Posts Tagged ‘Fascinating’
A Brief Overview Of Glial Priming, How It (Probably) Applies To (Some Cases Of) Autism, And Worrisome Speculation On A Model Of A Low Penetrant Effect
Posted October 26, 2012on:
Hello friends –
The concept of glial priming (and implicit double multi hits) is the nexus of developmental programming, low penetrant effects, and an altered microglial responsiveness, a blueprint for a change in function in the tightly entangled neuroimmune environment; sort of an all time greats theory mashup for this blog. The basic idea is that microglia can become sensitized to insults and subsequently respond to similar insults with greater robustness and/or for increased timespans later in life. Here is a snippet from Microglia in the developing brain: A potential target with lifetime effects on the primed glial phenotype:
There is a significant amount of evidence regarding what is often termed ‘‘priming’’ and ‘‘preconditioning’’ events that serve to either exacerbate or provide neuroprotection from a secondary insult, respectively. In these states, the constitutive level of proinflammatory mediators would not be altered; however, upon subsequent challenge, an exaggerated response would be induced. The phenomena of priming represent a phenotypic shift of the cells toward a more sensitized state. Thus, primed microglia will respond to a secondary ‘‘triggering’’ stimulus more rapidly and to a greater degree than would be expected if non-primed.
Glial priming may be the fulcrum on which much of the underlying early immune activation research balances, the machinery that drives environmental influences during development leading to irregular neuroimmune functionality through the lifespan. Even though this type of finding is not really unexpected when considered within the prism of programming effects in other systems and the perturbed immune milieu in many (all?) neurological disorders, it is still pretty cool.
The first paper that I read that specifically mentioned glial priming was Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of proinflammatory cytokine upregulation, (Somera-Molina KC , 2007) which totally kicked ass. They brought a lot of heat at design time of the study; (very powerful) seizures were induced /saline given in animals at postnatal day 15 and 45; at day 55 animals were analyzed and showed distinct increases in microglial activation, neurologic injury, and future susceptibility to seizures in the ‘two hit’ group (i.e., animals that got seizure inducing kainic acid instead of saline on both day 15 and 45). Even better, it was shown that a CNS available inhibitor of inflammatory cytokine production rescued the effect of the seizure. In other words, it didn’t matter if the animals had a seizure, what mattered was the presence or absence of an unmitigated inflammatory response associated with the seizure.
Treatment with Minozac, a small molecule inhibitor of proinflammatory cytokine upregulation, following early-life seizures prevented both the long-term increase in activated glia and the associated behavioral impairment.
That is an important step in understanding the participation of inflammation in seizure pathology. There were also observable effects (worse) in animals that got seizures just once, if they got induced on day 15 versus 45, and even worse symptoms for the “double hit” animals. That was pretty fancy stuff in 2007. The similarity in terms of seizure susceptibility really reminded me of another paper, Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats, which also showed altered susceptibility to seizures in animals subjected to seizures in early life, with the effect mediated through inflammation related cytokines. Here, however, the same effect observed, but with the addition of clinical evidence of chronically perturbed microglia phenotype in the treatment group. Nice!
The same group followed up with Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model (full version), with more of the same. Here is part of the Discussion:
First, in response to a second KA ‘hit’ in adulthood, there is an enhancement of both the upregulation of proinflammatory cytokines, microglial activation, and expression of the chemokine CCL2 in adult animals who had previously experienced early-life seizures. Consistent with the exaggerated proinflammatory cytokine and microglial activation responses after the second hit, these animals also show greater susceptibility to seizures and greater neuronal injury. Second, administration of Mzc to suppress of the upregulation of proinflammatory cytokines produced by early-life seizures prevents the exaggerated cytokine and microglial responses to the second KA hit in adulthood. Importantly, regulating the cytokine response to early-life seizures also prevents the enhanced neuronal injury, behavioral impairment, and increased susceptibility to seizures associated with the second KA insult. These results implicate microglial activation in the mechanisms by which early-life seizures lead to increased susceptibility to seizures and enhanced neurologic injury with a second hit in adulthood.
Not only that, but the authors speculated on the possibility of a rescue effect through neuroimmune modulation!
Our data support a role for activated glia responses in the mechanisms by which early-life seizures produce greater susceptibility to a second neurologic insult. The improved outcomes with Mzc administration in multiple acute or chronic injury models where proinflammatory cytokine upregulation contributes to neurologic injury (Hu et al., 2007; Somera-Molina et al., 2007; Karpus et al., 2008; Lloyd et al., 2008) suggest that disease-specific interventions may be more effective if combined with therapies that modulate glial responses. These results are additional evidence that glial activation may be a common pathophysiologic mechanism and therapeutic target in diverse forms of neurologic injury (Akiyama et al., 2000; Craft et al., 2005; Emsley et al., 2005; Hu et al., 2005; Perry et al., 2007). Therapies, which selectively target glial activation following acute brain injury in childhood, may serve to prevent neurologic disorders in adulthood. These findings raise the possibility that interventions after early-life seizures with therapies that modulate the acute microglial activation and proinflammatory cytokine response may reduce the long-term neurologic sequelae and increased vulnerability to seizures in adulthood.
(Please note, the agent used in the above studies, kainic acid, is powerful stuff, and the seizures induced were status epileptcus, a big deal and a lot different than febrile seizures. That doesn’t mean that febrile seizures are without effect, I don’t think we are nearly clever enough to understand that question with the level of detail that is needed, but they are qualitatively different and not to be confused.)
The idea of modulating glial function as a preventative measure seems especially salient to the autism community alongside the recent (totally great) bone marrow studies observing benefits to a Rett model and an early life immune activation model of neurodevelopment.
A lot of kids with autism go on to develop epilepsy in adolescence, with some studies finding prevalence in the range of 30%, which terrifies the shit out of me. Is a primed microglial phenotype, a sensitization and increased susceptibility to seizures one of the mechanisms that drive this finding?
After Somera-Molina, I started noticing a growing mention of glial priming as a possible explanation for altered neuroimmune mechanics in a lot of places. Much of the early life immune literature has sections on glial priming, Early-Life Programming of Later-Life Brain and Behavior: A Critical Role for the Immune System (full / highly recommended / Staci Bilbo!) is a nice review of 2010 data that includes this:
However, there is increasing support for the concept of “glial priming”, in which cells can become sensitized by an insult, challenge, or injury, such that subsequent responses to a challenge are exaggerated (Perry et al., 2003). For instance, a systemic inflammatory challenge in an animal with a chronic neurodegenerative disease leads to exaggerated brain inflammation compared to a control animal (Combrinck et al., 2002). The morphology of primed glial cells is similar to that of “activated” cells (e.g. amoeboid, phagocytic), but primed glial cells do not chronically produce cytokines and other pro-inflammatory mediators typical of cells in an activated state. Upon challenge, however, such as infection or injury in the periphery, these primed cells will over-produce cytokines within the brain compared to cells that were not previously primed or sensitized (Perry et al., 2002). This overproduction may then lead to cognitive and/or other impairments (Cunningham et al., 2005; Frank et al., 2006; Godbout et al., 2005).
Other studies included increased effects of pesticide exposure following immune challenge, Inflammatory priming of the substantia nigra influences the impact of later paraquat exposure: Neuroimmune sensitization of neurodegeneration, which includes, “These data suggest that inflammatory priming may influence DA neuronal sensitivity to subsequent environmental toxins by modulating the state of glial and immune factors, and these findings may be important for neurodegenerative conditions, such as Parkinson’s disease (PD).” Stress was also found to serve as a priming agent in Glucocorticoids mediate stress-induced priming of microglial pro-inflammatory responses, which studied the effect of stress mediated chemicals on inflammatory challenges; the authors get bonus points for using glucocorticoid receptor agonists and surgical procedures to eliminate glucocorticoid creation to observe a priming effect of stress on neuroimmune response.
Here is a terrifying but increasingly unsurprising study on how neonatal experience modifies the physical experience of pain in adulthood, recently published in Brain, Priming of adult pain responses by neonatal pain experience: maintenance by central neuroimmune activity
Adult brain connectivity is shaped by the balance of sensory inputs in early life. In the case of pain pathways, it is less clear whether nociceptive inputs in infancy can have a lasting influence upon central pain processing and adult pain sensitivity. Here, we show that adult pain responses in the rat are ‘primed’ by tissue injury in the neonatal period. Rats that experience hind-paw incision injury at 3 days of age, display an increased magnitude and duration of hyperalgesia following incision in adulthood when compared with those with no early life pain experience. This priming of spinal reflex sensitivity was measured by both reductions in behavioural withdrawal thresholds and increased flexor muscle electromyographic responses to graded suprathreshold hind-paw stimuli in the 4 weeks following adult incision. Prior neonatal injury also ‘primed’ the spinal microglial response to adult injury, resulting in an increased intensity, spatial distribution and duration of ionized calcium-binding adaptor molecule-1-positive microglial reactivity in the dorsal horn. Intrathecal minocycline at the time of adult injury selectively prevented both the hyperalgesia and early microglial reactivity associated with prior neonatal injury. The enhanced neuroimmune response seen in neonatally primed animals could also be demonstrated in the absence of peripheral tissue injury by direct electrical stimulation of tibial nerve fibres, confirming that centrally mediated mechanisms contribute to these long-term effects. These data suggest that early life injury may predispose individuals to enhanced sensitivity to painful events.
One of the primal drivers of behavior in any animal, pain, can be persistently modified at a molecular level! Have you ever known someone that seemed to have a higher pain tolerance than you? Maybe they did, and the training of their microglia (or yours) in early life might be why. The most basic physiologic responses can be organized through the crucible of early life events sensitizing microglia to the future environment. Multi hit wow!
The effect that befalls us all, getting older, has a ton of studies on the effect of aging on glial priming, with greatest, err, ‘hits’ including Immune and behavioral consequences of microglial reactivity in the aged brain, Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system (full),Immune and behavioral consequences of microglial reactivity in the aged brain (full), and the autism implication heavy Microglia of the Aged Brain: Primed to be Activated and Resistant to Regulation, and others. Broadly, these studies spoke of the same pattern, a primed neuroimmune response, except in this instance, the “hits” that predisposed towards altered microglial reactivity weren’t a vigorous insult during development, but just the hum drum activity of growing older. It wasn’t a hit so much, more like a then gentle force of a relentless tide, but the functional effect on microglia response was largely similar, responses to stimuli were changed and programming was observed. I do not believe that the underlying instrument of change in age related priming is understood, but the thought occurs to me that it could simply be an exhaustion effect; a lifetime of exposure to inflammatory cytokines gradually changes the microglial phenotype.
So what about autism?
First and foremost, it provides us a line of insight into the likelyhood of a causal relationship between an altered neuroimmune milieu and autism (or nearly any other neurological disorder); that is, the question of whether or not our continued and repeated findings of altered neuroimmune parameters in the autism population represent a participating force in autism, as opposed to an artifact, a function of something else, which is also causing autism, or perhaps a result of having autism. While these are still possible explanations, the findings of glial priming provide additional detail on available mechanisms to affect brain activity and behavior through neuroimmune modifications alone.
If nothing else, we now know that we need not rely on models with no underlying substrate except the lamentations of ‘correlation does not equal causation’ and the brash faith of another, as of yet undefined, explanation. These models tell us that immune mediated pathologies can be created (and removed!) in very well established animal models of behavioral disturbances with corollaries to autism findings.
For more direct links to autism, we can look at the autism immune biomarker data set and find evidence of primed peripheral (i.e., outside the CNS) programming, literal examples where the autism population responds with a different pattern than the control group including an increased response to some pathogen type agonists, increased immune response following exposure to pollutants, of even dietary proteins.
The pattern we see of an altered microglial phenotype in the autism population, a state of chronic activity, is certainly consistent with disturbed developmental programming; it does not seem unlikely to me that a priming effect is also present, the initial prime seems to be responsible for the programming. As far as I know, there are no studies that have directly attempted to evaluate for a primed phenotype in the microglia of the autism population; I’d be happy to be corrected on this point.
Thinking about the possibility of increased microglial responsiveness and possible cognitive effects of a sustained neuroimmune toggling got me wondering if this is one of the mechanisms of a change in behavior following sickness? Or, alternatively, for some of us, “Is This Why My Child Goes Goddamn Insane And Stims Like Crazy For A Week After He Gets Sick?”
If we look to a lot of the studies that have shown a priming effect, they share a common causative pathway as some cases of autism, an early life immune insult. For some examples, the interested reader could check out Neonatal programming of the rat neuroimmune response: stimulus specifc changes elicited by bacterial and viral mimetics (full paper), Modulation of immune cell function by an early life experience, or the often mentioned Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats (full paper). If there are some cases of autism that have an early life immune insult as a participating input, it is very likely a primed microglia phenotype is also present.
The studies on aging are bothering me, not only am I getting older, but the findings suggest that a priming need not necessarily mandate a distinct ‘hit’, it can be more like a persistent nudge. Our fetuses and infants develop in an environment with an unprecedented number of different nudges in the past few decades as we have replaced infection with inflammation. Acknowledging this reality, however, raises the troubling thought that our embrace of lifestyles associated with increased inflammation has reached a tipping point that we are literally training the microglia of our children to act and react differently; we aren’t waiting a lifetime to expose our fetuses and infants to environments of increased inflammation, we are getting started from the get go.
Even with all of that, however, there is a genuinely microscopic Google footprint if you search for “autism ‘glial priming’”. So, either I’m seeing phantoms (very possible), or the rest of the autism research community hasn’t caught on yet, at least in such a way that Google is notified.
Even if I am chasing phantoms, there is evidence of a widespread lack of understanding of the depth of the neuroimmune/behavioral crosstalk literature, even by the people who should be paying the most attention. This was brought to my attention by a post at Paul Patterson’s blog, where Tom Insel was quoted as finding the recent Patterson and Derecki findings ‘unexpected’.
A bone marrow transplant, which replaces the immune system, corrected both the immune response and the behavior. This finding, which was unexpected, is surprisingly similar to another recent paper reporting disappearance of the symptoms of Rett syndrome in mice following a bone marrow transplant.
Keep in mind, this is from the guy who is the head of the IACC! I can tell you one thing; while the studies were impressive, I don’t think that the findings were especially unexpected. The researchers took the time to give mice bone marrow transplants, and in Wild-type microglia arrest pathology in a mouse model of Rett syndrome, the authors utilized a variety of knockout mice and even partial body irradiation to illuminate the question of neuroimmune participation in disorder. This work was not initiated in a vacuum, they did not throw a dart at a barn door sized diagram of study methodologies and land on ‘bone marrow transplants with subsequent analysis of microglia population properties and behaviors, accounting for different exposure timeframes, radiation techniques, and genotypes’. These were efforts that had a lot of supporting literature in place to justify the expense and researcher time. [I really want to find time to blog both of those papers in detail, but for the record, I did feel the rescue effects are particularly nice touches.]
So given that the head of the IAAC was surprised to find that immune system replacement having an effect on behavior was ‘surprising’, I’m not all together shocked at the relative lack of links on ‘glial priming’ and autism, but I don’t think it will stay that way for too much longer. As more experiments demonstrating a primed phenotype start stacking up, we are going to have to find a way to understand if generation autism exhibits a primed glial phenotype. I don’t think we are going to like the answer to that question very much, and the questions that come afterwards are going to get very, very inconvenient.
Spelling it out a bit more, with bonus speculation, we should remember our recent findings of the critical role microglia are playing in shaping the neural network; our microglia are supposed to be helping form the physical contours of the brain, a once in a lifetime optimization of synaptic structures that has heavy investment from fetushood to toddlerhood. Unfortunately, it appears that microglia perform this maintenance while in a resting state, i.e., not when they have been alerted of an immune response and taken on a morphology consistent with an ‘activated state’. An altered microglia morphology can be instigated during infection, or perceived infection and consequent immune response. For examples of peripheral immune challenges changing microglial morphology, the neuroimmune environment and behavior some examples include: Peripheral innate immune challenge exaggerated microglia activation, increased the number of inflammatory CNS macrophages, and prolonged social withdrawal in socially defeated mice, Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system, or Long-term changes of spine dynamics and microglia after transient peripheral immune response triggered by LPS in vivo.
But what if we have a susceptible population, a population sensitized such that the effects of an immune challenge would result in an exaggerated and extended microglial response, effectively increasing the length of time the microglia would be ‘not resting’. What might be the changes in this population in response to a series of ‘hits’?
It does not seem to be a large logical leap to assume that if some of the altered brain physiology in autism is due to abnormal microglia function during the period of robust synaptic pruning, triggering the microglia to leave their resting state for an extended period in response could be a reasonable participant. Think of it as an exaggerated loss of opportunity effect, essentially a longer timeframe during which the microglia are not performing synaptic upkeep when compared to the microglia in an individual that is not sensitized. While our brains do show a lot of ability to ‘heal’, that does not mean that all things or times are created equally; there are some very distinct examples of time and spatially dependent neurochemical environments during early synapse development, environments that change as time goes on; i.e., Dynamic gene and protein expression patterns of the autism-associated met receptor tyrosine kinase in the developing mouse forebrain (full paper), or A new synaptic player leading to autism risk: Met receptor tyrosine kinase. In other words, recovering from a delay in microglial participation in synaptic pruning during development may not be as simple as ‘catching up’; if the right chemical environment isn’t available when the microglia get done responding, you might not be able to restart like a game of solitaire. The Met levels might be different, the neurexin levels might be different, a thousand other chemical rally points could be set that much of a nudge differently; in a system dependent on so many moving variables being just so, an opportunity missed is an opportunity lost. For good.
While the effects of a series of challenges and consequent obstructions of synaptic maintenance might not be acutely clear, I am becoming less and less convinced of the ‘safety’ of an observed lack of immediately obvious effects. I think that an intellectually honest evaluation of our recent ‘discoveries’ in many areas of early life disturbances (i.e., antibiotics and IDB risk, C-section and obesity risk, birth weight and cardiovascular risk) tell us that subtle changes are still changes, and many rise to the level of a low penetrant, environmentally induced effect once we get clever enough to ask the right question. And boy are we a bunch of dummies.
Taking all of this into consideration, all I can think is thank goodness we haven’t been artificially triggering the immune system of our infants for the past two decades while we were blissfully unaware of the realities of microglial maintenance of the brain and glial priming! What a relief that we did not rely on an assumption of lack of effect as a primary reason not to study the effect of an immune challenge. If we had done those things, we might start kicking ourselves when we realized out that our actions could be affecting susceptible subsets of children who were predisposed to reacting in difficult to measure but real ways that could literally affect the physical structure of their brains.
A Sense Of Relief After (Some Of) Your Phantoms Are Observed By Others, A Distillation of Humbling Complex Early Life Neuroimmune Literature: “Microglia in the developing brain: a potential target with lifetime effects”, and The Need For Dispassionate Analysis
Posted June 15, 2012on:
Hello friends –
I have a confession to make. The fact that a lot of very smart people have ignored or flat out laughed at my arguments bothers me sometimes. I have applied non-trivial, not to be rebated time and effort to put forth what I considered to be logical views, scientifically defendable and important ideas; and yet people I knew were otherwise rational, and in some cases, very intelligent, just hadn’t seemed to get what I was saying. Often this was within the context of a discussion argument of vaccination, but my larger concern, that of a non-imaginary, non-trivial increase in children with autism in the past decades, also usually falls on deaf ears. If “environmental changes” incorporate the chemical milieu of our mother’s wombs, the microbial world our infants are born into, or the ocean of synthetic chemicals we all swim through every day, we have no rational conclusion but that our environment has changed a lot in the past few decades. Considered within the context of the reality based model where the events of early life can be disproportionally amplified through the lifetime of an organism, clinging to the idea that there has been a stable incidence of autism seems dangerously naïve, at most charitable.
And yet, for the most part, many or most of the people who are alarmed are crackpots. There were times I questioned myself. Am I missing something? Am I chasing phantoms? Why aren’t any of these other smart people as worried as I am?
A while ago I got a copy of Microglia in the developing brain: A potential target with lifetime effects (Harry et all), a paper that tells me that if nothing else, I have some good company in pondering the potential for disturbances in early life to uniquely affect developmental outcome, in this instance through alterations to the neuroimmune system. If I am incorrect about the validity of a developmental programming model with lifetime effects, lots of prolific researchers are wrong about the same thing in the same way. Harry is a very thorough (and terrifying) review of the relevant literature. Here is the abstract:
Microglia are a heterogenous group of monocyte-derived cells serving multiple roles within the brain, many of which are associated with immune and macrophage like properties. These cells are known to serve a critical role during brain injury and to maintain homeostasis; yet, their defined roles during development have yet to be elucidated. Microglial actions appear to influence events associated with neuronal proliferation and differentiation during development, as well as, contribute to processes associated with the removal of dying neurons or cellular debris and management of synaptic connections. These long-lived cells display changes during injury and with aging that are critical to the maintenance of the neuronal environment over the lifespan of the organism. These processes may be altered by changes in the colonization of the brain or by inflammatory events during development. This review addresses the role of microglia during brain development, both structurally and functionally, as well as the inherent vulnerability of the developing nervous system. A framework is presented considering microglia as a critical nervous system-specific cell that can influence multiple aspects of brain development (e.g., vascularization, synaptogenesis, and myelination) and have a long term impact on the functional vulnerability of the nervous system to a subsequent insult, whether environmental, physical, age-related, or disease-related.
The body of Microglia in the developing brain: A potential target with lifetime effects has tons of great stuff. From the Introduction
The evidence of microglia activation in the developing brain of patients with neurodevelopmental disorders(e.g., autism) and linkage to human disease processes that have a developmental basis (schizophrenia) have raised questions as to whether developmental neuroinflammation actively contributes to the disease process. While much of the available data represent associative rather than causative factors, it raises interesting questions regarding the role of these ‘‘immune-type’’ cells during normal brain development and changes that may occur with developmental disorders. Within the area of developmental neurotoxicology, the potential for environmental factors or pharmacological agents to directly alter microglia function presents a new set of questions regarding the impact on brain development.
There is a short section on what is known about the colonization of the brain by microglia, it is a busy, busy environment, and while we are just scratching the surface, microglia seem to be involved in scads of uber-critical operations, many of which pop up in the autism literature. It is just being confirmed that microglia constitute a distinct developmental path that diverges as an embryo, two papers from 2007 and 2010 are referenced as reasons we now believe microglia are a population of cells that migrate into the CNS before birth and are not replaced from the periphery in adulthood. From there, the beautiful complexity is in full effect; as the microglia develop and populate the brain there are specific spatial and morphological conditions, microglia are first evident at thirteen weeks after conception, and do not reach a stable pattern until after birth. In fact, it appears that microglia aren’t done finishing their distribution in the CNS until the postnatal period, “With birth, and during the first few postpartum weeks, microglia disseminate throughout all parts of the brain, occupying defined spatial territories without significant overlap (Rezaie and Male, 2003) suggesting a defined area of surveillance for each cell.”
It occurred to me to wonder if there are differences in microglia settlement patterns in males and females in human infants, as has been observed in other models? Could a spatially or temporally different number of micoglia, or different developmental profiles of microglia based on sex be a participant in the most consistent finding in the autism world, a rigid 4:1 male/female ratio?
Speaking towards the extremely low replacement rates for microglia in adulthood, the authors wonder aloud on the possible effects of perturbations of the process of microglial colonization.
The slow turn-over rate for mature microglia raises an issue related to changes that may occur in this critical neural cell population. While this has not been a primary issue of investigation there is limited data suggesting that microglia maintain a history of previous events. Thus, if this history alters the appropriate functioning of microglia then the effects could be long lasting. Additionally, a simple change in the number of microglia colonizing the brain during development, either too many or too few, could have a significant impact not on only the establishment of the nervous system network but also on critical cell specific processes later in life.
Perhaps coincidentally (*cough*), we have abundant evidence of an altered microglial state and population in the autism population; while we do not know that these findings are the result of a disturbance during development, it is an increasingly biologically plausible mechanism, and thus far, I’ve yet to see other mechanisms given much thought, excepting the chance of an ongoing, undetected infection.
There is a brief section concerning the changes found in adult microglial populations in terms of density, form, and gene expression in different areas of the brain, “With further investigation into the heterogeneity of microglia one would assume that a significant number of factors, both cell membrane and secreted, will be found to be differentially expressed across the various subpopulations.” Nice.
There is a section of the paper on microglial phenotypes, there are a lot of unknowns and the transformation microglia undergo between functional states is even more nebulously understood during brain development. “It is now becoming evident that in the developing brain, many of the standards for microglia morphology/activation may require readdressing.” We haven’t even figured out what they’re doing in the adult brain!
There is a really cool reference for a study that shows altered microglial function dependent on the age of the organism.
In the adult rodent, ischemia can induce microglia to display either a more ramified and bushy appearance or an amoeboid morphology depending on the level of damage and distance from the infarct site(s). In the immature rodent, ischemia-induced changes in capillary flow or, presumably, altered CNS vascularization can retain the microglia in an amoeboid phenotype for longer and delay the normal ramification process (Masuda et al., 2011).
One way of looking at this would be to say that we should exercise extreme caution in trying to translate our nascent understanding of how mature microglia react when speculating on how immature microglia will act. To follow up on just how little we know, there is a long discussion about the shortcomings of a the term ‘activated’ microglia with some details on chemical profiles of broadly generalized ‘classically inflammatory, ‘alternatively activated’, ‘anti-inflammatory’, and ‘tissue repair’ phenotypes.
Next up is a dizzyingly list of brain development functions that microglia are known, or suspected to participate in. Without getting too deep in the weeds, of particular interest to the autism realm, that list includes neurogenesis and differentiation in the cortex [related: Courchesne, me], cell maturation via cytokine generation, axon survival and proliferation [related: Wolff, me], programmed cell death of Purkinje cells, clearance of ‘early postnatal hippocampul neurons’, and the ‘significant contribution to synaptic stripping or remodeling events’, i.e., pruning (Paolicelli / fractaltine), and even experience dependent microglia / neuron interactions. Taking all of this (and more) into consideration, the authors conclude “Thus, one can propose that alterations in microglia functioning during synapse formation and maturation of the brain can have significant long-term effects on the final established neural circuitry. “ Ouch.
Next up is a summary of many of the animal studies on microglial participation in brain formation, there is a lot there. Interestingly (and particularly inconvenient) is the finding that a lot of the functional actions of microglia during development appear to operate after birth. “Overall, the data suggest that microglial actions may be most critical during postnatal brain maturation rather than during embryonic stages of development.” Doh!
Early life STRESS gets some attention, and for once there is some good news if you look at it the right way. There is something about a very cool study from Schwarz (et all / Staci Bilbo!) involving drug challenge that peered deep into the underlying mechanisms of an environmental enrichment model; animals given a preferential handling treatment were found by two metrics to have differential microglia response in adulthood with (biologically plausible) observations, increased mRNA levels for IL-10 production, and decreased DNA methylation; i.e., less restriction on the gene that produces IL-10, and more messenger RNA around to pass off the production orders [totally beautiful!]. There is more including thyroid disruption (though in a way that I found surprising), and the observations of time dependent effects on immue disturbances. (super inconvenient)
There is so much data that keeps piling on that the authors end up with “Overall, the existing data suggest a critical regulatory role for microglia in brain development that is much expanded from initial considerations of microglia in the context of their standard, immune mediated responses.”
A terrifying concept that I haven’t found time to dedicate a post towards is microglia priming, which gets some attention in Harry.
There is a significant amount of evidence regarding what is often termed ‘‘priming’’ and ‘‘preconditioning’’ events that serve to either exacerbate or provide neuroprotection from a secondary insult, respectively. In these states, the constitutive level of proinflammatory mediators would not be altered; however, upon subsequent challenge, an exaggerated response would be induced. The phenomena of priming represent a phenotypic shift of the cells toward a more sensitized state. . . Exactly how long this primed state will last has not been determined; however, data from microglia suggest that it can extend over an expanded period of time. Preconditioning can also represent changes that would occur not only over the short term but may be long lasting.”
I happen to think that microglia priming is going to be a very important cog in the machinery for this journey when all is said and done; the evidence to support a preconditioning system is strong, and in parallel, the things we see different in autism (and elsewhere) is consistent with a different set of operations of microglia, AND we also have evidence the disturbances that would invoke microglial change are subtle but real risk factors for autism.
What comes next is a type of greatest hits mashup of very cool papers on developmental programming in the CNS.
Galic et al.(2008) examined age related vulnerabilities to LPS in rats to determine critical age periods. Postnatal injection of LPS did not induce permanent changes in microglia or hippocampal levels of IL-1b or TNFa; however, when LPS was given during the critical postnatal periods, PND 7 and 14, an increased sensitivity to drug induced seizures was observed in 8-week-old rats. This was accompanied by elevated cytokine release and enhanced neuronal degeneration within the hippocampus after limbic seizures. This persistent increase in seizure susceptibility occurred only with LPS injection at postnatal day 7 or 14 and not with injections during the first day of life or at PND 20. Similar long-lasting effects were observed for pentylenetetrazol-induced seizures when PND 11 or 16 rat pups were subjected to LPS and hyperthermic seizures (Auvin et al., 2009). These results again highlight this early postnatal period as a ‘‘critical window’’ of development vulnerable to long-lasting modification of microglia function by specific stimuli. Work by Bilbo and co-workers demonstrated LPS-induced deficits in fear conditioning and a water maze task following infection of PND 4 rats with Escherichia coli. In the young adult, an injection of LPS induced an exaggerated IL-1b response and memory deficits in rats neonatally exposed to infection (Bilbo et al., 2005). Consistent with the earlier work by Galic et al. (2008), an age dependency for vulnerability was detected with E. coli-induced infection at PND 30 not showing an increased sensitivity to LPS in later life (Bilbo et al., 2006).
In particular, Galic 2008, or Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats (full paper) was a very formative paper for me; it was elegant in design and showed alarming differences in outcome from a single immune challenge experience, if it occurred during a critical developmental timeframe. If you haven’t read it, you should.
This paper has a nice way of distilling the complexity of the literature in a readable way.
One hypothesis for developmental sensitivity is the heterogeneous roles for inflammatory factors and pro-inflammatory cytokines during development, including their timing-, region and situation-specific neurotrophic properties. Many of the proinflammatory cytokines are lower at birth with a subsequent rapid elevation occurring during the first few weeks of life. In an examination of the developing mouse cortex between PND 5 and 11, mRNA levels for TNFa, IL-1b, and TNFp75 receptor remained relatively constant while a significant increase in mRNA levels of CR3, macrophage-1 antigen (MAC-1), IL-1a, IL-1 receptor 1 (IL- )R1, TNFp55 receptor (TNFp55R), IL-6, and gp130 occurred (Fig. 2). This data suggests that an upregulation of interleukins and cytokine receptors may contribute to enhanced cytokine signaling during normal cortical development.
One hypothesis put forward using a model reliant on postnatal exposure to LPS suggests that these types of exposure may ‘‘reprogram’’ neuroimmune responses such that adult stress results in hyperactivation of the hypothalamic pituitary adrenal (HPA) axis (Mouihate et al., 2010) and corticosterone changes (Bilbo and Schwarz, 2009).While limited, the available data suggest that events occurring during development, especially postnatal development, have the potential to cause long term alterations in the phenotype of microglia and that this can be done in a region specific manner.
In what could, conceivably, be a coincidence, our available information on the autism brain also shows region specific changes in microglia populations, microglial activation profiles, and oxidative stress. I do not believe the findings reviewed in Microglia in the developing brain: A potential target with lifetime effects will be meaningless artifacts; the likelihood that our observations of an altered neuroimmune state in autism are not, at least, participatory has become vanishingly small.
Can these findings inform us on the incidence question? I was lurking on a thread on Respectful Insolence a while ago, and someone gave what I thought was a very succinct way of thinking about the changes that our species has encountered the past few decades; it went something like “we have replaced infection with inflammation”. That’s a pretty neat way of looking at how things have gotten different for humanity, at least lots of us, and especially those of us in the first world. We used to get sick and die early; now we live longer, but oftentimes alongside chronic disorders that share a common underlying biological tether point, inflammation.
Any dispassionate analysis of the available data can tell us that we have, indeed, replaced infection with inflammation; we suffer from less death and misery from infection, but more metabolic disorder, more diabetes, more hypertension, more asthma and autoimmune conditions than previous generations. We have largely replaced good fatty acids with poor ones in our diet. All of these conditions are characterized by altered immune biomarkers, including an increase in proinflammatory cytokines. Those are the facts that no one can deny; we have replaced infection with inflammation.
But when we look to the findings of Microglia in the developing brain: A potential target with lifetime effects, it becomes clear that our newfound knowledge of microglial function and crosstalk with the immune system raises some very troubling possibilities.
Lately it has been quite in vogue among a lot of the online posting about autism to at least mention environmental factors which could participate in developmental trajectory leading to autism; that’s a big step, an important and long overdue acknowledgement. If you pay close attention, you will notice that 99% of these admissions are handcuffed to the word “prenatal”. This is likely an attempt to deflect precise questions about the robustness of our evaluation of the vaccine schedule, but the big question, the incidence question, still hinges on fulcrum of the genetic versus environmental ratio ; that is a problem for the purveyors of the fairytale because the prenatal environment of our fetuses, the chemical milieu of their development, is qualitatively different compared to generations past. That chemical soup is their environment; and that environment has unquestionably changed in the past decades as we have replaced infection with inflammation.
Our previous analysis tells us that invoking inflammation outside the brain modifies microglial function inside the wall of the blood brain barrier; good or bad, no honest evaluation of the literature can argue against a lack of effect. What happens outside the brain affects what happens inside the brain. If, however, microglia are active participants in brain formation, as a swath of recent research indicates, can this fact give us insight into the incidence question?
Is a state of increased inflammation the pathway between maternal asthma, depression, stress, and obesity being associated with increased risk of autistic offspring? Have we replaced infection with inflammation plus?
What could be more lethal to the fairytale of a static tale of autism than a positive relationship between a lifestyle characterized by increased inflammation and the chances of having a baby with autism?
Are we totally fucked?
We cannot know the answers unless we have the courage to ask the difficult questions with methods powerful enough to provide good data, and it won’t be easy. The static rate of autism fairytale is a comforting notion; it expunges responsibility for the coronal mass ejection sized change to our fetuses developing environment, and while hiding behind the utterly frail findings of social soft scientists, we can happily place tin foil hats and accusations of scientific illiteracy on anyone who might be worried that our abilities have outstripped our wisdom. That is a terrible, cowardly way to approach the incidence question, what we should be doing is exactly the opposite, ridiculing the epidemic sized error bars in prevalence studies and demanding more answers from the hard scientists. Eventually we will get there and it will be a critical mass of information from studies like Harry that will propel decision makers to abandon the fairytale for a course regulated by dispassionate analysis.
The Interconnectedness of the Brain, Behavior, and Immunology and the Difficult to Overstate Flaccidity of The Correlation Is Not Causation Argument
Posted May 12, 2011on:
Hello friends –
I’ve gotten into a lot of discussions online about the vaccines and autism; generally with very poor, if not nonexistent, evidence of having changed any opinions, but relatively strong evidence ( p > .001) that persisting in making my arguments can get you called ‘an antivaccine loon’, ‘idiot’, someone who engages in ‘Gish Gallop’, or the worst insult I’ve received so far, ‘anti-science’. While I am really torn on the vaccine issue, I am certain that both peripheries of this debate are at least somewhat wrong in the conclusions that they’ve drawn from the available evidence. I do believe that lots of parents have witnessed a very real change in their children post vaccination, and I also don’t believe for a single second that vaccines are the cause of an epidemic of autism. It’s a mess and I’ve been poking around the Internet almost five years into journey autism and from my eyes, it hasn’t improved any in the past half decade. This is very sad.
That being said, while I do think we need to have a rational and dispassionate discussion about what our existing vaccine studies can and cannot tell us about autism, I’m really concerned about the fact that the vaccine wars seem to have inoculated otherwise intelligent people from any semblance of intellectual curiosity regarding the immunological findings in the autism realm. That’s a problem, because there are lots of things other than vaccines that can modify the immune response, various environmental agents and cultural changes that are relatively new, and ignoring immunological findings in autism because they happen to intersect with the function of vaccination is a huge, massive, supernova sized disservice to what history will view us poorly on, refusing to perform honest evaluation due to fear and the comfort of willful ignorance.
Here, in this post, I will make the case that this lack of curiosity on immunological findings in autism is either born of a lack of understanding on how much we know about the ties between the immune system and the brain, or alternatively, originates from a deep seated desire to avoid honest interactions. This isn’t to make the case that vaccines can cause autism, or even that the immunological disturbances observed in autism are causative, but rather that an obstinate refusal to consider these as possibilities is the sign of someone who cannot, or will not accept, the biological plausibility of immunologically driven behaviors despite a constellation of evidence.
One of the things that jumps out to me why the autism population might be a subgroup of the population susceptible to changes as a result of immune dysfunction (and thus, potentially adversely affected as a result of vaccination), is the sheer volume of evidence we now have available to us indicating an altered immune response, and indeed, an ongoing state of inflammation within the brain in the autism population, and most strikingly, repeated observations of a correlation between the degree of immune dysregulation as a propensity of an inflammatory state, and the severity of autism behaviors. Again and again we’ve seen that as markers indicative of an inflammatory state increase, so too, do severity of autism behaviors. Not only that, but there are instances wherein the decrease of components known to regulate the immune response decrease, autistic behaviors are more severe. Subtle shifts in either the start or the resolution of the immune response seems to affect autistic behavior severity in the same way. I know coincidences happen all the time, but that doesn’t mean that everything is a coincidence.
We also have a large number of studies that tell us that in vitro, similar levels of stimulation with a variety of agents cause exaggerated or dysregulated production of immune markers in the autism population.
A large percentage of the time that I mention these findings, usually within discussions with an origin in vaccination, someone decides to educate me on one of the most rudimentary scientific axioms:
Correlation does not equal causation.
It must be stated, the above statement is absolutely true. Unfortunately for the people for whom this accurate, but simplistic catchphrase comprises the entirety of their argument, it completely ignores a wealth of research that tells us in very unambiguous terms that there is incontrovertible evidence that crosstalk between the immune system and central nervous system can modify behavior. The research indicating a relationship between immune dysregulation and autism does not exist in a vacuum, but rather, is only a tiny fragment of evidence, mostly accumulated within the last few years, that tells us that the paradigm of the past decades, that of the brain as a immune privileged organ without communication to the immune system, is as antiquated as refrigerator moms and a one in ten thousand prevalence.
From a common sense, why didn’t I think of that standpoint, the best example of the interaction between the brain and the immune response is the old standard, just plain old getting sick. You live in the dirty world, you pick up a pathogen, you get sick, and suddenly you get lethargic and you start to run a fever. But is it the pathogen itself that is actually making you feel like staying in bed all day?
What is being learned is that it is not necessarily the microbial invader that is causing you to get tired and feel sore, but rather, that your decreased energy levels are centrally mediated through your brain, and the triggers for your brain to start a fever include molecules our bodies use for a wide range of communications, including immune based messaging, cytokines. Some of the most common cytokines in the research to follow include IL-6, IL-1B, and TNF-Alpha; so called ‘pro-inflammatory’ cytokines. Researchers have been plugging away at just how the immune response is capable of modifying behaviors, i.e., inducing, sickness behavior for a while now, at least in terms of autism research. From 1998, we have Molecular basis of sickness behavior:
Peripheral and central injections of lipopolysaccharide (LPS), a cytokine inducer, and recombinant proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) induce sickness behavior in the form of reduced food intake and decreased social activities. Mechanisms of the behavioral effects of cytokines have been the subject of much investigation during the last 3 years. At the behavioral level, the profound depressing effects of cytokines on behavior are the expression of a highly organized motivational state. At the molecular level, sickness behavior is mediated by an inducible brain cytokine compartment that is activated by peripheral cytokines via neural afferent pathways. Centrally produced cytokines act on brain cytokine receptors that are similar to those characterized on peripheral immune and nonimmune cells, as demonstrated by pharmacologic experiments using cytokine receptor antagonists, neutralizing antibodies to specific subtypes of cytokine receptors, and gene targeting techniques. Evidence exists that different components of sickness behavior are mediated by different cytokines and that the relative importance of these cytokines is not the same in the peripheral and central cytokine compartments.
The first sentence in this abstract references a practice that is extremely common in studying the immune system, intentionally invoking a robust immune response by exposing either animals, or cells in vitro, to the components that comprise the cell wall of certain types of bacteria; lipopolysaccharide, or LPS. LPS could be considered a sort of bacterial fingerprint, a pattern that our immune systems, and the immune system of almost everything, has evolved to recognize, and correspondingly initiates an immune response.
Because this is a conversation that frequently has an origin in vaccination, essentially the act of faking an infection, it is salient to remember that the animals or cell cultures aren’t really getting sick when exposed to LPS; there is no pathology associated with whatever type of bacteria might be housed within a cell membrane containing LPS. Usually, when the body is exposed to a gram negative bacteria, and the consequent LPS exposure, there are also effects of the bacteria that interact with the organism, but by only incorporating the alert signal for a bacterial invader, we can gain insight into the effect of the immune response itself; there isn’t anything else to cause any changes. This means that similarly to LPS administration, straight administration of these pro-inflammatory cytokines are similar to the result of getting sick with a pathogen, at least as far as the immune response is concerned.
In the above instance, administration of LPS, or simply cytokines, had been shown to be capable of causing reduced food intake and ‘decreased social activities’.
Later in 1998, Central administration of rat IL-6 induces HPA activation and fever but not sickness behavior in rats (full version), was published wherein the authors report that central administration (i.e., directly into the CNS), of cytokines in isolation (IL-6) or in combination (IL-6 + IL-1B) were capable of inducing altered HPA activation, fevers, and sickness behaviors. Effects of peripheral administration of recombinant human interleukin-1 beta on feeding behavior of the rat was published a few years later, and observed that peripheral administration (i.e., not the CNS) of IL-1B could affect how much a rat ate, with sucrose ingestion being consistently altered during periods of sickness.
Jumping ahead a few years, a review paper Expression and regulation of interleukin-1 receptors in the brain. Role in cytokines-induced sickness behavior reviewed how cytokines participate in sickness behavior, Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior examined the interactions of IL-6 and leptin in sickness behavior, and Behavioral and physiological effects of a single injection of rat interferon-alpha on male Sprague-Dawley rats: a long-term evaluation reported “these data suggest that a single IFN-alpha exposure may elicit long-term behavioral disruptions”.
Much more recently, Sickness-related odor communication signals as determinants of social behavior in rat: a role for inflammatory processes more elegantly found that behavior was modified by LPS exposure, and that this effect was neutralized by concurrent administration of the anti-inflammatory cytokine, IL-10. Similarly, Inhibition of peripheral TNF can block the malaise associated with CNS inflammatory diseases observed another distinct means by which interfering with the immune response could attenuate the effect of faux sickness, in part, concluding, “Thus behavioral changes induced by CNS lesions may result from peripheral expression of cytokines that can be targeted with drugs which do not need to cross the blood-brain barrier to be efficacious.” In other words, what is happening in the periphery, outside of the protective boundaries of the blood brain barrier, can none the less manipulate behaviors that are controlled by the brain.
There are tons, tons more studies like this, but the point should be clear by now; it is accepted that you can achieve some of the same behaviors the come alongside illness, such as fever and lethargy, without the presence of an actual bacteria or virus; all you need is for your brain to think that you are sick.
While it must be acknowledged that the behavioral disturbances observed in autism are a lot different than feeling the need to watch TV all day, these types of studies were among the first clues that the traditional view of the CNS as a separate entity within the gated community of the blood brain barrier needed revision.
Measuring how much sugar water a rat drank is great stuff, but the reality is that we have conservatively a gazillion studies telling us that disorders that manifest behaviorally have strong, strong ties to the immune system; and once we begin to understand the vast scope of these findings, the utter frailty of “correlation does not equal causation” becomes painfully clear to the intellectually honest observer.
The big problem I found myself with in crafting this posting was that the sheer volume of studies available really makes a complete illustration of the literature impossible; I started looking and pubmed nearly puked trying to return to me a listing of all of the things I wanted to summarize. So here is some of the best of the best; to keep things interesting, I thought I’d only include findings from 2007 or later as a mechanism to show just how nascent our understanding of the connections between the brain and the immune system really are.
Initially, we can start with a condition that nearly everyone agrees is diagnosed based on behavior, depression. It turns out, the number of findings establishing a link between immune system markers and depression is wide and deep.
Here’s a great one, Elevated macrophage migration inhibitory factor (MIF) is associated with depressive symptoms, blunted cortisol reactivity to acute stress, and lowered morning cortisol, which reports, that MIF can modify HPA axis function and is tied to depression; a particularly compelling finding considering well documented alterations in HPA axis metabolites in autism, and the fact that increased MIF has also been found in the autism population, and as levels increased, so too did autism severity.
Here is part of the abstract for Inflammation and Its Discontents: The Role of Cytokines in the Pathophysiology of Major Depression (full paper)
Patients with major depression have been found to exhibit increased peripheral blood inflammatory biomarkers, including inflammatory cytokines, which have been shown to access the brain and interact with virtually every pathophysiologic domain known to be involved in depression, including neurotransmitter metabolism, neuroendocrine function, and neural plasticity. Indeed, activation of inflammatory pathways within the brain is believed to contribute to a confluence of decreased neurotrophic support and altered glutamate release/reuptake, as well as oxidative stress, leading to excitotoxicity and loss of glial elements, consistent with neuropathologic findings that characterize depressive disorders.
Somewhere along the way, researchers discovered that some anti-depressants can exert anti-inflammatory effects, for examples of these findings we could look to Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll-like receptors, or Plasma cytokine profiles in depressed patients who fail to respond to selective serotonin reuptake inhibitor therapy, which concludes in part, “Suppression of proinflammatory cytokines does not occur in depressed patients who fail to respond to SSRIs and is necessary for clinical recovery”.
In Investigating the inflammatory phenotype of major depression: focus on cytokines and polyunsaturated fatty acids, the authors report that, “The findings of this study provide further support for the view that major depression is associated with a pro-inflammatory phenotype which at least partially persists when patients become normothymic.” A nice review of the evidence of immunological participation in depression can be found in The concept of depression as a dysfunction of the immune system (full paper).
Moving forward, we can look to schizophrenia, we have similar findings, including Serum levels of IL-6, IL-10 and TNF-a in patients with bipolar disorder and schizophrenia: differences in pro- and anti-inflammatory balance, which observed an imbalanced baseline cytokine profile in the schizophrenic group; findings very similar in form with An activated set point of T-cell and monocyte inflammatory networks in recent-onset schizophrenia patients involves both pro- and anti-inflammatory forces. Similarly, the findings from Dysregulation of chemo-cytokine production in schizophrenic patients versus healthy controls, (full paper) which states, in part:
Growing evidence suggests that specific cytokines and chemokines play a role in signalling the brain to produce neurochemical, neuroendocrine, neuroimmune and behavioural changes. A relationship between inflammation and schizophrenia was supported by abnormal cytokines production, abnormal concentrations of cytokines and cytokine receptors in the blood and cerebrospinal fluid in schizophrenia
Their findings include differentially increased and decreased production of chemokines and cytokines as a result of LPS stimulations in the case group. Of particular note, a similarly dysregulated immune profile of cytokine and chemokine generation has been found in the autism population in several studies.
We also have several trials of immunomodulatory drugs in the schizophrenic arena that further implicate the immune system in pathology, including Adjuvant aspirin therapy reduces symptoms of schizophrenia spectrum disorders: results from a randomized, double-blind, placebo-controlled trial, a ‘gold standard’ trial which found that, “Aspirin given as adjuvant therapy to regular antipsychotic treatment reduces the symptoms of schizophrenia spectrum disorders. The reduction is more pronounced in those with the more altered immune function. Inflammation may constitute a potential new target for antipsychotic drug development”. A similar clinical trial, Celecoxib as adjunctive therapy in schizophrenia: a double-blind, randomized and placebo-controlled trial , another gold standard trial, which also had findings in the same vein, “Although both protocols significantly decreased the score of the positive, negative and general psychopathological symptoms over the trial period, the combination of risperidone and celecoxib showed a significant superiority over risperidone alone in the treatment of positive symptoms, general psychopathology symptoms as well as PANSS total scores.” [Celecoxib is a cox-2 inhibitor; i.e., anti-inflammatory, i.e., immunomodulatory]
What about bi-polar disorder? More of the same, including, The activation of monocyte and T cell networks in patients with bipolar disorder, or Elevation of cerebrospinal fluid interleukin-1ß in bipolar disorder, which reports, in part, “Our findings show an altered brain cytokine profile associated with the manifestation of recent manic/hypomanic episodes in patients with bipolar disorder. Although the causality remains to be established, these findings may suggest a pathophysiological role for IL-1ß in bipolar disorder.”. These studies were published in April and March, 2011, respectively.
Brain tissue from persons with bi-polar disorder also showed increased levels of excitotoxicity and neuroinflammation in Increased excitotoxicity and neuroinflammatory markers in postmortem frontal cortex from bipolar disorder patients (full version), and authors report differential cytokine profiles depending on state of mania, depression, or remission in Comparison of cytokine levels in depressed, manic and euthymic patients with bipolar disorder.
Another disorder based solely around behavior, Tourette syndrome, has increasingly unsurprising findings. Polymorphisms of interleukin 1 gene IL1RN are associated with Tourette syndrome reports “The odds ratio for developing Tourette syndrome in individuals with the IL1RN( *)1 allele, compared with IL1RN( *)2, was 7.65.” (!!!) , and Elevated expression of MCP-1, IL-2 and PTPR-N in basal ganglia of Tourette syndrome cases is yet another example of observations of CNS based immune participation in a disorder that is diagnosed by behavior.
There are also some reviews that perform a cross talk of sorts between disorders; i.e., The mononuclear phagocyte system and its cytokine inflammatory networks in schizophrenia and bipolar disorder, or Immune system to brain signaling: Neuropsychopharmacological implications, published in May 2011, which has this abstract:
There has been an explosion in our knowledge of the pathways and mechanisms by which the immune system can influence the brain and behavior. In the context of inflammation, pro-inflammatory cytokines can access the central nervous system and interact with a cytokine network in the brain to influence virtually every aspect of brain function relevant to behavior including neurotransmitter metabolism, neuroendocrine function, synaptic plasticity, and neurocircuits that regulate mood, motor activity, motivation, anxiety and alarm. Behavioral consequences of these effects of the immune system on the brain include depression, anxiety, fatigue, psychomotor slowing, anorexia, cognitive dysfunction and sleep impairment; symptoms that overlap with those which characterize neuropsychiatric disorders, especially depression. Pathways that appear to be especially important in immune system effects on the brain include the cytokine signaling molecules, p38 mitogen-activated protein kinase and nuclear factor kappa B; indoleamine 2,3 dioxygenase and its downstream metabolites, kynurenine, quinolinic acid and kynurenic acid; the neurotransmitters, serotonin, dopamine and glutamate; and neurocircuits involving the basal ganglia and anterior cingulate cortex. A series of vulnerability factors including aging and obesity as well as chronic stress also appears to interact with immune to brain signaling to exacerbate immunologic contributions to neuropsychiatric disease. The elucidation of the mechanisms by which the immune system influences behavior yields a host of targets for potential therapeutic development as well as informing strategies for the prevention of neuropsychiatric disease in at risk populations.
All of the conditions above, depression, schizophrenia, bi-polar, and tourettes are diagnosed behaviorally; it is only in the last few years that the medical dimension of these disorders were even understood to exist. None of the studies that I referenced above are more than five years old; the idea that behavioral disorders were so closely entangled with the immune system is very, very new. It should be noted that I intentionally left out disorders that also have reams of evidence of immune participation, but which are more degenerative in nature; i.e., Alzheimer’s, ALS, Parkinson’s. When discussing autism, I also left out studies involving aberrant presence of auto-antibodies, of which there are many.
One of the things that I have learned in trying to refine my thought processes during my time on the Internet is that rarely does a single study tell us much about a condition; but the converse also holds true, if we have many studies with different methodologies or measurement end points, but they all reach similar conclusions, then the likely-hood that the findings are accurate is much, much greater. All of the studies I have listed above tell us something similar; that the immune system is clearly, unmistakably playing a part in a lot of conditions classically considered neurological and diagnosed behaviorally. It isn’t enough to nitpick flaws in a single one of the studies in order for ‘correlation does not equal causation’ to make meaningful headway into the implications of these studies; instead, all of the studies above, and lots more, have to be wrong in the same way if we would like to return to a place where we can keep our heads in the sand, hoping for coincidences and bleating out catchphrases in the face of clinical findings. That isn’t going to happen. Given this reality, we should not and cannot ignore the growing evidence of immune abnormalities in the autism population, no matter how inconvenient following that trail of evidence might become.
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.
Implications for Autism Or Just Interesting? “Epigenetic and immune function profiles associated with posttraumatic stress disorder”
Posted June 25, 2010on:
Hello friends –
One of my tangential pubmed alerts notified me of this study the other day: Epigenetic and immune function profiles associated with posttraumatic stress disorder
The biologic underpinnings of posttraumatic stress disorder (PTSD) have not been fully elucidated. Previous work suggests that alterations in the immune system are characteristic of the disorder. Identifying the biologic mechanisms by which such alterations occur could provide fundamental insights into the etiology and treatment of PTSD. Here we identify specific epigenetic profiles underlying immune system changes associated with PTSD. Using blood samples (n = 100) obtained from an ongoing, prospective epidemiologic study in Detroit, the Detroit Neighborhood Health Study, we applied methylation microarrays to assay CpG sites from more than 14,000 genes among 23 PTSD-affected and 77 PTSD-unaffected individuals. We show that immune system functions are significantly overrepresented among the annotations associated with genes uniquely unmethylated among those with PTSD. We further demonstrate that genes whose methylation levels are significantly and negatively correlated with traumatic burden show a similar strong signal of immune function among the PTSD affected. The observed epigenetic variability in immune function by PTSD is corroborated using an independent biologic marker of immune response to infection, CMV—a typically latent herpesvirus whose activity was significantly higher among those with PTSD. This report of peripheral epigenomic and CMV profiles associated with mental illness suggests a biologic model of PTSD etiology in which an externally experienced traumatic event induces downstream alterations in immune function by reducing methylation levels of immune-related genes.
Essentially the authors took a bunch of people that are more likely to experience stressful situations and PTSD, urban Detroit residents, who amazingly report PTSD symptoms at twice the level that previous studies have found in analysis of larger areas. [Apparently, getting physically attacked is more common there, which gives rise to PTSD even more than ‘other traumatic event types’, and was reported by 50% of the participants from a larger study which formed the population pool of this study. (!!)] With this population base, blood was drawn and methylation profiles were analyzed between participants who reported PTSD symptoms (n=23) and those who ‘only’ had ‘potentially traumatic events’ (PTE). PTSD and ‘controls’ where matched by race, age, sex, and blood profiles.
Once methylation levels were identified, a functional annotation clustering analysis was performed, which I believe is similar a pathway analysis; essentially a bioinformatic tool to gain insight into which biological functions were being manipulated as a result of differential methylation of the genome. This is a powerful new tool in discerning what is happening in autism and elsewhere, and I expect it will provide some surprising answers in the future. Here is their text on what they found:
Consistent with previous findings from gene expression (4, 5) and psychoeneuroimmunologic studies (3), each of the top three FACs determined from uniquely unmethylated genes among PTSD-affected individuals shows a strong signature of immune system involvement. This signature includes genes from the innate immune system (e.g.,TLR1 andTLR3), as well as from genes that regulate innate and adaptive immune system processes (e.g., IL8, LTA, and KLRG-1). In contrast, pathways and processes relevant to organismal development in general—and neurogenesis in particular—figure prominently among the genes uniquely unmethylated in the PTSD-unaffected group (e.g., CNTN2 and TUBB2B; Fig. S2). Notably, similar clusters were obtained using an alternative approach based on genes differentially methylated between the two groups at P < 0.01, with annotations in the top five FACs that include signal, cell proliferation, developmental process, neurologic system process, and inflammatory response
Keeping in mind that reduced methylation results in increased gene expression, if we take a look at Table 1, some of the parallels to autism jump out a little more robustly:
In the ‘Uniquely Unmethylated’ (i.e., higher expression), area, we find that participants affected by PTSD had showed greater enrichment in genes related to the immune response, and specifically the inflammatory response and innate immune response. Our evidence for similar immunological profiles in the autism realm is deep, and includes multiple observations of an active immune response in the CNS, highly significant over expression of genes related to immune function in the CNS, several observations of known upregulators of the innate immune response that are associated with inflammatory conditions, and multiple studies finding an exaggerated innate immune response in vitro when compared to controls. The correlations with developmental process and neuron creation are pretty straightforward.
In the ‘Uniquely Methylated’ area (i.e., lower expression), the sensory perception differences hit close to home, and xenobiotic metabolism has been implicated by several studies.
Going further, the researchers attempted to evaluate for correlations between the number of potentially traumatic experiences and the methylation profile, and somewhat unsurprisingly found that as the number of experiences increased, the methylation differentials showed wider variation.
Here again we see a distinct signature of immune-related methylation profiles among the PTSD-affected group only. More specifically, we see methylation profiles that are suggestive of immune activation among persons with more PTE exposure in the genes that are significantly negatively correlated with increasing number of PTEs—a pattern reflective of that observed for the uniquely unmethylated genes in this same group (Table 1).
From the discussion section:
Among the many analyses performed in this work, the immune related functions identified in the PTSD-affected group were consistently identified only among gene sets with relatively lower levels of methylation (Tables 1 and 2). Demethylation has previously been shown to correlate with increased expression in several immune system–related genes (reviewed in ref. 22), including some identified here [e.g., IL8 (23)]. In contrast, methylation profiles among the PTSD-unaffected are distinguished by neurogenesis-related functional annotations. Neural progenitor cells have previously been identified in the adult human hippocampus (24); however, stress can inhibit cell proliferation and neurogenesis in this brain region (reviewed in ref. 25), and recent work suggests that adult neurogenesis may be regulated by components of the immune system (reviewed in ref. 26). Thus, immune dysfunction among persons with PTSD may be influenced by epigenetic profiles that are suggestive of immune activation or enhancement and also by an absence of epigenetic profiles that would be consistent with the development of normal neural-immune interactions (27).
Among the genes uniquely methylated in the PTSD-affected group, it is striking that the second most enriched cluster—sensory perception of sound—directly reflects one of the three major symptom clusters that define the disorder (Fig. 3B). Genes in this FAC thatmay be particularly salient to this symptom domain include otospiralin (OTOS),which shows decreased expression in guinea pigs after acoustic stress (28) and otoferlin (OTOF), mutations in which have been linked to nonsyndromic hearing loss in humans (29). Exaggerated acoustic startle responses, often measured via heart rate or skin conductance after exposure to a sudden, loud tone, have been well documented among the PTSD affected (30) and are indicative of a hyperarousal state that characterizes this symptom domain. Notably, prospective studies have demonstrated that an elevated startle response is a consequence of having PTSD, because the response was not present immediately after exposure to trauma but developed with time among trauma survivors who developed the disorder (30, 31).
My son had some very severe auditory related problems earlier in his life, and still occasionally struggles with either sudden loud noises, or some very specific noises, such as some dog barks, or the sound of an infant crying. Previously the only physiology based attempt at an explanation I’d heard of for this type of response involved fine grained brain architecture and consequent filtering and/or overexcitation problems. The idea that sound sensitivities in particular can be obtained environmentally is of particular interest to the autism community.
From the common sense angle, I find this completely fascinating; we’ve known for a long time that living with consistent stress is bad for you with a variety of nasty endpoints, but this type of finding narrows down the means by which this happens. In the far off future, perhaps targeted methyl affecting drugs could be considered for people who experience extremely stressful events, as sort of a ‘PTSD vaccine’ [hehe] could be developed.
From an ASD perspective, increased feeling of anxiety, or just generally being ‘stressed out’ is a consistent finding both in research and from what I’ve read of readings from people with autism on the Internet. I’ve seen several explanations, with sensory based problems being mentioned several times. From a biological standpoint we seem to have a growing body of evidence of an abnormally regulated stress response in the autism cohort. An internet friend of mine, Loftmatt, has written extensively on his thoughts concerning the increase in stress in modern society and the mechanisms by which this could be contributing to our apparent observations of an increase in autism. This study would seem to provide insight towards a possible mechanism by which a frequent state of stress could lead to some of our immunological findings in the autism realm; a possibility I hadn’t considered previously when trying to detangle a means by which our observations of immune activation were not participating in autistic behavior. The thought of a feedback loop also strikes me looking at this, something causes a feeling of extreme stress, which leads to abnormal methylation levels and genetic expression, which leads to increased physiological (and behavioral?) alterations, and even more stress.
I may try to poke through the supplementary materials to see if any specific genes or pathways found to be differentially regulated have parallels in some of the other studies we’ve seen recently such as Garbett or Hu, although this may be somewhat of a crapshoot unless I could figure out how to actually submit gene lists to GSEA and read the responses.
And we may need to consider the possibility that these types of effects can be trans-generational. One of the most fascinating studies I’ve seen on epigentics involved exactly that, a multi-generational effect of famine in Holland, wherein the grandchildren of women who were pregnant during a time of famine bore striking differences in a variety of endpoints compared to children whose grandmothers were not pregnant during that time.
The more we learn, the more complicated the world becomes.
Posted June 11, 2010on:
Hello friends –
I ran across this one on accident the other day (why wasn’t it in one of my pubmed alerts?):
Previous studies have demonstrated an association between preterm delivery and increased risk of special educational need (SEN). The aim of our study was to examine the risk of SEN across the full range of gestation.
Methods and Findings
We conducted a population-based, retrospective study by linking school census data on the 407,503 eligible school-aged children resident in 19 Scottish Local Authority areas (total population 3.8 million) to their routine birth data. SEN was recorded in 17,784 (4.9%) children; 1,565 (8.4%) of those born preterm and 16,219 (4.7%) of those born at term. The risk of SEN increased across the whole range of gestation from 40 to 24 wk: 37–39 wk adjusted odds ratio (OR) 1.16, 95% confidence interval (CI) 1.12–1.20; 33–36 wk adjusted OR 1.53, 95% CI 1.43–1.63; 28–32 wk adjusted OR 2.66, 95% CI 2.38–2.97; 24–27 wk adjusted OR 6.92, 95% CI 5.58–8.58. There was no interaction between elective versus spontaneous delivery. Overall, gestation at delivery accounted for 10% of the adjusted population attributable fraction of SEN. Because of their high frequency, early term deliveries (37–39 wk) accounted for 5.5% of cases of SEN compared with preterm deliveries (<37 wk), which accounted for only 3.6% of cases.
Gestation at delivery had a strong, dose-dependent relationship with SEN that was apparent across the whole range of gestation. Because early term delivery is more common than preterm delivery, the former accounts for a higher percentage of SEN cases. Our findings have important implications for clinical practice in relation to the timing of elective delivery
[Full paper from link. Emphasis is mine]
Essentially the authors evaluated gestational lengths with a fine tooth comb to discern if ‘early’, though not technically ‘pre-term’ delivery was associated with a ‘special education need’ (SEN), which in this case embodies a range of developmental problems including dyslexia, autism, or even physical problems like deafness or vision problems.
What the authors found was that there were subtle, but real effects in the likelyhood of having a special education need for non full term births that was dose dependent, but even included children that would not necessarily be considered early by existing standards.
Our study demonstrated a strong trend of decreasing risk of SEN with advancing gestational age at birth. The key finding of the present analysis is that this trend continued across gestational ages classified as term. Although the risk of SEN was highest among infants who were delivered preterm (<37 wk gestation), these accounted for only 5.1% of deliveries. Therefore, only a relatively small proportion of SEN (3.5%) could be attributed to preterm delivery. By contrast, 39.6% of infants were delivered between 37 and 39 wk gestation. Therefore, whilst these early term infants had only a moderately increased risk, 5.3% of SEN cases could be attributed to early term delivery.
The authors claim that the finding of effects at early, but not pre-term gestational lengths is one that is largely missing from existing studies, which have not taken these date ranges into consideration, or the ones that did, were not studying for cognitive problems, and indeed, excluded children with these criteria. Curiously, they also report an increase in SEN in children who had extra gestational periods, i.e., > 41 weeks in some studies.
The authors make absolutely no speculation as to what might be driving increased special education needs as the result of premature or early birth.
Looking at their results, one of the most striking things is that the impact did not alter if elective (i.e. C-Section) versus non-elective births were used as a variable. But this has deep ramifications for the autism storyline, which holds that if there are environmental factors that can contribute to autism, they are prenatal, and indeed, are often thought to involve insults very early in the prenatal period. In this case, we know that a genetic or environmental force isn’t contributing to the early birth, because it didn’t matter if the birth was spontaneous or not. The only area for an effect is postnatal. That is a big, big difference in the narrative.
Is this a matter of some just in time epigenetic programming happening in the womb that doesn’t get a chance to finish up in early births? Alternatively it could be that early birth allows for environmental exposures that the infant is not quite prepared to deal with. Or it could be both, or neither, or an illusory finding, but if these findings can be replicated, it raises a lot of questions about the sacred line between prenatal and postnatal environmental influences.
Unfortunately, the raw data for this project doesn’t seem to be available online; it might be really nice to see if there were patterns to be observed had particular salience to our population of interest.