Posts Tagged ‘Inconvenient Finding’
Adventures in Expected Findings, Fascinating Complexity, Feedback Loops and Tragic Hypocrisy – ‘Mitochondrial Dysfunction In Autism’
Posted December 31, 2010on:
Hello friends –
The mitochondria discussion in the autism community reminds me a lot about the political discussion in the United States; I know it is important, but it is just so hard for me to care enough to get involved; it mandates walking the plank into an environment dripping in hypocrisy, where highly complicated problems are reduced to black and white meme friendly soundbytes, and discussions that seem a lot more like billboards on different sides of the road than people wanting to discuss anything. It started with the case of Hannah Poling, the little girl who experienced a dramatic and sudden developmental regression following her vaccinations at age 18 months, a case wherein the federal government conceded that vaccines through likely interaction with a pre-existing defect in mitochondrial function were likely the cause of her developmental trajectory and ‘autism like features’.
On some parts of the Internet, you’d think that every single child with an autism diagnosis experienced a drastic, overnight regression in development that Hannah Poling did; despite abundant, clear as the day common sense evidence that the onset of autism is gradual in the overwhelming majority of instances. For the most part, I don’t think it was a spin job. I just don’t think they get it. Although, I must admit, I do believe that there are a very small, but real, minority of parents who have witnessed similar things with their children. Hannah Poling is not unique.
On the other hand, lots of other places you could find people whose online existence is part and parcel with the notion that our real autism rates are static, that the inclusion of less severe children was burgeoning our observed rates of increases, and yet, found the intellectual dishonesty to question if Hannah Poling had autism or not, as if suddenly, in this one particular instance, a diagnostic report of having ‘features of autism’ as opposed to ‘autism’ was meaningful. As if that fucking mattered.
On the one side there is the failure to recognize any semblance of nuance, of complexity, and on the other, a startling hypocrisy and lack of curiosity.
A few weeks ago (maybe a few months ago, by the time I finally get this post published, at my rate), a paper came out that reported, among other things, children with autism were more likely to have mitochondrial dysfunction, mtDNA overreplication, and mtDNA deletions than typically developing children. That paper, of course, is Mitochondrial Disorder In Autism, a new winner in the field of simple to understand, straightforward titles. The good news is that Mitochondrial Disorder In Autism is another portrait of beautiful and humbling complexity with something to offer an open mind. Maddeningly, my real world email address received an embargo copy of the paper, which is somehow protected from copy paste operations, meaning most parts from that paper here will be manually transcribed, or more likely, paraphrased.
This is a cool paper, it sheds light on the possible participation of a widely observed phenomena in autism, increased oxidative stress, gives us additional evidence that the broader incidence of mitochondrial dysfunction is significantly very higher in the autism population, and an possible illustration of a feedback loop.
Very briefly paraphrased (damn you, embargo copy!), the authors used samples of peripheral cells of the immune system, lymphocytes, to test for mitochondrial dysfunction. This is a big step, it allowed the researchers to bypass the traditional method of muscle biopsy, which is both invasive and painful. It is reminiscent of using lymphoblastoid cells as proxies for neural cells in genetic expressions studies; the type of small, incremental data that can get lost in the headline, but has potentially broad applications.
In Mitochondrial Dysfunction in Autism, according to the authors, lymphocytes were considered sufficient surrogates because they are power hungry and derive a significant portion of their energy needs from oxidative phosphorylation; i.e, mitochondrial function. It was small study, ten children with autism and ten controls; I’m not clear why such a small sample was used, perhaps the laboratory time and/or dollar requirements involved with detecting mitochondrial dysfunction, even in peripheral cells, mandated that such small numbers be used. (?) Perhaps funding could not be obtained for a larger study without some preliminary results, and as is mentioned several times in the text, these findings should be replicated if and when possible.
Two types of changes to mtDNA were evaluated for, the ratio of the total number of mtDNA to nuclear DNA (i.e., ‘normal DNA’), and the presence of deletions of parts of mtDNA. These changes are a lot different than what we normally think of in genetic studies, and here’s my short story (barely longer than my understanding) of how.
Each mitochondria has a variable number of mtDNA copies, usually estimated at between 2 and 10. The understanding on what a relatively higher, or lower number of copies of mtDNA means for an organism is ongoing and nascent; for example, findings of associations with lower mtDNA levels in elderly women and cognitive decline, or finding that mtDNA copy number associate positively with fertility, both of which were published in 2010 (there are, conservatively, a brazillion other studies with a broad range of topics). Highly salient for our purposes, however, are findings cited by this article, Oxidative Stress-related Alteration of the Copy Number of Mitochondrial DNA in Human Leukocytes, which reports that cells experiencing oxidative stress had increased number of mtDNA copies. In Mitochonddrial Dysfunction in Autism the authors report an increase in the number of mtDNA copies in the autism group.
Secondarily, the authors also looked for differences in mtDNA structure, but again in this instance, not in the way that we frequently think about genetic studies; they were not looking for an A replaced G mutation that exists in every gene, in every cell, in the individual, but rather, different structural components that were indicative of damage within the copies of mtDNA. Thus, it wasn’t so much a case of a blueprint gone wrong, as much of case by case differences in mtDNA; potentially the result of exposure to reactive oxygen species during replication.
Changes in both copy number of mtDNA (increased), and structure (mostly deletions) were observed in the autism group.
Up and above changes to mtDNA, several biomarkers of direct and indirect mitochondrial dysfunction were measured, including lactacte to pyruvate ratios, (which have been observed abnormal previously in autism and speculated to be resultant from mitochondrial problems), mitochondrial consumption of oxygen, and hydrogen peroxide production, a known signal for some types of mitochondrial dysfunction. Several of the biomarker findings were indicative of problems in mitochondrial function in the autism group, including impaired oxygen consumption, increased hydrogen peroxide production, and as noted by other researchers, higher pyruvate levels, with a consequent decreased lactate to pyruvate ratio compared to controls.
These findings were described by the authors like this:
Thus, lymphocytic mitochondria in autism not only had a lower oxidative phosphorylation capacity, but also contributed to the overall increased cellular oxidative stress.
In plainer English, not only was the ability to produce energy reduced, but the propensity to create damaging byproducts, i.e., oxidative stress, i.e., ROS was increased. Talk about a double whammy! There have been a lot of studies of increased oxidative stress in the autism population, one of the first was Oxidative stress in autism: increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin–the antioxidant proteins, with other titles including, Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism, Oxidative stress in autism, Brain Region-Specific Changes in Oxidative Stress and Neurotrophin Levels in Autism Spectrum Disorders (ASD) and many, many others. Could mitochondrial dysfunction be the cause of increased oxidative stress in autism? Could oxidative stress by the cause of mitochondrial dysfunction in autism? Could both be occurring?
Oxidative stress deserves a free standing post (or a few), but at a high level refers to the creation of damaging particles, called reactive oxygen species by our bodies during the course of many biological operations; including generating energy (i.e., the function of mitochondria). The graceful management of these particles is essential for normal functioning; too little containment and there can be damage to cellular structures like cell membranes, or DNA. You can measure these types of damage, and a wide swath of studies in the autism realm have found that on average, children with autism exhibit a state of increased oxidative stress when compared to children without that diagnosis. A great variety of conditions other than autism, but which you’d still generally rather not have, are also characterized by increased oxidative stress, as are things that you can’t really help having, like getting old.
(It should be noted, however, that in an illustration of humbling complexity, we are now learning that containing free radicals by all means possible may also not necessarily be a good idea; our bodies utilize these chemicals as signals for a variety of things that aren’t immediately obvious. For example, there is preliminary evidence that too much antioxidants can cancel out, the benefits of exercise; our bodies were using the effects of exercise as a signal to build more muscle, likewise, we have evidence that oxidative stress plays a part in apotosis, or programmed cell death, and interfering with that may not be a good idea; in fact, it could, participate in carcinogenisis. There is no free lunch.)
Mitochondrial Dysfunction in Autism speculates that oxidative stress and mitochondrial dysfunction could be linked, either by increased oxidative stress leading to problems in mtDNA replication (i.e., the observed mtDNA problems are a result of aggressive attempts at repair, repair to damage induced by the presence of reactive species), or by deficiencies in the ability to remove ROS; i.e., decreased glutathione levels as observed by James. This really speaks towards the possibility of a feedback loop, something leads to an increase in oxidative stress that cannot be successfully managed, which causes mitochondrial damage, which leads to problems in mtDNA replication, which in turn, leads to dysfunction, and increased oxidative stress. Again, from the paper:
Differences in mtDNA parameters between control children and those with autism could stem from either higher oxidative stress or inadequate removal of these harmful species. The increased reactive oxygen species production observed in this exploratory study is consistent with the higher ratio of oxidized NADH to reduced glutathione in lymphoblastoid cells and mitochondria from children with ASD, supporting the concept that these cells from children with autism present higher oxidative stress. Increased reactive oxygen species production induced by mitochondrial dysfunction could elicit chronic oxidative stress that enhances mtDNA replication and possibly mtDNA repair.Collectively, these results suggest that cumulative damage and oxidative stress over time may (through reduced capacity to generate functional mitochondria) influence the onset or severity of autism and its comorbid symptoms.
(My emphasis). More on why a little later.
There is a lengthy section of the paper regarding the limitations of the study, including a relatively small sample set, racial differences between the participants, and the possibility that the number of evaluations made could impact the strength of some associations. Detangling the arrow of causality is not possible from this paper, and likely involves different pathways in different patients. None the less, it is additional confirmation of something gone awry in the power processing centers of cells in people with autism.
This is a pretty small study, from a number of subjects perspective, and the pilot nature of the study is somewhat of a problem in trying to determine how much caution we must use when attempting to generalize the findings to a larger population. However, on the other hand, if we look towards earlier findings, some of which were linked above, the reports in Giulivi should not really be that surprising. In fact, we should have been amazed if they hadn’t observed mitochondrial problems.
Here is why:
We have voluminous observations of a state of increased oxidative stress in the autism population; Chauhan 2004, Zoroglu 2004, James 2004, Ming 2005, Yao 2006, James 2009, Sajdel-Sulkowska 2009, Al-Mosalem 2009, De Felice 2009, Krajcovicová-Kudlácková M 2009, El-Ansari 2010, Mostafa 2010, Youn 2010, Meguid 2010, and Sajdel-Sulkowska 2010, all are clinical trials that reported either increased levels of oxidative stress markers, decreased levels of detoxification markers, or both, in the autism group. There is no way, absolutely no way that children with autism have less oxidative stress, or the same oxidative stress than children without that diagnosis, barring some mechanism by which all of the above studies are wrong in exactly the same direction. There is just too much evidence to support an association, and as far as I know, (?) no evidence to counter balance that association. [Please note that the above studies are for biomarker based studies only, I left out several genetic studies with similar end game conclusions; i.e., alleles known to be associated with increased oxidative stress and/or mitochondrial function are also associated with an autism diagnosis.]
We also have just a large body of clinical evidence that tells us that as oxidative stress and mitochondrial function are closely linked, as oxidative stress increases, so too do problems with mitochondrial function and/or replication; Richter 1998, Beckman 1998, Lu 1999, Lee 2000, Wei 2001, Lee 2002, Liu 2003, Liu 2005, Min Shen 2008 are useful examples. Unless all of these studies, and many more, are incorrect in the same way, and the underlying physical foundations of why oxidative stress would lead to mitochondrial function are also incorrect, we must conclude that a state of increased oxidative stress, as observed repeatedly in autism, leads to a degradation of mitochondrial function.
It turns out, there also a growing body of evidence linking oxidative stress and/or mitochondrial dysfunction to other conditions with a neurological basis (Rezin 2009), such as schizophrenia, (Prabakaran 2004, Wood, 2009, Martins-de-Souza 2010, Verge 2010, Bitanihirwe 2011) or bi-polar disorder (Andreazza 2010, Clay 2010, Kato 2006, Kaikuchi 2005). Here is the abstract for Oxidative stress in psychiatric disorders: evidence base and therapeutic implications:
Oxidative stress has been implicated in the pathogenesis of diverse disease states, and may be a common pathogenic mechanism underlying many major psychiatric disorders, as the brain has comparatively greater vulnerability to oxidative damage. This review aims to examine the current evidence for the role of oxidative stress in psychiatric disorders, and its academic and clinical implications. A literature search was conducted using the Medline, Pubmed, PsycINFO, CINAHL PLUS, BIOSIS Preview, and Cochrane databases, with a time-frame extending to September 2007. The broadest data for oxidative stress mechanisms have been derived from studies conducted in schizophrenia, where evidence is available from different areas of oxidative research, including oxidative marker assays, psychopharmacology studies, and clinical trials of antioxidants. For bipolar disorder and depression, a solid foundation for oxidative stress hypotheses has been provided by biochemical, genetic, pharmacological, preclinical therapeutic studies and one clinical trial. Oxidative pathophysiology in anxiety disorders is strongly supported by animal models, and also by human biochemical data. Pilot studies have suggested efficacy of N-acetylcysteine in cocaine dependence, while early evidence is accumulating for oxidative mechanisms in autism and attention deficit hyperactivity disorder. In conclusion, multi-dimensional data support the role of oxidative stress in diverse psychiatric disorders. These data not only suggest that oxidative mechanisms may form unifying common pathogenic pathways in psychiatric disorders, but also introduce new targets for the development of therapeutic interventions.
Given all of this, one might consider casting an extremely skeptical eye towards the argument that the observations in Mitochondrial Dysfunction in Autism are insufficiently powered to reach any conclusions about an association; at this point, I think it is fair to say that what should have been surprising finding would have been a lack of mitochondrial dysfunction in autism. We need to rethink some foundational ideas about the relationship between oxidative stress, mitochondrial function, other neurological disorders, and/or assume that a dozen studies are all incorrect in the same way before the small number of participants and other limitations of this study should cause us to cast too much doubt on the findings. The findings in Mitochondrial Dysfunction in Autism are not due to random chance.
All that being said, there are still lots of questions; the most intriguing ones I’ve seen raised in other discussions on this paper would include, Is the mitochondrial dysfunction physiologically significant? and secondly, What has caused so many children with autism to exhibit these physiological differences?
I’ll admit it, early on in my online/autism persona lifetime, I’d have viewed the first question as largely deserving of a healthy dose of (hilariously delivered) sarcasm. But the reality is that this is a more difficult question to answer than it would seem on the surface. The reasons I’ve seen posited that this might be valid sound pretty good at first glance, i.e., the brain is the most prolific user of energy in the body, and problem with energy creation there are pretty simple to equate to cognitive problems. And this might be what is happening, I don’t believe we have enough information reach any conclusions. I will note, however, with no small amount of amusement, that the online ‘skeptical’ community had no problem with this exact argument in discussing what happened to Hannah Poling, as long as it was exceptionally rare.
Specifically speaking towards the problems of physiological significance, we haven’t any direct evidence one way or the other that the mitochondrial dysfunction observed in muscle biopsy or lymphocytes is present in the CNS of people with autism, and this is an important distinction; it is known that there are large differences in mitochondrial need and function between tissue type, and it is almost always dangerous to assume that because you see something outside the privileges of the blood brain barrier, that you will see the same thing within it. Therefore, we should remember that it is possible that the brains are unaffected, while the peripheral cells are.
However, we do have some indirect evidence to suggest that there are mitochondrial function problems in the CNS in the autism population. Based on studies that have measured oxidative stress levels in the brain, specifically Brain Region-Specific Changes in Oxidative Stress and Neurotrophin Levels in Autism Spectrum Disorders (ASD) we have preliminary evidence that areas of the brain are affected by high levels of oxidative stress. Furthermore, we have a multitude of studies regarding an ongoing immune response in the brain in autism, and we know that the immune response can generate oxidative stress, and indeed, interact with some of the results of oxidative stress, potentially participating in a feedback loop.
In short, we know that inflammation, oxidative stress, and mitochondrial function are closely linked; considering the fact that we have evidence of two of these processes being altered in the CNS in autism, barring an unforeseen mechanism by which this association is not in place in the brain, an exceedingly unlikely situation given our observations in other cognitive domains, it seems probable that some degree of mitochondrial dysfunction occurs in the brain as well as the periphery. If this is sufficient to cause autism will require more studies; some evaluations correlating behavioral severity and / or multiple evaluations over time would be good starting points. as well, of course, as direct CNS evaluation.
The second question, towards relevance of these findings, the reason such a large percentage of children with autism appear to have characteristics of mitochondrial dysfunction is even more difficult to detangle. The potential of a feedback loop existing between oxidative stress and mitochondrial function was problematic enough, but it seems likely there could be other participants, for example, the immune system. There are repeated observations of an exaggerated immune response, from genetic predispositions to known toll like receptor promoters, circulating levels of endogenous factors associated with a vigorous immune response, baseline levels of cytokines and chemokines, and cytokine values resulting from direct toll like receptor activation. Is the over active inflammatory response observed in autism causing the mitochondrial dysfunction through an increase in oxidative stress? Is the increased oxidative stress causing an ongoing inflammatory response? Studies evaluating for a relationship between these parameters would help to answer these questions.
For a real world example of why such a relationship might be possible, we can take a look at a paper that landed in my inbox around the same time that Mitochondrial Dysfunction in Autism did, Dopaminergic neuronal injury in the adult rat brain following neonatal exposure to lipopolysaccharide and the silent neurotoxicity. This paper is another that shows some very difficult to predict outcomes as a response to an early life immune challenge. Here is the abstract:
Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague–Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1b and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity.
Briefly, the researchers challenged the animals with an immune stimulator shortly after birth, and then went on to observe chronic microglial activation and inhibited mitochondrial function into adulthood. Behavioral problems included hyperactivity and stereotyped tasks (though these behaviors appeared to reverse in adulthood. Subsequent challenge with methamphetamine in adulthood resulted in increased locomotive and stereotyped behaviors in the treatment group.
Check that out! These animals never actually got sick, their immune system had only been fooled into thinking that it was under pathogen attack, and yet, still showed chronic activation of the neuroimmune system and impaired mitochondrial function in dendrites into adulthood! ). In a sense, it might be appropriate to say, then, that the behaviors were not a state of stasis. Talk about an inconvenient finding.
There is also the possibility that exposure to chemicals, such as pesticides, may be able to cause mitochondrial dysfunction.
Finally, during the time it took me to put this post together, several other reviews of Mitochondrial Dysfunction in Autism landed online in places that purport to be bound by objective and dispassionate evaluation of the science of autism; Respectful Insolencence, LBRB, and Science2.0 all had posts (probably others too). [The masochists out there that go through the discussion threads will note that several of the thoughts in this posting were experimented with in responses to these threads, ideas which were largely, or entirely, ignored.] If you were to read these other reviews (I would recommend that you do), you might come away with the impression that Mitochondrial Dysfunction in Autism consisted of nothing more than criteria for selecting participants and limitations of the study. The calls for caution in running wild with these findings are there, and I largely agree with this sense of caution, as is the admission that this is an area that should be studied more intently, but nowhere was there any acknowledgement of the consistency between these findings and the repeated observations of increased oxidative stress in autism and the biological reality that oxidative stress is linked with mitochondria function, nowhere was there any mention of the fact that the findings were in alignment with deficiencies in detoxification pathways as observed multiple times in autism, nowhere was there anything regarding our voluminous evidence of impaired mitochondrial function in a veritable spectrum of cognitive disorders. Did the online skeptical community get a different copy of the paper that I did? Perhaps, were they unaware of the repeated reports of increased oxidative stress in autism, and the incontrovertible evidence of an association between oxidative stress and mitochondrial dysfunction? Is there a chance that their pubmed results regarding mitochondria and disorders like schizophrenia or bi-polar disorder are different than mine?
I am afraid that this is what the vaccine wars and wrangling over the meaning of neurodiversity have done to us; the skeptical community absolutely went “all in” on the premise that the Hannah Poling concession was founded on a very, very rare biological condition. They have sunk one hundred and ten percent of their credibility behind the notion that thimerosal based studies and MMR based studies are sufficient to answer the question of if vaccines can cause autism, or if we must, features of autism. And now, with converging evidence from several directions pointing towards a confluence of mitochondria impairment and oxidative stress in autism and other neurological conditions, speaking towards the meat of Mitochondrial Dysfunction in Autism is more than just eating crow, it is akin to blaspheming, for if diagnosable mitochondrial disorder affects a meaningful fraction of children with autism, and mitochondrial dysfunction a much larger percentage, the foundations behind the meme of the vaccine question as one that needs no further evaluations begins to fall apart. That is a legitmately scary proposition, but one that is going to have to be reckoned with sooner or later; the only difference is that the more time passes, the greater the credibility strain on the mainstream medical establishment when, eventually, it is admitted, that we need to come up with good ways to generate quality information on vaccinated and unvaccinated populations.
Similarly there is remakarble opposition in some quarters to the idea of imparied detoxificiation pathways, or indeed, a state of increased oxidative stress in some of the same places. I think the underlying reason for this is that some of these early findings were used by some DAN doctors to promote things like chelation, almost certainly the wrong treatment for the overwhelming majority of children on whom it was performed; and in a well intentioned zeal to discount some of these practioners, as well as the outrage over statements by some (i.e., ‘toxic children’), the reality of the situation; that our children are more likely to have increased oxidative stress, do have less glutiathione, became acceptable facts to bypass in the rush to hurl insults or wax poetic. We can acknowlege that children with autism have these conditions while simultaneously expressing concern, or outrage, at the notion that this makes them poisonous; but ignoring the physiological reality of our findings does nothing to help anyone. The data is the data.
This is all too bad. In fact, it is worse than too bad; there is no reason, absolutely no reason that a discussion on mitochondrial impairment must focus exclusively on the vaccine question, in fact, just the opposite. There are lots of ways to achieve an endpoint of mitochondrial dysfunction, and lots of things besides vaccines that can be problematic for people with this problem. (including, of course, actual infection!) But we have become so polarized, so reliant on hearing the same soundbyte laden diatribes, that any sense of nuance on the question immediately labels on as ‘anti vaccine’, ‘anti science’ (even worse!), or for that matter, ‘pro-vaccine’ or shill. The questions raised by Mitochondrial Dysfunction in Autism are important and aren’t going to go away, no matter how inconvenient the follow up findings may be.
Posted February 22, 2010on:
Hello friends –
One of the most frequent omissions in the pre-eminent autism debate is the very, very different immune response that our population of interest seems to have in comparison with children without a diagnosis of autism. A 2009 paper from the MIND institute is a good example of this type of finding, “Differential monocyte responses to TLR ligands in children with autism“.
Some background is critical here to understand this paper. The very first step in the initation of an immune response is the identification of an invading pathogen as foreign to the body, an intruder, and subsequently marshalling other immune system cells to launch a counterattack on the foreign attacker. The components of the immune system that are responsible for this are the Toll Like Receptors, or TLRS. [The Wiki link, at left, has a very nice table of the known TLRs and the triggering molecular structure, immune system cells that express the TLR, and signaling mechanisms. ] At a very detailed molecular level, these proteins have developed the ability to discriminate different classifications of microbial pathogens; in other words, some TLRs can identify cell structures common to bacteria, some TLRs identify signatures associated with viruses, and so on. It is the TLRs that launch the first phase of the immune response, the innate immune response, and there is increasing evidence that TLRs also play a role coordinating the adaptive immune response. For our purposes, it is sufficient to understand that Toll Like Receptors are the critical starting point of the generation of innate immune cytokines that we see abnormal in so many studies in autism.
From the abstract:
Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1b, IL-6, IL-8, TNFa, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1b, IL-6, and TNFa responses following TLR 2, and IL-1b response following TLR stimulation in monocyte cultures from children with ASD (p < 0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1b, IL-6, GM-CSF, and TNFa responses in monocyte cell cultures from children with ASD compared with controls (p < 0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.
So, at a high level we can see that in the test tube, blood from children with autism generates a different immune response than blood from children without autism, and further, that this differentiation seems to be TLR specific. In a surpizingly common finding, we observe an increase in pro-inflammatory cytokines such as IL-1B, IL-6, and TNF-Alpha, all of which have many other findings in autism, seizures, and other neurological conditions. More curious, to my mind, is the decreased response to TLR9, another TLR responsible for orchestrating the immune response to some types of bacterial invaders.
From the discussions section:
Our results indicate notable differences in cytokine production following TLR stimulation in monocyte cell cultures from ASD children including increased pro-inflammatory cytokine production following exposure to the TLR 2 ligand, LTA with increased production of IL-1b, IL-6, and TNFa (3.3-, 3.1-, and 2.9-fold increases, respectively) relative to TD controls. In addition, there was an almost twofold increase in IL-1b responses following TLR 4 stimulation with its ligand LPS. Our current findings are consistent with previous reports of enhanced innate immune activity in ASD (Croonenberghs et al., 2002; Jyonouchi et al., 2001), and further indicates that a dysfunctional innate immune response may occur in a number of individuals with ASD.
TLR2 and TLR4 are both involved with sensing and responding to bacteria; I’m not up to speed currently to give a good description of the specific bacterial populations; for example, TLR4 is responsible for sensing gram negative bacteria, which refers to a specific protein structure on some types of bacteria. The paper then goes on to describe some of the other known findings involving TLRs or their outputs for autism or other neurological conditions.
Pro-inflammatory cytokines, IL-1b, IL-6, and TNFa, which are predominantly derived from cells of the monocyte lineage, are of special interest in the study of neuroimmunological contributions to psychiatric disorders. These cytokines can act both locally and centrally to increase neuroinflammatory responses and/or to affect brain function such as the induction of serotonin from the hypothalamus; changes that may affect behavioral responses (Dunn, 2006). Of the TLR ligands analyzed in this study, those specific to induce TLR 2 signaling, elicited the most profound pro-inflammatory response in monocyte cell cultures derived from children with ASD. TLR 2 is constitutively expressed on the surface of microglial cells (Bsibsi et al., 2002; Kielian et al., 2005; Olson and Miller, 2004) and deficiencies in TLR 2 but not TLR 4, reduce T cell recruitment, microglial proliferation, and cytokine/chemokine expression in a neonatal murine model (Babcock et al., 2006). Previous animal studies have demonstrated that TLR 2 stimulation, leading to pro- inflammatory cytokine production, is sufficient to induce neuroinflammation and the neuronal degeneration that is characteristic of bacterial meningitis, and that TLR 2 deficient animals are protected from such changes (Hoffmann et al., 2007). In a murine EAE model of multiple sclerosis, the clinical disease course and severity of the condition correlated with increased brain expression of CD14 and TLR 2 transcripts, suggesting that there is an increase in or upregulation of microglial cells and monocytes in this model, and that TLR signaling may be actively involved in neuroinflammation and autoimmune development (Zekki et al., 2002). The induction of an inflammatory cytokine storm, initiated by monocyte activation, could produce downstream effects leading to the generation of neuroinflammatory and/or autoimmune responses. An autoimmune sequelae such as the generation of anti-neuronal antibodies to a wide variety of targets have been described in individuals with ASD and may be a consequence of responses originally started by inappropriate innate immune activity (Cabanlit et al., 2007; Connolly et al., 2006, 1999; Croen et al., 2008; Kozlovskaia et al., 2000; Silva et al., 2004; Singh and Rivas, 2004b; Singh et al., 1997a,b; Wills et al., 2009).
Of particular interest here is the discussion that TLR seems to play a very important role in the immune response in the CNS, and in fact, animals bred without TLR2 expression fail to develop a neuroinflammatory state when induced in normal rodents. Given what we know from Vargas, Li, Chez, and Garbett, we seem to be observing an ongoing immune response in the CNS in autism, the fact that TLR2 seems to respond more robustly in the autism population would seem to be a piece of the puzzle as to why this might be occurring. In a very real way, for reasons still unclear, people with autism are predisposed to respond more robustly using mechanisms already associated with neuroinflammatory conditions.
Following is a section focusing on a variety of research involving prenatal immune challenges and subsequent behavioral outcomes in the offspring. Then there is a section that has a lot of very cautiously placed ‘ifs’, ‘maybe’s, and ‘possibles’ that still raises a lot of intriguing possibilities.
In this study, we demonstrated that there is differential signaling in monocytes through different TLRs in children with ASD compared to TD controls. For instance, while LTA induced an increased pro-inflammatory IL-1b, IL-6, and TNFa response and LPS induced increased IL-1b in ASD compared to TD, exposure to poly I:C or flagellin produced similar responses between cases and controls, and CpG produced a significantly lower monocyte response in ASD compared to TD. This may mean that signals generated through different TLR by the recognition of distinct PAMPS expressed by specific bacteria or viruses may lead to differential innate immune activity in ASD. For example, in the current study, signaling through TLR 9 by CpG stimulation was notable for resulting in significantly lower IL-1b, TNFa, MCP-1, and GM-CSF release in ASD compared with TD. Typically, TLR 9 ligand recognition induces downstream anti-viral responses, mainly through interferon a/b production (Kawai and Akira, 2007). The clinical significance of this is unknown but may suggest that children with ASD respond poorly to TLR 9 stimulation that may lead to an ineffective anti-viral interferon response and may to inappropriate responses which could lead to infection, chronic inflammation and tissue destruction and could hence expose the individual to increased levels of autoantigens.
In contrast, signaling through TLR 2 and TLR 4 leads to the marked release of pro-inflammatory cytokines. The pronounced increase in the production of these cytokines in response to LTA and LPS ligation warrants further investigation to elucidate the signaling cascade generated from TLR 2 and TLR 4. A previous report indicated that in the first month of life, children that later develop ASD have more infections than their counterparts (Rosen et al., 2007). These previous findings documenting the presence of increased bacterial and viral infections in conjunction with our observations that children with ASD are hyper-responsive to LTA and LPS stimulation could suggest that aberrant signaling through TLR 2 and TLR 4 may participate in this disorder. Inappropriate stimulation of innate immune responses during critical neurodevelopmental junctures, such as early childhood, could contribute to alterations in neurodevelopment and potentially lead to changes characteristic of ASD (Rosen et al., 2007).
I haven’t read Rosen 2007 yet, but it is on my list. [Does anyone have a copy?]
This altered innate immune response may have widespread effects on the activation and response of other immune cells and may also impact on neuronal activity given the extent of cytokine receptors present on neuronal and glial cells (Gladkevich et al., 2004). Furthermore, altered innate responses may ultimately play a role in the initiation and perpetuation of autoimmune responses that are present in some individuals with ASD. Our observations might also reflect genetic alterations in TLR signaling pathways, or pathways that control
monocyte function, such as the MET pathway, and ultimately lead to monocyte activation and cytokine production. MET is a pleiotropic receptor tyrosine kinase and is a key negative regulator of immune responses (Beilmann et al., 1997, 2000; Ido et al., 2005; Okunishi et al., 2005) that exerts its effects through engagement of its ligand, hepatocyte growth factor (HGF). Notably, MET signaling induces a tolerogenic phenotype in innate immune cells without affecting their antigen presenting capabilities (Okunishi et al., 2005; Rutella et al., 2006). Interestingly, the gene encoding MET carries a common polymorphism, the rs1858830 ‘C’ allele, which is functional and increases the relative risk for autism approximately 2.25-fold (Campbell et al., 2006). Thus, the MET ‘C’ variant may predispose to the absence of down-regulation of innate immune cell activation in ASD, and that the combination of a MET polymorphism and increased response to TLR ligands could combine to increase susceptibility to loss of self-tolerance and increased immune responsiveness.
The MET stuff is very cool and isn’t going away; I need to do some more reading on it, but having a particular downregulating allele for MET increases your risk of autism in a subtle, but real fashion. The resultant molecule from MET, HGF, serves a lot of different functions, including neuron formation, gastrointestinal repair, and, as noted above, as an immunoregulator. The allele is still relatively common, close to one half of everyone has it, but it is, nonetheless, over represented in the autism population. It would seem that you need something else, (probably a lot of something elses) at a genetic level to really start increasing your risk of autism; and above the authors speculate that an inherited downregulatory immune control in conjunction with an upregulated immune response could be a example of multiple low penetrance genes interacting to more greatly increase risk of developing autism.
There are more papers on TLR responsiveness in autism, and other neurological conditions that I’d like to get too eventually, but this one is the most recent, and as a result benifits greatly from a larger base of knowledge from a variety of related areas. I’d like to read a lot of the papers listed as references here; they are all pieces of the puzzle, its just tough to see how they fit in.