Archive for January 2011
Adventures in Expected Findings Part II – Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism
Posted January 27, 2011on:
Hello friends –
Hot on the heels of Mitochondrial Dysfunction in Autism, another study on mitochondrial function in the autism population was just released, this time giving us insight into what is happening inside the gated community behind the blood brain barrier. How potentially inconvenient. Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism came out the other day; I’ve yet to receive a full copy (one has been promised to my real world email), but the abstract is juicy enough to warrant a small posting.
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. (my emphasis)
A few things immediately jump out at me. Firstly, the Chauhan’s are authors of this paper, who have been around the autism / oxidative stress block since the get go, as authors of the very nice Oxidative stress in autism: increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin–the antioxidant proteins, a really nice paper that was one of the first I saw that broke the autism groups into classic and regressive phenotypes with findings of increased oxidative stress in the latter.
Secondly, one of the biggest concerns with Mitochondrial Dysfunction in Autism when it was released a few weeks ago was, whether or not the findings taken from lymphocytes, cells outside of the brain, could be reliably used as proxies for what is happening within the CNS. Based on the findings in Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism it would seem that, at least in children, there is an increased frequency of mitochondrial problems in the brain. Of course, if we acknowledge the reality of the interconnectedness of immune activation, oxidative stress, mitochondrial impairment and what we already know about the CNS in autism, these findings shouldn’t really be all that surprising. None the less, it is nice to have some direct evidence of this.
Unfortunately, we still don’t know what is causing the problems with mitochondria function in the brain; it is possible, though exceedingly unlikely that all of the participants in this study also had a diagnosable electron chain disorder (I haven’t gotten a full copy of the paper yet). I think it is possible that there is a feedback loop in place involving the immune response, oxidative stress, and mitochondria that for some reason our children’s physiology cannot shake loose from.
The very small sample size of the children in this study, five, is an unfortunate reality for nearly all brain based studies in the autism world. Though I’ve yet to read the full paper, my prediction is that it is liberally peppered with cautious language regarding interpreting the findings widely without further confirmation. That is probably pretty good thinking.
But, if we look closely, and we taken notice of the where of mitochondrial problems in the autism group was observed, we may have evidence of participatory processes. Specifically, Chauhan found decreased electron chain transport measurements in the cerebellum, frontal cortex, and temporal cortex.
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.
There have been a few other studies (that I know of) that have looked for brain region specific abnormalities that might be of interest to u. Brain Region-Specific Changes in Oxidative Stress and Neurotrophin Levels in Autism Spectrum Disorders (ASD), which found increased markers of oxidative stress in the cerebellum:
Consistent with our earlier report, we found an increase in NT-3 levels in the cerebellar hemisphere in both autistic cases. We also detected an increase in NT-3 level in the dorsolateral prefrontal cortex (BA46) in the older autistic case and in the Wernicke’s area and cingulate gyrus in the younger case. These preliminary results reveal, for the first time, brain region-specific changes in oxidative stress marker 3-NT and neurotrophin-3 levels in ASD.
Interesting note: the ‘Wernicke’s area’ of the brain plays a large part in language skills, and in fact, damage to the Wernicke’s area can cause a type of aphasia.
The number of studies that tie together oxidative stress and mitochondrial function are many and numerous to the point of cumbersomeness, I have a short list of them on a previous post about mitochondria function in autism, here.
Two of the really nice neuroimmune studies in the autism realm, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism, and Immune Transcriptome Alterations In the Temporal Cortex of Subjects With Autism both provide evidence of an ongoing immune response in some of the specific areas of the CNS where Chauhan found impaired mitochondrial function, the cerebellum and the temporal cortex.
We demonstrate an active neuroinflammatory process in the cerebral cortex, white matter, and notably
in cerebellum of autistic patients.
The neuroglial activation in the autism brain tissues was particularly striking in the cerebellum, and the changes were associated with upregulation of selective cytokines in this and other regions of the brain.
Expression profiling of the superior temporal gyrus of six autistic subjects and matched controls revealed increased transcript levels of many immune system related genes. We also noticed changes in transcripts related to cell communication, differentiation, cell cycle regulation and chaperone systems.
Detangling if these findings are related, and if so, the direction of causality is for another series of studies to discern. Calls towards the possibility that relationships like this are spurious are common, but I hate to invoke coincidences for no good reason other than coincidences do occur. My suspicion is that the immune findings and impaired mitochondria findings are related, but a cautious suspicion is all that is warranted at this time. I do believe that the relationship between immune activation and mitochondria function is being evaluated now; though I do not know if it is being addressed directly in the CNS, which would be ideal.
Curiously from my perspective, however, is the finding that young adults and adults with ASD in Chauhan did not exhibit decreased electron chain function. The original microglia paper from Vargas, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism found extensive evidence of an ongoing immune response in the CNS of people with autism into adulthood. From the standpoint of a theory wherein an immune response were driving the mitochondrial impairment due to increased oxidative stress, the findings in Chauhan of normal mitochondria function are contradictory to what was found in Vargas. (?)
A few other thoughts occurred to me as I considered the age differences found in Chauhan. If mitochondrial dysfunction is part of the pathogenic force driving behaviors associated with autism, it is possible that a decrease as adulthood is reached conforms with a general improvement in adaptation many people seem to report. Alternatively, if we are actually observing a true increase in the number of people with behaviors that can be classified as autistic, that is, the number of children with autism is a new phenomena, the age findings in Chauhan could be artifacts of different underlying causes of autism in the adults versus the children. I’m a big believer in a wide range of physiological roads to the end point of autistic behaviors, so such a situation doesn’t really bother me conceptually, though it is very, very problematic to put to any kind of designed experiment.
Lastly, for a while now I’ve been putting some thought towards something that’s really been bugging me about the neuroimmune findings in autism when put in context with other ‘classic’ neurological diseases that also exhibit a strong immune component; i.e., Alzheimer’s or Parkinson’s, both of which have strong immune findings as well, but are more strikingly degenerative in nature when compared to autism. Generally you talk about a child with autism gradually getting better, or in some cases reaching a plateau; but very rarely (or never) is there the steady and unforgiving decrease in function that you see in diseases like Alzheimer’s. I’m struggling with this reality and how our findings fit in. I’m not sure how, or if, the age differences in Chauhan are meaningful towards this apparent paradox, but my pattern recognition unit sure is trying to tell me something, I just can’t tell if it’s sending me on (another) snipe hunt or not.
When the entire paper lands in my inbox, I may write another post about it. I’m interested in seeing if any other blogs pick up on this paper or not and what their take on it is. I’m still sort of in the dark on the machinations of the press cycle as it relates to autism news, but this paper doesn’t seem to have gotten the press release treatment that Mitochondrial Dysfunction in Autism did, even though its findings are just as interesting.
Low Penetrance Environmental Impacts, Gene Environmental Interactions, and the Depressingly Bad Jokes that Infiltrate Autism Discussions
Posted January 8, 2011on:
Hello friends –
There’s been something at the back of my mind for a while now regarding the potential for environmental influences to participate in autism, and indeed, a true rise in the number of children that have developmental problems that I’ve been struggling with articulating elegantly. The right course came to me while reading threads where the recent autism risk as proximity to highways paper was discussed. I’m actually not too big on the paper, it is very preliminary, uses some terms that are kind of confusing, and at very best, should be used as a guide for more targeted studies. For anyone who didn’t see it when it came out, essentially it reported a small increase of risk of having a baby with autism as the pregnant mother lived closer to some types of highways.
What I liked about this study is that at the core, there was a twinge of a biologically plausible mechanism, specifically, exposure to pollutants during development and consequent interference with neural development. Examples given in the text including possible endocrine disrupting effects of some types of automotive exhaust, and studies showing altered glutamate expression and associated plasticity defects resulting from pollutants.
What I didn’t like about the study is that it didn’t include any biomarkers and seemed relatively soft on the definitional terms. It was essentially a GIS placement and association lookup; lots of data and easy to find phantoms. A methodologically similar study by Bearman was released a few months previously; purporting to assign a very specific percentage of autism increase (16%) to the spatial proximity of other parents with children with autism, with the idea being that those chatty parents convinced their close neighbors to get their child diagnosed, while those people who more than 500 meters from a child with autism, and therefore don’t talk to as many people, failed to get their child diagnosed. I came down pretty hard on Bearman and don’t see much difference to apply less skepticism here. I will note, however, with no small amount of amusement, that when Bearman was discussed, no one seemed too concerned about the lack of control for urbanicity in the ‘skeptical’ realm. Big surprise.
The skeptics took the freeway paper apart, or in some instances, took apart a reporter or blogger who was spinning the findings as stronger than they were. I was more or less in agreement with the skeptics ideas on this one; this paper certainly was not sufficiently strong to make any conclusive statements and as usual, some headlines got it way wrong.
On the other hand, according to my underlying principles of subtle change still being meaningful, the humbling complexity of poking around with systems like embryonic development, and the difficult to overstate gulf between what we know and what we think we know about the effects of our reckless introduction of a galaxy of sythentic chemicals into the environment our infants are born into, this study fit in pretty nicely; at the very least as a reason to perform bioinformatic analysis of pregnant women to test for biomarkers of exhaust exposure and cognitive outcomes a few years down the ‘road’.
It didn’t take long before the gross over simplifications started rolling in though; i.e., ‘If this study is valid, we should have seen the rise in autism when the Interstate program was initiated in the 1950’s!’ [cue laugh track], or ‘I guess I have genes that made me live near an Interstate’. [cue whoot whoot track] It occurred to me that the Interstate jokes are a good illustration of what is largely wrong with nearly every single discussion on environmental participation you stumble into on the Internet. On one hand, the notion that unless an environmental study has sufficient power to prove a causal relationship for autism, or indeed, can be shown to be unable to account for all autism cases, it is safe to be mocked, or for the more academically minded, accused of being the result of data dredging. Similarly, anything showing a glimmer of plausibility that isn’t a genetic finding can lends itself towards showing how worthless the genetic angle is. These are useful cards to play if your goal is to bash environmental causation theories (and thereby, vaccination causation theories), or if your goal is to bash genetic theories; but ultimately are wastes of time if we want to understand a condition with the murky history and multifaceted manifestations of autism. The crux of what really bothered me about both sides of the Internet joke is that they each ignore meaningful information that can be offered from the other side. It is worse than dumb, it is wasteful.
Stepping away from the environmental end for a moment, I think it is safe to say that everyone is beginning to realize that the hunt for high impact genetic changes that can explain more than a tiny fraction of our autism cases is an abject failure. While there are some genetic changes, like Fragile X, that confer extremely high risk of autism, the absolute number of people with such changes is relatively simple to determine, and they comprise a vanishingly small subset of the children with autism. What we do seem to be finding is that there are lots of genetic changes that confer a small risk of having autism, the so called, low penetrance genetic changes. The idea here is that if you have many, (maybe as many as a dozen or more) low penetrance genes, the cumulative effects build up until a physiological end point is reached wherein autistic behaviors manifest. I actually like the idea behind low penetrance genes a lot; it makes a lot of our finings of genetics make sense, and I absolutely believe in a strong genetic participation in autism.
Remember, at the end of the day, genes are nothing more than blueprints for building proteins. Most genetic alterations don’t involve complete additions, or removals, of proteins, but rather, creation of a little less, or a little more of a protein, or perhaps, creation of proteins that are just a tiny bit different than ‘normal’, sort of like autism itself. While the environment these proteins enter, or are regulated into entering, starts influencing the eventual biological outcome in the most immediate sense imaginable, the end points of genetics, these proteins and their precise structures are indisputably important in what is happening in everything our bodies do; including, in some instances, have autism.
Consider the tightly orchestrated formation of the microscopic chasms between neurons, the process of synaptogenesis. Dozens (or hundreds) of chemicals dance together in order to form the structures in our brains that exchange chemical messengers, neurotransmitters, that literally form the foundation of neuron to neuron communication, and thus, cognition; the physical constructs of thought. It is a biological cauldron that we are just beginning to comprehend, the mind formingly intricate, time dependent interplay of a chemical deck of cards orders of magnitude more complicated than sequencing the genome.
The evidence for altered synapses, and modified synaptic function in autism, and most (all?) other developmental disorders is impossible for an intellectually honest observer to deny. Some of the most commonly found genetic alterations in people with autism involve genes known to participate in the formation, maintenance, or functioning of synapses. For example, neurexin , shank, and neuroligin, are some well known, or at least, well reported reported genes intimately attached to synapse function also found associated with autism, and our list should also include calcium expression and adhesion genes (and many, many others). Each of these genes or processes contribute to the synapse in subtle, but different ways, at different times, and yet we can see that interferences anywhere down the functional class of chemicals is associated with autism. Yet, very few people, (I’ve read of none), have been found to have a neurixin allele, a shank allele, and a neruoligin mutation. And there are some people who have the same mutations, but do not exhibit autistic behavior. There are also a great many people that have no known mutations in any of these genes, and still, receive an autism diagnosis. What does this tell us?
It should tell us that while there are lots of genetic ways that synapse function can be altered in such a way that autistic behaviors bubble up to the diagnosis endpoint, but more importantly, the critical question need not necessarily revolve around what genes you have, but rather, is synapse function manipulated? Furthermore, we should be able to conclude that simply having a single modifier (i.e., one shank mutation) go wrong isn’t a guarantee of an autism diagnosis, and thus; the participation of individual mutations is real, but small. [I would also argue that it is likely that there are a great number of as of yet, undetected genetic misprints that contribute in the same real, but subtle ways.]
Another more accessible example of a low penetrance gene is the MET gene, which produces a protein known to interact with a lot of important processes involved in autism, including brain formation, immune system functioning, and intestinal repair. There have been a lot of high quality studies on the MET mutations in the past few years including those that report higher incidences of MET mutations in children with autism and gastrointestinal problems, higher findings of MET alleles in autism, association to communication phenotypes and MET expression, replication of above studies, evidence of interaction with other genes known to be associated with autism, decreased expression in post mortem brain tissue, and animal studies showing differential, time dependent expression of MET. (and many, many others).
The kicker towards this discussion, howeever, is that the changes to the MET gene are really, very, very common. Nearly one half of everyone has the low MET production gene, but even still, many more people with autism have it. So, while it is clearly implicated, other changes are obviously necessary for that particular genetic change to result in autism. What we are learning about the systems affected by MET, or lots of the genes implicated in autism, is that very subtle changes towards critical processes are sufficient to modify the course of development. Somewhat counter intuitively, I would argue that the implication of this is compelling evidence (or terrifying news) for those of us with worries about the possibility of an environmentally driven increase in the number of people with an autism diagnosis; indeed, it argues that just like genetics, we must admit the reality that if genes can be low penetrance, so too, then, can environmental impacts.
For example, back to brain formation. We know that the neurexin proteins participate in forming our synapses. But we have evidence that hypo
thyroidism can lead to structural changes during development, and we also know that there is increasing evidence that endocrine disruptors can interferre with thyroid metabolism, or for that matter, a wide range of findings on endocrine disruptors and cognitive function. Or if we look to pesticides, we have evidence that developmental exposure to diazonon can modify neurotransmitter function, with similar findings are available for other classes of pesticides. Similarly with heavy metals.
The skeptics would claim with some legitimacy that there are significant dose dependency problems to be addressed before we should start pointing to every experimental slice of evidence of potential harm and claiming that the sky is falling. But. What if, in fact, we need only perturb the process of brain development a little bit, and with a little help from other, low penetrance genes or other exposures, developmental trajectories begin to alter? This would seem to be precisely what we are learning from the genetic angle; it isn’t one big thing incorrectly designed, it is lots of small things. And while our genetic code has, for the most part, remained stable; our environment today is vastly overpopulated with chemicals capable of minor, but real, effects when compared to yesteryears past.
The search for a single environmental impact with the ability to explain a significant portion of autism diagnosis is as futile as the hunt based on genetics. This makes for a far messier landscape, but also one that fits my terrifying, over arching principle of the Fairytale of a static (or near static) rate of autism, that our uncontrolled experiment of introducing synthetic chemicals into our environment coupled with widespread social changes with real physiological impacts, a set of experiments absolutely unprecedented in the history of living things on planet Earth, that changes to our offspring are unavoidable. To suggest otherwise, strikes me as either the height of arrogance, or the depths of ignorance.
Going back to the freeway study for a minute, I ran into a paper while writing this piece that involves pollutants, interaction with the MET gene, gene x environment interactions, and low penetrance impacts that I think has salience towards this discussion.
Here is the abstract:
Gene by environment interactions (G × E) are thought to underlie neurodevelopmental disorder, etiology, neurodegenerative disorders, including the multiple forms of autism spectrum disorder. However, there is limited biological information, indicating an interaction between specific genes and environmental components. The present study focuses on a major component of airborne pollutants, polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene [B(a)P], which negatively impacts cognitive development in children who have been exposed in utero. In our study, prenatal exposure of Cpr(lox/lox) timed-pregnant dams to B(a)P (0, 150, 300, and 600 μg/kg body weight via oral gavage) on embryonic day (E14-E17) consistent with our susceptibility-exposure paradigm was combined with the analysis of a replicated autism risk gene, the receptor tyrosine kinase, Met. The results demonstrate a dose-dependent increase in B(a)P metabolite generation in B(a)P-exposed Cpr(lox/lox) offspring. Additionally, a sustained persistence of hydroxy metabolites during the onset of synapse formation was noted, corresponding to the peak of Met expression. Prenatal B(a)P exposure also downregulated Met RNA and protein levels and dysregulated normal temporal patterns of expression during synaptogenesis (!). Consistent with these data, transcriptional cell-based assays demonstrated that B(a)P exposure directly reduces human MET promoter activity. Furthermore, a functional readout of in utero B(a)P exposure showed a robust reduction in novel object discrimination in B(a)P-exposed Cpr(lox/lox) offspring. These results confirm the notion that common pollutants, such as the PAH B(a)P, can have a direct negative impact on the regulated developmental expression of an autism risk gene with associated negative behavioral learning and memory outcomes.
I have to say, finding this paper was a bit of tragic humor for me; it was published in December 2010, with zero fanfare from the press, as opposed to the confounder heavy, Residential Proximity to Freeways and Autism in the CHARGE study, study, which had a thousand similar articles in Google News. But here we find a superb example of what gets bandied around a lot when in quick passing but rarely with any meat behind the discussion; a real life, experimentally sound version of a gene environment interaction that integrates biologically plausible mechanisms that is able to describe what is observed physiologically in autism with dose responses. Beautiful. But, it gets even better. It just so happens, the classifications of agents in use in this study, polycyclic aromatic hydrocarbons, are generated, in some instances, by car exhaust. In fact, in Detection of polycyclic aromatic hydrocarbon exposure from automobile exhaust fumes using urinary 1-hydroxypyrene level as an index, the authors conclude in part that “Automobile exhaust fume exposed subjects have a higher risk to be exposed to PAHs than the non-exposed subjects”. Go figure.
Whatever the problems with the freeway CHARGE study, they pale in comparison to the problems that the notion that because we didn’t observe increases in autism when the Interstate system was constructed, the findings must be spurious. Similarly, genetic predisposition is an indisputable fact; and knowing which genes are implicated in autism can help us intelligently target environmental factors that might be changing our infants.