passionless Droning about autism

Posts Tagged ‘Inflammation

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 seizuresConsistent 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 BrainPriming 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 hornIntrathecal 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, paincan 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.


–          pD

Hello friends –

A study with a beautifully terse title, Microglia in the Cerebral Cortex in Autism landed in my inbox the other day.  It adds to the growing literature showing perturbations in neuroimmune system in the autism population, this time by measuring the number of microglia in different parts of the brain.  Here is the abstract:

We immunocytochemically identified microglia in fronto-insular (FI) and visual cortex (VC) in autopsy brains of well-phenotyped subjects with autism and matched  controls, and stereologically quantified the microglial densities. Densities were determined blind to phenotype using an optical fractionator probe. In FI, individuals with autism had significantly more microglia compared to controls (p = 0.02). One such subject had a microglial density in FI within the control range and was also an outlier behaviorally with respect to other subjects with autism. In VC, microglial densities were also significantly greater in individuals with autism versus controls (p = 0.0002). Since we observed increased densities of microglia in two functionally and anatomically disparate cortical areas, we suggest that these immune cells are probably denser throughout cerebral cortex in brains of people with autism.

[Note: You don’t see p-values of .0002 too often!]  This paper is at a high level largely similar to another recent paper, Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism (discussed on this blog, here).  The authors were clever here, they intentionally used two very anatomically different, and spatially separated parts of the brain to evaluate for microglia population differences, a sort of bonus slice to learn more about the population of microglia in the brain.

The specific measurement technique in use, staining for specific antibodies, does not give us information regarding the activated/non activated state of the microglia, a determination which must be made with evaluations of morphology, though several other studies have measured this directly, and many more provide indirect evidence of a chronic state of activation of microglia.   Not only did the author s report an increase in population density in the autism group, the number of microglia was also positively correlated between sites; i.e., a patient with more microglia in the visual cortex was also more likely to have more microglia in the fronto-insular.

These findings demonstrate that, at the time of death, there were significantly higher microglial densities in the subjects with autism compared to the control subjects, and that this change in microglial density is widespread throughout the cerebral cortex in autism. The microglial  densities in FI and VC in the same subject were significantly correlated (both measures were available in 10 controls and 8 autistic subjects for a total of 18 subjects) with Pearson’s r2 = 0.4285, p = 0.0024 (Fig. 6). This indicates that the elevation in density is consistent between these areas, and probably throughout the cortex, in both subjects with autism and controls.

Also of interest, in the control group microglia densities tended to decrease with age, but this change was not seen in the autism population.

There is some discussion about a big problem in the autism research world, a very real and meaningful dearth of available tissue samples, this study shared five patients with Morgan, and one from Vargas.  [Note: Sign up to help.  Morbid but necessary.]

The authors went on to ask the exact same question I had, “How and when does the increased density of autistic microglial arrays arise, and how is it maintained?”  Unfortunately, while there aren’t any good answers, I was still a little disappointed with the analysis.  There is a quick rundown of a variety of neuroimmune and peripheral immune findings in autism, and some thoughts on ‘sickness behavior’ with the implicit interconnectedness of the immune state and behaviors, and some discussion on some of the many animal models of maternal immune activation in autism.

In an stroke of amazing serendipity, the authors wonder aloud towards the possibility of a type of distracted worker effect of microglia on neural networks, sort of a bank shot on the autism paradox I struggled with in my previous post when I said,

Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain?  Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?

The authors of Microglia in the Cerebral Cortex in Autism state

In contrast, microglia can also phagocytize synapses and whole neurons, thus disrupting neural circuits. For example,when the axons of motor neurons are cut, the microglia strip them of their synapses (Blinzinger and Kreutzberg 1968; Cullheim and Thams 2007; Graeber et al. 1993). Another example of the disruption of circuitry arises from the direct phagocytosis of neurons. Neurons communicate with microglia by emitting fractalkine*, which appears to inhibit their phagocytosis by microglia. Deleting the gene for the microglial fractalkine receptor (Cx3cr1) in a mouse model of Alzheimer’s disease has the effect of preventing the microglial destruction and phagocytosis of layer 3 neurons that was observed in these mice in vivo with 2-photon microscopy (Furhmann* et al. 2010). In particular, Cx3cr1 knockout mice have greater numbers of dendritic spines in CA1 neurons, have decreased frequency sEPSCs and had seizure patterns which indicate that deficient fractalkine signaling* reduces microglia-mediated synaptic  pruning, leading to abnormal brain development, immature connectivity, and a delay in brain circuitry in the hippocampus (Paolicelli* et al. 2011). In summary, the increased density of microglia in people with autism could be protective against other aspects of this condition, and that a possible side-effect of this protective response might involve alterations in neuronal circuitry.

Oh hell yeah.  (* concepts and papers discussed on this blog, here)

Going back to the big dollar question, How and when does the increased density of autistic microglial arrays arise, and how is it maintained?”, the possibility of an ongoing infection was raised as a one option, “The increase of microglial densities in individuals with autism could be a function of neuroprotection in response to harmful microorganisms.”  Vargas had a dedicated section towards a failure to find agents of the peripheral immune system that are consistent with infiltration from the peripheral immune system commonly observed during acute infection, I do not think other papers have looked for that per se, but will cede to someone with better data.  (?)   There was a very weird paper from Italy that pointed to a possible polyomavirus transmission from the father in the autism group, though this study was not referenced in Microglia in the Cerebral Cortex in Autism. [Note:  I showed my wife this paper, and she told me, “Good job with the autism gametes.”  Nice.]  Could a virus cause autism, is a nice discussion on this that includes blog and personal favorites, Fatemi, Patterson, and Persico discussing the possibilities and limitations of the study.  Great stuff!

While I must admit the possibility that the chronically activated microglia in autism are working on purpose, the irony gods mandate that I wonder aloud if certain segments of the autism Some-Jerk-On-The-Internet population will cling to the possibility that autism is caused by a disease in order to disavow a causative role for neuroinflammation?  Those are some tough choices.

There is a discussion on the myriad of ways that microglia could directly participate in autism pathogenesis, starting the discussion off right to the point, “By contrast, there are diseases that arise from intrinsic defects in the microglia themselves which can cause stereotypic behavioral dysfunctions.”  There is a short discussion of Nasu-Hakola disease, something I’d never heard of, which has evidence of an increase in cytokines as a result of genetically driven microglial deficiencies, and shows striking behavioral manifestations.

The possibility of some areas of the brain being more susceptible to alterations than others is there too, “Thus, while changes in microglial density appear to be widespread in brains of autistic individuals, some areas may be more vulnerable than others to its effects.”   Considering this idea alongside the extremely heterogeneous set of symptoms assigned to autism, a curious question to ponder becomes; if neuroinflammation is a participatory process in the behavioral manifestation of autism, could some of the variability in autistic behaviors be explained by spatially specific gradients of microglial activity?  Going further, considering the still largely mysterious migration of microglia into the brain during development, could the temporal origin of microglial activation in autism be a determinant in the eventual behavioral manifestations?  These are tricky questions, and I don’t think that our current methodological capacities are sufficient to start thinking about forming a model for analysis.

One concept I was surprised to not receive attention was a developmental programming model, where animal studies tell us that if something happens during critical developmental timeframes, the effect can propagate into adulthood.    In fact, one study, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain (Bland et all)  exposed four day old animals to e-coli, which found, among other things, “significantly more microglia in the adult DG of early-infected rats”, something seemingly of considerable salience to the current findings, especially considering the known risk factors of early infections as autism risk factors.  In Bland, no external agent other than an infection during early life was necessary; this is the essence of the developmental programming model, even after the infection was long since cleared, patterns of physiology were imprinted, the animals recovered from e-coli but were changed from the experience.  This my biggest issue with the possibility of an as of yet undefined, and continued evidence free pathogen or process that is causing the immune abnormalities we see in autism, it mandates we ignore existing biologically plausible models that fit well within known risk factors for autism.  Why?

Another area this paper was curiously silent on is the data regarding differences in males and females in the timeframes of microglial migration into the brain, something I’d like to learn much more about soon.  As an example, Sex differences in microglial colonization of the developing rat brain [yet another by blog favorite, Staci Bilbo] reported “the number and morphology of microglia throughout development is dependent upon the sex and age of the individual, as well as the brain region of interest” among other findings broadly consistent with a beautiful complexity.  This is interesting fodder for a discussion concerning possibly the most persistent finding in autism, a very high male to female ratio that has a series of possible explanations [somewhat discussed on this blog, here].

So we know more, but still have only increased our knowledge incrementally.  It is increasingly likely that an increased number of microglia in many areas of the brain is characteristic of autism, but the whys, hows, whens, wheres, and whoms still hold many mysteries.  The more things change, the more they stay the same.

–          pD

Hello friends –

I’ve had a couple of interesting papers land in my pubmed feed the past few weeks that seem to be tangentially touching on something that has been at the back of my mind for a long time; namely, the repeated findings of a state of an ongoing immune response in the CNS of the autism population, coupled with a behavioral state that is either static, or in many cases, showing gradual improvement over time.  [Discussions of ongoing immune response in the brain in autism, here, here, or here].  This is exactly the opposite of what I expected.  Most of the conditions I had generally associated with a state of neuroinflammation, i.e., Alzheimer’s or Parkinson’s show a behavioral profile opposite to autism over time, i.e., a deterioration of skills and cognitive abilities.   The diagnosis for these conditions is never a straight line or a gradual curve upwards, but a dispassionately reliable trajectory of a downward spiral.

This is something that has been really bugging me a lot as a riddle, I’ve mentioned it here in comments, and other places on the Internet.  While outright signs of neuroinflammation are clearly associated with conditions you would rather not have, as opposed to have, we must admit that the available evidence tells us that  we cannot just wave our hands, say ‘neuroinflammation!’, and know much more than the broad strokes.  [Note: In my early days of my AutismNet life, my view was somewhat less nuanced.]  I think that part of what was bothering me is the result of an oversimplified model in my mind’s eye, but I’d formed that model on top of a set of measurements that had empirical precision but underpowered understandings, alongside a more fundamental lack of knowledge.

We know a little more now.

The first paper that really got me thinking along these lines was Synaptic pruning by microglia is necessary for normal brain development, (discussed on this blog, here), which provided evidence of microglial involvement in the ‘pruning’ of synapses, an important step in brain development thought to streamline neural communication by optimizing neuron structure.  This was the first paper I’d read that hinted at microglia participation in ‘normal’ brain function; it was only very recently that microglia were considered to have any role in non pathological states.  Another paper, Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease, also implicated microglia in synaptic pruning.

Then I got myself a copy of The role of microglia at synapses in the healthy CNS: novel insights from recent imaging studies. It is a review of several recent studies on the non-excited life of microglia.

In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.

This review by Tremblay was published in 2012, evidence of the nascent nature of our available data on microglial involvement in the normal brain environment; Tremblay states that part of the reason this type of finding is so recent is the relative difficulty of measuring microglia in non excited states.  They were the electrons of brain measurements; our previous attempts to measure them were capable of causing them to change morphology.

The roles of ‘resting’ or immunologically quiescent microglia have remained relatively unknown (also see Tremblay et al., 2011). This is largely due to the difficulties of studying microglia in their non-activated state. Microglia respond promptly to any changes occurring in their environment, and therefore experimental ex vivo and in vitro preparations inevitably result in transformation of their normally prevailing behavior.


Anyway, some new whizbang technologies (i.e., in vivo two-photon laser scanning microscopy)[?] are allowing researchers to peer into the ho-hum everyday activities of ‘non activated’ microglia, and what they are finding is that the term ‘activated microglia’ might be a bit of a misnomer, microglia have been participating in brain function all along, it is just that our filters were insignificantly powered to detect some of their actions until very recently.   Several studies have shown that so called ‘resting’ microglia are constantly evaluating their environment with protusions that seemed to operate rather quickly in relationship to other types of neurons.

This unexpected behavior suggested that resting or surveillant microglia may continuously survey the brain parenchyma as part of their immune function, which would justify the substantial expenditure of energy required to continuously maintain microglial dynamics in the normal brain, without excluding the possibility of an additional, distinct contribution to normal brain physiology

Several papers are reviewed that utilized a couple of highly technical methods, including double roll your own transgenic mouse models to visualize the interactions of microglia in a non excited state and synapses.  Specific areas of the brain were measured in different studies, microglia were observed transiently engaging with neurons and seemed to target some dendrites for removal.  The authors speculate that this could be a mechanism by which neuronal network maintenance, plasticity, could be affected.

In the mature healthy CNS, neuronal networks are continuously remodeled through the formation, modification and elimination of synaptic structures (see Fortin et al. (2011) for molecular mechanisms of structural plasticity) in relation with behavioral and sensory experience.


To determine a possible role of surveillant microglia in the structural remodeling of synaptic structures under normal physiological conditions, Tremblay et al. (2010b) also examined the size changes of spines and terminals before, during and after microglial contacts. Spines contacted by microglial processes during imaging (30–120 min sessions) were found to be smaller initially than those which remained non-contacted. Spines, but not terminals, also underwent transient increases in size during microglial contact, with smaller spines showing the most pronounced changes. Surprisingly, chronic imaging over 2 days further revealed a statistically significant difference in the elimination rate of microglia-contacted spines: spines contacted by microglia were more frequently eliminated than non-contacted spines (24 versus 7%; P  0.05), and in all cases, only the small spines were seen to disappear. These observations suggest that despite an apparently random sampling of the parenchyma, microglial processes specifically target a subset of small, structurally dynamic and transient dendritic spines.

There is also some description of studies that seemed to indicate that the microglial/synapse interactions could be modified through environmental stimulus, two experiments were described involving sensory deprivation and consequent changes in microglia activity.  Other experiments described changes in microglial surveillance as a result of induced changes in neuronal excitability by chemical agonists or antagonists of glutamate receptors.  [Perhaps this is the basis of the curious findings in Neuroprotective function for ramified microglia in hippocampal excitotoxicity?]

In their concluding statements, Tremblay provides a good description of just how little we know, and in a style that I love, poses open questions for the newer rounds of literature to address.

Since the recent studies have barely scratched the surface (of the brain in this case), the modalities of microglial interactions with excitatory and inhibitory synapses throughout the CNS, much as their functional significance and particular cellular and molecular mechanisms still remain undetermined. For example, in which contexts do quiescent microglia directly phagocytose axon terminals and dendritic spines, use other mechanisms such as proteolytic remodeling of the extracellular space, or refrain from intervening?  How do surveillant microglia recognize and respond to the various molecular signals in their environment, including dynamic changes in neurotransmission and neuronal activity at individual synapses? How do these immune cells cooperate with other glial cells, as well as peripheral myeloid cells, in maintaining or shaping neuronal architecture and activity? And, as in the case of microglial memory of past immune challenges (see Bilbo et al., 2012), do surveillant microglia somehow remember their previous behavioral states, the flux of information processing in the brain, or the structural changes of synaptic elements in recent and not so recent windows of intervention?

The last sentence there, I think, is especially salient considered within a context of developmental programming.

So what we’ve learned is that decades after the discovery of microglia cells as the immune regulators in the CNS, they appear to also be participating in more fundamental maintenance of the neural structure of our brains; there is increasing evidence of direct relationships in synaptic and axonal removal as well as roles in neurotransmission and the regulation of excitability.   Is more on the horizon?

But what about autism and our apparent autism paradox of a static or improving behavioral state alongside conditions of immune activation within the CNS?

Well, I have also been thinking about two brain scanning studies that have come out not too long ago, Neuron Number in Children With Autism (Courchesne et all) , which found increased numbers of neurons in the autism cohort, and Differences in White Matter Fiber Tract Development Present From 6 to 24 Months in Infants With Autism (Wolff et all) which found that the autism group showed denser bundled of white matter, so called wiring, between different parts of the brain.  In both of these studies mention is made of the fact that it was possible that their findings, increased cell numbers could be the result of inappropriate removal of excess neurons during development.

Apoptotic mechanisms during the third trimester and early postnatal life normally remove subplate neurons, which comprise about half the neurons produced in the second trimester. A failure of that key early developmental process could also create a pathological excess of cortical neurons.


For example, differences in structural organization prior to a period of experience-dependent development related to social cognition (52–54) may decrease neural plasticity through limitations on environmental input, preventing typical neural specialization (52). These alterations could have a ripple effect through decreasing environmental responsiveness and escalating invariance*, thus canalizing a specific neural trajectory that results in the behavioral phenotype that defines ASDs. In typical development, the selective refinement of neural connections through axonal pruning (55) along with constructive processes such as myelination (56) combine to yield efficient signal transmission among brain regions. One or both of these mechanisms may underlie the widespread differences in white matter fiber pathways observed in the current study. 

* 😦

So, we have growing evidence of microglial participation of neural maintenance alongside growing evidence of impaired maintenance in the autism cohort.

Can our autism paradox be explained by microglia converging in the center of these related lines of thought?  Is the answer to our riddle that the ongoing immune response in the brain is not sufficiently powered, or targeted, to cause increasing loss of abilities, but instead, was enough to keep critical, once in a lifetime chances for brain organization from occurring?  Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain?  Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?

That is a pretty neat idea to consider.

Even without the Courchesne and Wolff, the findings that specifically mention impaired network maintenance as possible culprits, the findings of active participation of ‘non-active’ microglia in brain optimization and normal processes is a very problematic finding for another autism canard, the idea that findings of neuroinflammation may not be pathological.  The intellectually honest observer will admit that the crux of this defense lay in vaccine count trial testimony presented by John Hopkin’s researchers after their seminal neuroinflammation paper was published.  Unfortunately, the vigor with which this testimony is trotted out online does not match the frequency with which such ideas actually percolate into the literature.

But with the data from Tremblay, Paolicelli, and others, such an idea becomes even more difficult to defend, we must now speculate on a mechanism by which either microglia could be in an excited state and continue to perform streamlining of the neural structure, or insist that it is possible that microglia were not excited during development, and something else happened to interfere with neuron numbers, and then, subsequently the microglia became chronically activated.

This is unlikely, and unlikelier still when we consider that anyone proposing such a model must do so with enough robustness to overcome a biologically plausible pathway supported by a variety of studies.  And that is only if there was anything underneath the vapor!  Make no mistake, if you ever press someone to actually defend, with literature, the mechanisms by which a state of chronic neuroinflammation might be beneficial in autism, or even the result of something else that also causes autism, no further elucidation of that mechanism is ever forthcoming.  There isn’t anything there.

At some point, it becomes incumbent of people wishing to make an argument that they propose a biologically plausible mechanism if they wish to continue to be taken seriously.  If they cannot, if the literature cannot be probed to make such a case with more empirical support than it might be, the notion so add odds with available evidence should be summarily discarded, unless and until a transcendent set of findings is presented.  There should always be room for more findings in our worldview, but precious limited space for faith in the face of contradictory findings.

–          pD

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:

Table 1

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).
Lastly, the participants were scanned for antibodies to CMV, a persistent herpesvirus found in almost all humans, and can be used as a biomarker to indicate compromised immune function.  Significant differences in antibodies were observed between the two groups.

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.


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