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 subtypesSome 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 phosphorylationOxidative 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 AutismThe 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.
Primary Mitochondrial Disease and Secondary Mitochondrial Dysfunction: Importance of Distinction for Diagnosis and Treatment
Dr Frye likes his B vitamins. On his list are B vitamins 1,2,3,5,6,7,9 and 12
“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 , 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 . 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 . 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) , 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
Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)-induced toxicity of isolated rat liver mitochondria.
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.
Mitochondrial Complex 1 Activity Measured by Spectrophotometry Is Reduced across All Brain Regions in Ageing and More Specifically in Neurodegeneration
Mildronate DosageInterestingly, the neuroprotective dose of Mildronate is much lower than the usual dose.
Mildronate and its Neuroregulatory Mechanisms: Targeting the Mitochondria, Neuroinflammation, and Protein Expression
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.
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 . 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 . KD reduced the mutation load of a heteroplasmic mtDNA deletion in a cybrid cell line from a Kearns–Sayre syndrome patient , was shown to increase the expression levels of uncoupling proteins and mitochondrial biogenesis in the hippocampus of mice and rats  and , and increased mitochondrial GSH levels 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 . However, other reports showed that KD can have the opposite effect, and worsens the mitochondrial defect invivo, for instance in the Mterf2−/− , or the Mpv17–/−mouse models .
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 .
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  and .
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.
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.
ConclusionSome 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