Dopamine as Dysfunctions 

We saw in great detail with the neurotransmitter GABA that the autism dysfunctions are usually related to the function and make-up of the neurotransmitter receptors, rather than the amount of GABA itself. Targeting these dysfunctions does indeed deliver results for many people with autism and Asperger’s.

Potentially this might be the case with dopamine, but it looks much less likely.

I did look at the following paper which seeks to link the genes of dopamine receptors (DRD1, DRD2, DRD3, DRD4, DRD5), dopamine-synthesizing enzyme DDC, dopamine transporter (DAT) and dopamine-catabolizing enzymes COMT and MAO to the several hundred known autism genes.

Using bioinformatics, in some they found a link and in others they did not.

The graphic below looks nice, but I am not sure it tells us much useful.  To me it looks much better to go direct to the autism gene and then see how to selectively modulate it. I do not think you can assume that the associated dopamine gene/receptor is the unifying problem across dysfunctional autism genes.  It would be great if it was.  

Autism spectrum disorder (ASD) is a debilitating brain illness causing social deficits, delayed development and repetitive behaviors. ASD is a heritable neurodevelopmental disorder with poorly understood and complex etiology. The central dopaminergic system is strongly implicated in ASD pathogenesis.

Genes encoding various elements of this system (including dopamine receptors, the dopamine transporter or enzymes of synthesis and catabolism) have been linked to ASD. Here, we comprehensively evaluate known molecular interactors of dopaminergic genes, and identify their potential molecular partners within up/down-steam signaling pathways associated with dopamine. These in silico analyses allowed us to construct a map of molecular pathways, regulated by dopamine and involved in ASD. Clustering these pathways reveals groups of genes associated with dopamine metabolism, encoding proteins that control dopamine neurotransmission, cytoskeletal processes, synaptic release, Ca2+ signaling, as well as the adenosine, glutamatergic and gamma-aminobutyric systems. Overall, our analyses emphasize the important role of the dopaminergic system in ASD, and implicate several cellular signaling processes in its pathogenesis.

Fig. 3. Reconstruction of biomolecular pathways related to dopaminergic genes associated with ASD (also see Fig. 2 and Table 2 for details). Known biological interactions between protein products of various genes are shown as complexes or denoted by arrows (sharp – activation, dull – inhibition). Proteins encoded by genes associated with ASD are marked with red (other colors are used here for illustration purposes only, to better distinguish visually between multiple different proteins within the dopaminergic pathways). Clustering of proteins into distinct functional groups is shown by dashed lines.

The strongest evidence for the role of dopamine genes in neuropsychiatric disorders is not in schizophrenia or autism, but in ADHD. As you can see in the paper below, even there the association is weak.


Although twin studies demonstrate that ADHD is a highly heritable condition, molecular genetic studies suggest that the genetic architecture of ADHD is complex. The handful of genome-wide scans that have been conducted thus far show divergent findings and are, therefore, not conclusive. Similarly, many of the candidate genes reviewed here (i.e. DBH, MAOA, SLC6A2, TPH-2, SLC6A4, CHRNA4, GRIN2A) are theoretically compelling from a neurobiological systems perspective, but available data are sparse and inconsistent. However, candidate gene studies of ADHD have produced substantial evidence implicating several genes in the etiology of the disorder. The literature published since recent meta-analyses is particularly supportive for a role of the genes coding for DRD4, DRD5, SLC6A3, SNAP-25, and HTR1B in the etiology of ADHD.

Yet, even these associations are small and consistent with the idea that the genetic vulnerability to ADHD is mediated by many genes of small effect.


In the ideal world you would take a sample of spinal fluid and measure 5-HIAA, to look for low brain serotonin and measure HVA for low brain dopamine.

For low serotonin you would give 5-HTP, with Dr Ramaekers suggesting 1mg/kg.

For low dopamine you would give levodopa or carbidopa.

In the real world even blood draws can be problematic so most people will never have their spinal fluid analyzed. Perhaps one day in the future this will be standard practice after an autism diagnosis, with numerous test being run at the same time and justifying this invasive procedure.   Many blood tests tell you little about brain disorders because the blood brain barrier means that the levels outside the brain will be completely different to those inside the brain. Measuring spinal fluid should be a good proxy for inside the brain.

The research suggests that 1mg/kg of 5-HT could have a long term beneficial effect, particularly if given from a very early age, in those with low serotonin in their brains, which is a large group of autism.

There are 5 types of dopamine receptors and in some genetic disorders the receptors’ response can be up/down regulated.  That would trigger a chain reaction with the non dopamine neurotransmitter receptors that are known to interact with that type of dopamine receptor.

There are associations between some autism genes and some dopamine genes, but it looks much more fruitful to target the autism genes themselves.

Avoid drug induced Parkinson’s Disease and other drug induced disorders, by very selective use of drugs.

Tuesday, 21 February 2017

Mitochondrial Disease and Autsim

Today’s post was originally intended to look at some further methods used to enhance cognitive function. Unlike people with typical mild cognitive impairment (MCI), some people with autism exhibit highly variable cognitive function, one way this is visible is in their hand writing quality. We previously saw that in cases of PANDAS/PANS, deterioration of hand writing is also seen during acute episodes. 
One possible cause of cognitive decline is mitochondrial dysfunction.  This is a highly complex subject in its own right and so I decided to start with a post introducing mitochondrial disease and dysfunction.

Mitochondria are tiny organelles found in almost every cell in the body. These organelles are responsible for creating 90% of cellular energy necessary to maintain life and support growth. Mitochondrial disease occurs when mitochondria in the cells fail to produce enough energy to sustain cell life. When enough cells cease to function properly organs, motor functions, and the neurological system can become impaired.
Mitochondrial disease is often misdiagnosed due to the fact many of the symptoms are synonymous with other, more common, diseases.
In more scientific terms mitochondrial disease refers to a wide ranging group of disorders resulting in defective cellular energy production due to abnormal oxidative phosphorylation (OXPHOS), which is explained a little later.

Primary Mitochondrial Disease (PMD) vs Secondary Mitochondrial Dysfunction (SMD)
I received a comment a while back from a parent who said that tests had ruled out mitochondrial disease.  It is actually a very grey area, where it is much easier to rule it in, than out. It looks like most people with autism have some mitochondrial dysfunction, albeit perhaps minor compared to those with an identified error in a critical gene, which is today relatively easy to diagnose.

Primary Mitochondrial Disease (PMD) is inborn; people with PMD gave a genetic variance that makes them vulnerable to a loss of mitochondrial function.  This loss may not begin until later in life and may increase in severity.
PMD is extremely rare in the general population, but is thought to occur in about 5% of cases of autism.
Primary mitochondrial disease (PMD) is diagnosed clinically and ideally, but not always, confirmed by a known or indisputably pathogenic mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) mutation. The PMD genes either encode oxphos proteins directly or they affect oxphos function by impacting production of the complex machinery needed to run the oxphos process.
Secondary mitochondrial dysfunction (SMD) is much more common than PMD. SMD can be caused by genes encoding neither function nor production of the oxphos proteins and accompanies many hereditary non-mitochondrial diseases. SMD may also be due to non-genetic causes such as environmental factors.
SMD has been documented in a variety of autoimmune processes including multiple sclerosis and lupus.
Aging contributes to oxidative stress in virtually all organs and tissues in the body and increases the risk for SMD.
Altered mitochondrial fusion/fission dynamics have been found to be a recurring theme in neurodegeneration. There is evidence of mitochondrial dysfunction in neurodegenerative diseases such as Alzheimer's and Parkinson's.
A significant number of metabolic disorders include SMD as a part of their phenotypes.
Abnormal biomarkers of mitochondrial function are very common in autism.  Depending on whose data you consider, you can say that SMD is present in a substantial minority or even a majority of cases.
Ideally you would use genetic testing to try to distinguish between PMD and SMD. This is important, since their treatments and prognoses can be quite different. However, even in the absence of the ability to distinguish between PMD and SMD, treating SMD with standard treatments for PMD can be effective.

Diagnosis of PMD, SMD and specific subtypes
Some researchers/clinicians make the issue of diagnosis sound very clear cut, whereas others see it as a subjective diagnosis associated with some “ifs” and “maybes”.

Mitochondrial dysfunction can affect the whole body or be organ specific. You can take a muscle biopsy for analysis but not a brain biopsy.
There are a small number of well-known specialists who diagnose mitochondrial dysfunction. They all have their own favoured treatments and they do vary.  

Oxidative phosphorylation
Oxidative phosphorylation (or OXPHOS in short) is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy.  This takes place inside mitochondria.

Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such as superoxide and hydrogen peroxide, which lead to propagation of free radicals, damaging cells and contributing to disease.
The five enzymes required have simplified names: complex I, complex II, complex III, complex IV, and complex V.
In the mitochondria, converting one molecule of glucose to carbon dioxide and water produces up to 36 ATPs. This does also require the presence of oxygen, in the absence of oxygen a different, much less efficient process is followed.  This is where some sportsmen seek to cheat by increasing the amount of oxygen in their blood.
Adenosine triphosphate (ATP) is a small molecule used in cells as a coenzyme. It is often referred to as the "molecular unit of currency" of intracellular energy transfer.

ATP transports chemical energy within cells for metabolism. Most cellular functions need energy in order to be carried out: synthesis of proteins, synthesis of membranes, movement of the cell, cellular division, transport of various solutes etc. ATP is the molecule that carries energy to the place where the energy is needed.

When ATP breaks into ADP (Adenosine diphosphate) and Pi (phosphate), energy is liberated.
By analyzing the level of certain byproducts of the five major steps between glucose and ATP you can determine which of the five enzyme complexes might be deficient.
Many poisons and pesticides target one of the enzyme complexes.  Inhibition of any step in this process will halt the rest of the process. One of these poisons, 2,4-Dinitrophenol, was actually used as an anti-obesity drug in the 1930s.

Complex I to V in Autism
The clinicians who like genetic testing look for concrete evidence of Primary Mitochondrial Disease (PMD). Other clinicians look for tell-tale signs in the level of chemicals like lactate and pyruvate to make diagnosis; this might suggest that a specific enzyme complex is deficient.

So if you have a diagnosis of say complex 1 deficiency, you can then go into the detail of that step in the process.  Here is gets rather complicated because 51 different genes encode components of complex 1.  Any one of them being down regulated could impair the level of complex 1.  

The researchers obtained blood samples from each child and analyzed the metabolic pathways of mitochondria in immune cells called lymphocytes. Previous studies sampled mitochondria obtained from muscle, but the mitochondrial dysfunction sometimes is not expressed in muscle. Muscle cells can generate much of their energy through anaerobic glycolysis, which does not involve mitochondria. By contrast, lymphocytes, and to a greater extent brain neurons, rely more heavily on the aerobic respiration conducted by mitochondria.

The researchers found that mitochondria from children with autism consumed far less oxygen than mitochondria from the group of control children, a sign of lowered mitochondrial activity. For example, the oxygen consumption of one critical mitochondrial enzyme complex, NADH oxidase, in autistic children was only a third of that found in control children. 

Complex I was the site of the most common deficiency, found in 60 percent of autistic subjects, and occurred five out of six times in combination with Complex V. Other children had problems in Complexes III and IV.

Levels of pyruvate, the raw material mitochondria transform into cellular energy, also were elevated in the blood plasma of autistic children. This suggests the mitochondria of children with autism are unable to process pyruvate fast enough to keep up with the demand for energy, pointing to a novel deficiency at the level of an enzyme named pyruvate dehydrogenase.

"The various dysfunctions we measured are probably even more extreme in brain cells, which rely exclusively on mitochondria for energy," 
"Children with mitochondrial diseases may present exercise intolerance, seizures and cognitive decline, among other conditions. Some will manifest disease symptoms and some will appear as sporadic cases," said Cecilia Giulivi, the study's lead author and professor in the Department of Molecular Biosciences in the School of Veterinary Medicine at UC Davis. "Many of these characteristics are shared by children with autism."

It looks like Dr Kelley, formerly at Johns Hopkins, has the largest following by those treating autism secondary to mitochondrial disease (AMD). Treatment includes augmentation of residual complex I activity with carnitine, thiamine, nicotinamide, and pantothenate, and protection against free radical injury with several antioxidants, including vitamin C, vitamin E, alpha-lipoic acid, and coenzyme Q10.
Dr Frye is a prolific publisher, unlike Dr Kelley, and their therapies do differ.  The table below is from one of Dr Frye’s papers.

Dr Frye likes his B vitamins. On his list are B vitamins  1,2,3,5,6,7,9 and 12

Dr Kelley is a big believer in the benefit of carnitine:

“Mutation in one or more subunits of mitochondrial complex I in AMD also is suggested by the often immediate response to carnitine, which activates latent complex I by the same NDUSF7/phosphatase-kinase system that activates pyruvate dehydrogenase.  Although immediate behavioral improvement with carnitine treatment in a child with regressive autism makes complex I deficiency the most likely cause, the similar effect of carnitine to activate latent pyruvate dehydrogenase complex recommends consideration of pyruvate dehydrogenase deficiency in the child with atypical autism and substantial postprandial lactic acidemia.”

“Supplemental carnitine enhances the conversion of acyl-CoAs to free CoA + acylcarnitines, thereby raising the intramitochondrial free CoA/acyl-CoA ratio and activating the phosphatase that reverses the inhibitory phosphorylation of NDUFS7.  Pharmacological amounts of pantothenic acid increase the synthesis of free CoA in mitochondria [22], which increases further the free-CoA/acyl-CoA ratio.  Raising the free-CoA/acyl-CoA ratio recruits more functional complex I units to compensate for the partial deficiency of complex I.  Because complex I is the rate limiting step in the mitochondrial respiratory chain for most substrates, each percentage increase in complex I activity should be followed by a substantial fraction of that percentage increase in mitochondrial ATP synthesis  

A problem with carnitine is very low bioavailability. 

Carnitine is important for cell function and survival primarily because of its involvement in the multiple equilibria between acylcarnitine and acyl-CoA esters established through the enzymatic activities of the family of carnitine acyltransferases. These have different acyl chain-length specificities and intracellular compartment distributions, and act in synchrony to regulate multiple aspects of metabolism, ranging from fuel-selection and -sensing, to the modulation of the signal transduction mechanisms involved in many homeostatic systems. This review aims to rationalise the extensive range of experimental and clinical data that have been obtained through the pharmacological use of L-carnitine and its short-chain acylesters, over the past two decades, in terms of the basic biochemical mechanisms involved in the effects of carnitine on the various cellular acyl-CoA pools in health and disease.

4.3. L-Carnitine: a conditional drug?

The potential limitation of L-carnitine-based “mitochondrial” therapy may be overcome through the attainment of supraphysiological concentrations of L-carnitine in plasma and target organs, so as to elicit the desired pharmaco-metabolic response. In target organs such as liver, heart, and skeletal muscle, the intracellular L-carnitine pool is in the high micromolar to low millimolar range, whereas in the plasma it is in the low micromolar range [124]. In addition, taking into account that physiological plasma levels of L-carnitine almost saturate the high-affinity L-carnitine transporters, relatively high L-carnitine
plasma exposures are required to significantly achieve organ Lcarnitine
increases. Under these conditions, it is possible that Lcarnitine moves into the intracellular milieu via passive diffusion and/or a low-affinity carnitine transporter [125]. However, the increase of L-carnitine plasma exposure upon L-carnitine oral administration, even when using high doses (e.g. more than 2 grams per day) [124], is quite modest, since L-carnitine has a very poor absorption and bioavailability, a very high renal clearance, and active uptake into tissues by a high-affinity transporter [124,125]. Intravenous administration of L-carnitine might overcome such a problem, particularly for acute/short-term treatment of hospitalized patients. However, this route of administration may present difficulties, particularly when kidney function is intact, because the efficient tubular reabsorption process ensures that more than 95% of L-carnitine filtered by glomeruli is retained [124,126]. Moreover, since renal tubular
reabsorption occurs via an active transporter, once the transporter
is saturated the excess of exogenous L-carnitine is readily excreted.
A Carnitine Analog Perhaps?
I did write a post about Meldonium/Mildronate, a drug that was made famous by the Russian tennis star Maria Sharapova.  This drug was developed in Latvia.

One of its effects is thought to be increasing the size of blood vessels and therefore improving blood flow; this increases exercise endurance.
This fact was very well known in the old Soviet Union and Meldonium was widely used by their soldiers fighting in Afghanistan.  At high altitudes there is less oxygen in the air you breathe and ultimately less in your blood and this compromises the ability of infantry soldiers.
The western world’s military have long used  acetazolamide/Diamox which makes your blood more acidic and this  fools the body into thinking it has an excess of CO2, and it excretes this imaginary excess CO2 by deeper and faster breathing, which in turn increases the amount of oxygen in the blood.   
Other than sportswomen and soldiers, Meldonium is used to treat coronary artery disease, where problems may sometimes lead to ischemia, a condition where too little blood flows to the organs in the body, especially the heart. Because this drug is thought to expand the arteries, it helps to increase the blood flow as well as increase the flow of oxygen throughout the body.
Meldonium also appears to have neuroprotective properties particularly relevant to the mitochondria.  At one point I thought this was just the Latvian researchers clutching at straws trying to push their drug as a panacea.
Rather, I think perhaps its core action may include making the mitochondria work a little better, by increasing complex 1. This might also increase stamina and it should also improve cognition in some.

Mildronate has a very similar structure to carnitine.

 Previously, we have found that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a small molecule with charged nitrogen and oxygen atoms, protects mitochondrial metabolism that is altered by inhibitors of complex I and has neuroprotective effects in an azidothymidine-neurotoxicity mouse model

The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT-induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronate's regulatory action. The results showed that mildronate protected mitochondria from AZT-induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT-caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronate's effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions.

Previously we demonstrated that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a representative of the aza-butyrobetaine class of compounds, protects mitochondrial metabolism under conditions such as ischemia. Mildronate also acted as a neuroprotective agent in an azidothymidine-induced mouse model of neurotoxicity, as well as in a rat model of Parkinson's disease. These observations suggest that mildronate may stimulate processes involved in cell survival and change expression of proteins involved in neurogenic processes. The present study investigated the influence of mildronate on learning and memory in the passive avoidance response (PAR) test and the active conditioned avoidance response (CAR) test in rats. The CAR test employed also bromodeoxyuridine (BrdU)-treated animals. Hippocampal cell BrdU incorporation was then immunohistochemically assessed in BrdU-treated, CAR-trained rats to identify proliferating cells. In addition, the expression of hippocampal proteins which could serve as memory enhancement biomarkers was evaluated and compared to non-trained animals' data. These biomarkers included glutamic acid decarboxylase 65/67 (GAD65/67), acetylcholine esterase (AChE), growth-associated protein-43 (GAP-43) and the transcription factor c-jun/activator protein-1 (AP-1). The results showed that mildronate enhanced learning/memory formation that coincided with the proliferation of neural progenitor cells, changing/regulating of the expression of biomarker proteins which are involved in the activation of glutamatergic and cholinergic pathways, transcription factors and adhesion molecule.

The data from our study suggest that mildronate may be useful as a possible cognitive enhancer for the treatment of patients with neurodegenerative diseases with dementia.

Mildronate Dosage
Interestingly, the neuroprotective dose of Mildronate is much lower than the usual dose.

Summary. This review for the first time summarizes the data obtained in the neuropharmacological studies of mildronate, a drug previously known as a cardioprotective agent. In different animal models of neurotoxicity and neurodegenerative diseases, we demonstrated its neuroprotecting activity. By the use of immunohistochemical methods and Western blot analysis, as well as some selected behavioral tests, the new mechanisms of mildronate have been demonstrated: a regulatory effect on mitochondrial processes and on the expression of nerve cell proteins, which are involved in cell survival, functioning, and inflammation processes. Particular attention is paid to the capability of mildronate to stimulate learning and memory and to the expression of neuronal proteins involved in synaptic plasticity and adult neurogenesis. These properties can be useful in neurological practice to protect and treat neurological disorders, particularly those associated with neurodegeneration and a decline in cognitive functions.

Concluding Remarks

The obtained data give a new insight into the influence of mildronate on the central nervous system.

This drug shows beneficial effects in the regulation of cell processes necessary for cell integrity and survival, particularly by targeting mitochondria and by stabilizing the expression of proteins involved in neuroinflammation and neuroregeneration. These properties can be useful in neurological practice to protect and treat neurological disorders, such as Parkinson’s disease, diabetic neuropathies, and ischemic stroke. Moreover, because mildronate improves learning and memory, one may suggest mildronate as a multitargeted neuroprotective/ neurorestorative drug with its therapeutic utility as a memory enhancer in cognitive impairment conditions, such as neurodegenerative diseases, schizophrenia, and other pathologies associated with a decline in awareness.

The present review summarizes our previously obtained data which demonstrated the influence of mildronate on mitochondrial processes and the expression of nerve cell proteins involved in the essential pathways for cell survival and functioning. Besides, the effectiveness of mildronate at much lower doses of 20 and 50 mg/kg in comparison with the traditionally recommended doses typical for cardioprotection (100 and 200 mg/kg) has been demonstrated.

Bypass the need for Complex 1 by ketosis?

Almost all the research on mitochondrial disease assumes that you want to convert glucose to ATP.
If a person has an inability to produce enough complex 1 they might be better off switching from glycolysis (glucose as fuel) to ketosis (ketones from fat as fuel).
There are posts in this blog describing the ketogenic diet, which has been widely used for decades to treat epilepsy.

Ketosis is a metabolic state in which some of the body's energy supply comes from ketone bodies in the blood, in contrast to a state of glycolysis in which blood glucose provides most of the energy.

Ketosis is a nutritional process characterized by serum concentrations of ketone bodies over 0.5 mM, with low and stable levels of insulin and blood glucose. It is almost always generalized with hyperketonemia, that is, an elevated level of ketone bodies in the blood throughout the body. Ketone bodies are formed by ketogenesis when liver glycogen stores are depleted (or from metabolising medium-chain triglycerides). The main ketone bodies used for energy are acetoacetate and β-hydroxybutyrate, and the levels of ketone bodies are regulated mainly by insulin and glucagon. Most cells in the body can use both glucose and ketone bodies for fuel, and during ketosis, free fatty acids and glucose synthesis (gluconeogenesis) fuel the remainder.

As is often the case, opinion is mixed on the ketogenic diet and mitochondrial disorders. It seems to make some people better and have no effect on others.  This is likely because they do not have precisely the same mitochondrial disorder.

2.6. Dietary manipulations

Several approaches based on dietary measures have been attempted, with controversial results. Ketogenic diet (KD), i.e. a high-fat, low-carbohydrate diet, has been proposed to stimulate mitochondrial beta-oxidation, and provide ketones, which constitute an alternative energy source for the brain, heart and skeletal muscle. Ketone bodies are metabolized to acetyl-CoA, which enters the Krebs cycle and is oxidized to feed the RC and ultimately generate ATP via OXPHOS. This pathway partially bypasses complex I via increased synthesis of succinate, which donates electrons to the respiratory chain via complex II. Increased ketone bodies have also been associated with increased expression of OXPHOS genes, possibly via a starvation-like response [80]. Starvation is a stressing condition to the cell, which results in activation of many transcription factors and cofactors (including SIRT1, AMPK, and PGC-1α) that ultimately increase mitochondrial biogenesis [80]. KD reduced the mutation load of a heteroplasmic mtDNA deletion in a cybrid cell line from a Kearns–Sayre syndrome patient [81], was shown to increase the expression levels of uncoupling proteins and mitochondrial biogenesis in the hippocampus of mice and rats [82] and [83], and increased mitochondrial GSH levels [84] in rat brain. These phenomena could contribute to explain the anticonvulsant effects of KD. In a preclinical trial on the deletor mouse, KD slowed the progression of mitochondrial myopathy [85]. However, other reports showed that KD can have the opposite effect, and worsens the mitochondrial defect invivo, for instance in the Mterf2−/− [86], or the Mpv17–/−mouse models [87].

Similar to KD, a high fat diet (HFD) was shown to have a protective effect on fibroblasts with complex I deficiency and be effective in delaying the neurological symptoms of the Harlequin mouse, a model of partial complex I defect associated with a homozygous mutation of AIFM1, encoding the mitochondrial apoptosis inducing factor [88].

Similar results could in principle be achieved using other compounds that release succinate in mitochondria. An example is triheptaoin, an anaplerotic compound inducing a rapid increase of plasmatic C4- and C5-ketone bodies, the latter being a precursor of propionyl-CoA, which is then converted into succinyl-CoA. Treatment with triheptaoin has been reported to dramatically improve cardiomyopathy in patients with VLCAD deficiency and myopathic symptoms in CPT2 deficiency patients [89] and [90]. 

Ketogenic diet

The ketogenic diet is a high-fat diet that effectively treats some forms of medically refractory epilepsy [7,8, Class I]. Recent animal research has suggested that the ketogenic diet may be beneficial in optimizing mitochondrial function [9, Class III].
Because many mitochondrial disease patients have secondary fatty acid oxidation disorders, there are limited data on use and safety of the ketogenic diet in patients with these conditions. Only a single report has looked at the lack of efficacy of the ketogenic diet in children with electron transport chain defects and intractable seizures [10, Class IV].
The ketogenic diet is the standard of care for pyruvate dehydrogenase deficiency, but it is contraindicated in patients with known fatty acid oxidation disorders and pyruvate carboxylase deficiency.

Experimental Therapies


o   At present there is no effective cure for mitochondrial diseases.

o   Generalist and tailored therapies are emerging at the pre-clinical level.

o   Some therapies are effective in disease models and ready for translation to patients.

o   Other approaches warrant more work at the pre-clinical level.

Some people’s autism does indeed appear to have been solely caused by the lack of mitochondrial enzymes.  These dysfunctions can be inherited or acquired.

As Dr Kelley suggests, a baby might be born with a 50% reduction in complex 1 and develop normally. Following a viral infection, or other insult, before the brain has substantially matured a further reduction in complex 1 occurs and this tips the balance to where mitochondria cannot function sufficiently. Siblings may have exactly the same biochemical markers, but continue normal development because they avoided the damaging insult that triggered regression at a critical point in the brain’s maturation.
The data does point to mitochondrial dysfunction being present beyond just those with regressive autism, so a little extra complex 1 may be in order for them too.

Of the five enzyme complexes, complex 1 appears to be the most important because it is “rate limiting”, meaning it is usually the enzyme with the least unused capacity.  It becomes the bottleneck in the energy production chain. Many other diseases and aging feature a decline in complex 1 which may account for some people’s loss of cognitive function.
Is mildronate a carnitine analog with better bioavailability? Are its cognitive enhancing effects due to increased blood flow, improved complex 1 availability or perhaps both?  We can only wait till the Latvians do some experiments on schizophrenia and autism.  The good news is that the dose at which the mitochondrial effects occur is five times less than the anti-ischemia dose.

I can see that the dose for athletes is twice the dose for ischemia. So it would seem that tennis players who have used mildronate for ten years, at ten times the mitochondrial dose, might provide some useful safety information.

As you can see from the packaging, the drug must be popular with cyclists too.

Suggested further reading, or indeed re-reading:

Richard I. Kelley, MD, PhD
Division of Metabolism, Kennedy Krieger Institute Department of Pediatrics, Johns Hopkins Medical Institutions