Mitochondrial Dysfunction in Autism

22 Dec

A recent paper from the MIND Institute, published in the Journal of the American Medical Association (JAMA) entitled Mitochondrial Dysfunction in Autism caused a bit of a stir. One which is far beyond what is supported by the paper’s conclusions or data, I will add.

The study is very small: 10 autistic children and 10 controls. The authors used a very nonstandard methodology. Perhaps the best summary of this study so far can be found on the Simons Foundation blog SFARI (Defects in mitochondria linked to autism). Deborah Rudacille discusses the methodology and brings in quotes from the study’s lead author (Cecilia Giulivi) as well as established experts in the field of mitochondrial disease and autism such as Jay Gargas.

Before I get too far ahead of myself, here is the abstract:

Context Impaired mitochondrial function may influence processes highly dependent on energy, such as neurodevelopment, and contribute to autism. No studies have evaluated mitochondrial dysfunction and mitochondrial DNA (mtDNA) abnormalities in a well-defined population of children with autism.

Objective To evaluate mitochondrial defects in children with autism.

Design, Setting, and Patients Observational study using data collected from patients aged 2 to 5 years who were a subset of children participating in the Childhood Autism Risk From Genes and Environment study in California, which is a population-based, case-control investigation with confirmed autism cases and age-matched, genetically unrelated, typically developing controls, that was launched in 2003 and is still ongoing. Mitochondrial dysfunction and mtDNA abnormalities were evaluated in lymphocytes from 10 children with autism and 10 controls.

Main Outcome Measures Oxidative phosphorylation capacity, mtDNA copy number and deletions, mitochondrial rate of hydrogen peroxide production, and plasma lactate and pyruvate.

Results The reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (normalized to citrate synthase activity) in lymphocytic mitochondria from children with autism was significantly lower compared with controls (mean, 4.4 [95% confidence interval {CI}, 2.8-6.0] vs 12 [95% CI, 8-16], respectively; P = .001). The majority of children with autism (6 of 10) had complex I activity below control range values. Higher plasma pyruvate levels were found in children with autism compared with controls (0.23 mM [95% CI, 0.15-0.31 mM] vs 0.08 mM [95% CI, 0.04-0.12 mM], respectively; P = .02). Eight of 10 cases had higher pyruvate levels but only 2 cases had higher lactate levels compared with controls. These results were consistent with the lower pyruvate dehydrogenase activity observed in children with autism compared with controls (1.0 [95% CI, 0.6-1.4] nmol × [min × mg protein]?1 vs 2.3 [95% CI, 1.7-2.9] nmol × [min × mg protein]?1, respectively; P = .01). Children with autism had higher mitochondrial rates of hydrogen peroxide production compared with controls (0.34 [95% CI, 0.26-0.42] nmol × [min × mg of protein]?1 vs 0.16 [95% CI, 0.12-0.20] nmol × [min × mg protein]?1 by complex III; P = .02). Mitochondrial DNA overreplication was found in 5 cases (mean ratio of mtDNA to nuclear DNA: 239 [95% CI, 217-239] vs 179 [95% CI, 165-193] in controls; P = 10?4). Deletions at the segment of cytochrome b were observed in 2 cases (ratio of cytochrome b to ND1: 0.80 [95% CI, 0.68-0.92] vs 0.99 [95% CI, 0.93-1.05] for controls; P = .01).

Conclusion In this exploratory study, children with autism were more likely to have mitochondrial dysfunction, mtDNA overreplication, and mtDNA deletions than typically developing children.

As the abstract states, the MIND Institute study methodology involved: “Mitochondrial dysfunction and mtDNA abnormalities were evaluated in lymphocytes from 10 children with autism and 10 controls”. Lymphocytes (a type of white blood cell). Children were concecutively recruited and genetically unrelated. Mitochondrial function was tested first, and given the results seen, children were brought back for a second blood draw where mitochondrial DNA (mDNA) and nuclear DNA (nDNA) were examined.

As shown in the figure below, they found that the autistic children had different mitochondrial activity levels than their controls. Note that “low” activity is not referenced to any standardized norms, but to the 10 control children.

They also performed genetic testing. Table 3 from the paper is reproduced below:

They show that, by their methodology, 7 of their 10 autistic kids have some form of genetic signature for mitochondrial dysfunction. 2 of 10 of their controls meet their criteria as well.

The Simons blog quotes the study author, Prof. Giulivi on this choice:

“Lymphocytes do not rely as heavily on mitochondria as the brain does,” she says, “so if this is happening in cells that don’t use mitochondria as much, it’s likely to be happening in cells that rely more heavily on mitochondria, like neurons.”

They also quote Dr. Fernando Scaglia, of the Baylor Clinic:

However, the unconventional decision to use lymphocytes should have been validated, says Fernando Scaglia, associate professor of molecular and human genetics at Baylor College of Medicine in Houston. “I’m not saying that studies done in lymphocytes are useless,” says Scaglia, an expert in inherited metabolic disease. “But they should be validated in other tissue.”

and Prof. Gargas of the University of California at Irvine:

“Lymphocytes are fine to study chromosomal DNA, but they are a horrible source for studying mitochondrial DNA,” he says.

Cells have hundreds of mitochondria, each with multiple copies of the DNA. In people with mitochondrial disease, some cells have healthy DNA and others have the mutated copies, he notes. In a blood sample, defective lymphoctyes tend to get lost among rapidly proliferating healthy cells.

“The best source for studying mitochondria are post-mitotic cells such as muscle,” he says. “That way you are sampling the set of cells the child was born with.”

In the end, if we stick to the idea that this is a very preliminary report and relies on a new unproven methodology at that, we can consider the study as posing interesting questions. Is mitochondrial dysfunction more prevalent in autistics than the general population? Are there ways to test this in a faster, less intrusive manner than is often used? If we take this study in context, there may be some value. Unfortunately as Seth Mnookin has already pointed out, this study has already been used to promote ideas that are clearly outside of the study and conclusions. This is the unfortunate world of autism research: it is hard for people to push the boundaries and risk being wrong. Not because it may cause the researchers some embarrassment, but because there are a multitude of people waiting to misuse information and mislead.

5 Responses to “Mitochondrial Dysfunction in Autism”

  1. daedalus2u December 22, 2010 at 15:44 #

    Sullivan, the data in the table doesn’t show any changes in mitochondrial DNA. What is shows is differences in the ratio of how much mitochondrial DNA there is compared to how much nuclear DNA there is.

    Essentially this is a measure of how many mitochondria there are in the cell because each mitochondrion has its own little bit of mitochondrial DNA and the nucleus has its big bit of nuclear DNA.

    These measurements are only relevant to the cells they looked at. Since they looked at lymphocytes, this is about how many mitochondria there are in those blood cells. Lymphocytes don’t require mitochondria to make ATP, lymphocytes can operate anaerobically by doing glycolysis. That is necessary because lymphocytes might need to operate in an anaerobic infected tissue compartment. What lymphocytes use mitochondria for is to make hydrogen peroxide to destroy extracellular bacteria by using that H2O2 along with extracellular myeloperoxidase.

    The comment by Prof Gargos is correct (and I think doesn’t go far enough). Lymphocytes come from blood stem cells and have a pretty short lifetime. They turnover pretty fast and there are cases of people with actual mitochondrial DNA defects in lymphocytes, losing those defects as they age because the stem cells without the defect replicate more and eventually replace those with the defects.

    DNA damage in a lymphocyte isn’t a big deal because the lymphocyte isn’t going to replicate itself. It will simply die when it gets worn out. DNA damage is how lymphocytes get worn out.

    The measurements they show don’t demonstrate that there is a “mitochondrial defect” in those children. Differences yes, but not necessarily “mitochondrial defects”. The differences are more likely to be due to differential regulation of mitochondria in lymphocytes as a part of the global physiological differences in autism, rather than being due to any “mitochondrial defect”.

  2. passionlessDrone December 22, 2010 at 16:37 #

    Hi Sullivan –

    Nice post. I’ve been working on a post on this topic for a while, but keep getting lost in the details and I’m slow to start with.

    Anyway, I had some thoughts on your posting.

    As shown in the figure below, they found that the autistic children had different mitochondrial activity levels than their controls. Note that “low” activity is not referenced to any standardized norms, but to the 10 control children.

    I’m not sure how much hay we should really make of this. It is possible that they found a control group with very well functioning mitochondria, but it is unlikely. And it also doesn’t do anything to speak towards the other biomarker studies we have indicating the same thing; or the other population study that found very similar values.

    To my mind, the statements by Gargas point towards an ongoing, acquired problem with mitochondrial function in autism, as opposed to a maternally inherited problem.

    Cells have hundreds of mitochondria, each with multiple copies of the DNA. In people with mitochondrial disease, some cells have healthy DNA and others have the mutated copies, he notes. In a blood sample, defective lymphoctyes tend to get lost among rapidly proliferating healthy cells.

    And yet, it was relatively simple to find defective lymphocytes in the autism population. Why? One reason this might be possible is that there is an ongoing process that is causing problems, as opposed to maternally inherited mtDNA problems. One suspect might be increased oxidative stress. We’ve got lots of reasons to suspect this is the case in autism, near a dozen studies finding biomarkers of increased oxidative stress in that population. And, we also have good evidence that as oxidative stress increases, so too do problems with mitochondria.

    For example, this paper was referenced in Giulivi, Oxidative stress-related alteration of the copy number of mitochondrial DNA in human leukocytes found that as indices of oxidative stress increased, so too did the copy number of mtDNA.

    Three oxidative indices including the incidence of 4,977 bp deletion of mtDNA (P = 0.016) and 8-OHdG content in leukocytes (P = 0.003) and TBARS in plasma (P = 0.001) were all positively correlated with the copy number of mtDNA in leukocytes. Taken these findings together, we suggest that the copy number of mtDNA in leukocytes is affected by oxidative stress in blood circulation elicited by the alteration of plasma antioxidants/prooxidants and oxidative damage to DNA

    This type of finding correlates nicely with the autism paper and the breadth of autism / oxidative stress papers we’ve already seen.

    We also have a ton of recent evidence of the same thing in other cognitive problems, for example, schizophrenia and bi-polar. Here is a review paper: Mitochondrial dysfunction and pathology in bipolar disorder and schizophrenia (there are many, many others)

    Bipolar disorder (BPD) and schizophrenia (SZ) are severe psychiatric illnesses with a combined prevalence of 4%. A disturbance of energy metabolism is frequently observed in these disorders. Several pieces of evidence point to an underlying dysfunction of mitochondria: (i) decreased mitochondrial respiration; (ii) changes in mitochondrial morphology; (iii) increases in mitochondrial DNA (mtDNA) polymorphisms and in levels of mtDNA mutations; (iv) downregulation of nuclear mRNA molecules and proteins involved in mitochondrial respiration; (v) decreased high-energy phosphates and decreased pH in the brain; and (vi) psychotic and affective symptoms, and cognitive decline in mitochondrial disorders

    The questions of physiological significance and / or causal direction are valid and require lots more study. CNS and/or correlations to behavioral severity would be nice data points to gather. But for Giulivi to be wrong, we need to start questioning wide swaths of research from areas outside of autism. For all the hand wringing about the pilot nature of this study and the potential problems of using peripheral cells as proxies, what should have been surprising is if they’d failed to observe mitochondrial problems in autism.

    – pD

  3. passionlessDrone January 26, 2011 at 18:06 #

    Hello friends –

    One of the biggest concerns with Giulivi was the usefulness of periphery cells as proxy views into the CNS. Another paper was recently released that may help understand this:

    Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism

    Mitochondria play important roles in generation of free radicals, ATP formation, and in apoptosis. We studied the levels of mitochondrial electron transport chain (ETC) complexes, i.e., complexes I, II, III, IV, and V, in brain tissue samples from the cerebellum and the frontal, parietal, occipital, and temporal cortices of subjects with autism and age-matched control subjects. The subjects were divided into two groups according to their ages: Group A (children, ages 4-10 years) and Group B (adults, ages 14-39 years). In Group A, we observed significantly lower levels of complexes III and V in the cerebellum (p < 0.05), of complex I in the frontal cortex (p < 0.05), and of complexes II (p < 0.01), III (p < 0.01), and V (p < 0.05) in the temporal cortex of children with autism as compared to age-matched control subjects, while none of the five ETC complexes was affected in the parietal and occipital cortices in subjects with autism. In the cerebellum and temporal cortex, no overlap was observed in the levels of these ETC complexes between subjects with autism and control subjects. In the frontal cortex of Group A, a lower level of ETC complexes was observed in a subset of autism cases, i.e., 60% (3/5) for complexes I, II, and V, and 40% (2/5) for complexes III and IV. A striking observation was that the levels of ETC complexes were similar in adult subjects with autism and control subjects (Group B). A significant increase in the levels of lipid hydroperoxides, an oxidative stress marker, was also observed in the cerebellum and temporal cortex in the children with autism. These results suggest that the expression of ETC complexes is decreased in the cerebellum and the frontal and temporal regions of the brain in children with autism, which may lead to abnormal energy metabolism and oxidative stress. The deficits observed in the levels of ETC complexes in children with autism may readjust to normal levels by adulthood.

    It’s a small study, brain tissue is hard to come by, but still there are some interesting findings here; particularly the spatial component and deteriorating effect of age.

    Food for thought.

    – pD

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