passionless Droning about autism

Archive for the ‘Intriguing’ Category

I’ve been thinking a lot lately about the beauty and trials of the tightly coupled systems, the interconnected pathways that keep popping up when pubmed tells me something that might be of interest on journey autism.  One theme bubbling to the top of my thoughts is that there is a large set of inputs capable of tweaking the areas we see altered in autism; broken isn’t necessarily appropriate, but the research increasingly tells us that a delicately balanced set of connected processes is readily changed, and the way that the physics work out, there is no way to change just one thing when you have a polygamous marriage of chemical systems.

Imagine a orchestra where all of the musicians were physically bound to one or more of their counterparts, a system of wires, pulleys, springs and levers such that the musicians are actually participating in the playing of each other, not soccer players doing synchronized flips so much as a set of violin-em-cello-em robots, connected to play their instruments in unison, wind them up and create a symphony.  Different orchestras might have a tighter wire from one member to another, or an older spring, but when they worked together, you could tell what composition they were playing.  In this analogy, you cannot have the drummers start beating harder and faster without also changing how hard the French horn players blow.  The situation only gets more complicated if some of our musicians were connected to several other musicians simultaneously.  There would still be music if the cellist couldn’t keep a steady rhythm, but it would be different music, not just a different cello.

The communication between a lot of our “systems”, immune, endocrine, stress response and central nervous systems are a lot like musicians in the orchestra, interdependent and intimately connected.

The funny thing is, this same message is being blared to me, and to you, all the time, damn near every time you turn on the TV, but it is hidden in plain sight by legislatively mandated doublespeak.  Consider how many advertisements each of us have seen for pharmaceutical drugs where the number of complications and contra-indicated conditions far, far exceed the number of desired effects?

Here is a list of common side effects of Viagra:

Diarrhea, dizziness, flushing, headache, heartburn, stuffy nose, upset stomach

So right off the bat, besides what we are looking for, we can see it is common to expect Viagra to also affect your GI system, immune system, and/ or brain function.  These are the types of things that are “common”.  (One wonders how Viagra would sell if it always caused headaches and diarrhea, and sometimes transiently ameliorated erectile dysfunction? )  A list of ‘severe’ side effects includes memory loss and a sudden decrease in hearing or vision.  Even after decades of work by a lot of exceptionally smart people and hundreds of billions of dollars, the interlocked complexity of our bodies are continuing to prove very difficult to adjust in only the way we’d like, and seemingly minor perturbations in one area can pop up in very unpredictable fashion in other functions.

Trying to put my mind around the implications of this in regards to autism often leaves me with a sense of being profoundly humbled and woefully underprepared, not unlike a lot of my experiences with autism in the real world.  Secondarily, again with great similarity to personal experience, I (eventually) come to the (re-)realization that we should rejoice in opportunities to be challenged and learning more about something makes us richer in ways more important than dollars.

A superb example of all of this and more landed in my inbox the other day, Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus (Williamson et all).  [Also on this paper, blog favorite, Staci Bilbo]

Williamson reported that animals given a so called ‘enriched environment’ exhibited significantly decreased immune responses in certain portions of the brain following immune challenge, with reduced levels of several chemokines and cytokines in the hippocampus in the treatment group. (A previous discussion about environmental enrichment on this blog can be found here)   In this instance, the treatment group got to spend twelve hours a day in a different area, a housing unit with “a running wheel, a PVC tube and various small objects and toys”, while the control group of animals stayed in their drab, Soviet era proletariat cages all day and all night long.  Here is the abstract:

Neurogenesis is a well-characterized phenomenon within the dentate gyrus (DG) of the adult hippocampus. Environmental enrichment (EE) in rodents increases neurogenesis, enhances cognition, and promotes recovery from injury. However, little is known about the effects of EE on glia (astrocytes and microglia). Given their importance in neural repair, we predicted that EE would modulate glial phenotype and/or function within the hippocampus. Adult male rats were housed either 12h/day in an enriched environment or in a standard home cage. Rats were injected with BrdU at 1week, and after 7weeks, half of the rats from each housing group were injected with lipopolysaccharide (LPS), and cytokine and chemokine expression was assessed within the periphery, hippocampus and cortex. Enriched rats had a markedly blunted pro-inflammatory response to LPS within the hippocampus. Specifically, expression of the chemokines Ccl2, Ccl3 and Cxcl2, several members of the tumor necrosis factor (TNF) family, and the pro-inflammatory cytokine IL-1ß were all significantly decreased following LPS administration in EE rats compared to controls. EE did not impact the inflammatory response to LPS in the cortex. Moreover, EE significantly increased both astrocyte (GFAP+) and microglia (Iba1+) antigen expression within the DG, but not in the CA1, CA3, or cortex. Measures of neurogenesis were not impacted by EE (BrdU and DCX staining), although hippocampal BDNF mRNA was significantly increased by EE. This study demonstrates the importance of environmental factors on the function of the immune system specifically within the brain, which can have profound effects on neural function.

Total interconnectedness kick ass!

Considering the wide ranging and predominantly ‘rather-not-have-than-have’ properties of ‘extra’ TNF-alpha and IL-1beta in the CNS, this is a pretty interesting finding.  Not only that, animals ‘protected’ through environmental enrichment also showed increased levels of growth factors known to be altered in autism, again in the hippocampus.  In a very real and measurable sense, it was possible to shuffle the neuroimmune cocktail of the brain by changing things like the availability of quality leisure time.  As we have seen in other areas, altering the chemical milieu of immunomodulatory factors in the brain isn’t trivial, and is increasingly associated with a variety of conditions classically diagnosed through the study of behaviors.

It should be noted that there were unexpected, and generally negative findings from this study, namely, a relative lack of biomarkers indicative of increased neurogenesis in the environmental enrichment group; something that I think took the authors by a bit of surprise.

There is a short discussion on the possibilities on why the findings of differential neuroimmune responses were found only in the hippocampus, with reference being made to previous studies indicating that this area of the brain has been found to be more susceptible to a variety of insults.

There were some other findings that struck me as particularly intriguing; something that has been hinted at previously in other studies (or transcripts), but not yet well described, likely due to the fact that the area is still largely unknown to us.  Specifically, the authors reported a state of glial activation, somewhat the opposite of what they expected.

The data instead suggest that EE changes the phenotype of glia, altering their activation and attenuating their pro-inflammatory response to peripheral LPS, although this remains to be directly tested. Interestingly, the blunted neuroinflammatory response within the DG of EE rats occurring concomitant with the increase in classical glial ‘‘activation’’ markers runs counter to our initial prediction. However, we believe these data simply highlight the fact that little is known about the function of these markers. Moreover, there is a growing literature that distinguishes classical versus alternative activation states in microglia, the latter of which is associated more strongly with repair (Colton, 2009; Colton and Wilcock, 2010).

And

Thus, it is possible that EE shifts microglia into an alternatively activated phenotype, an intriguing possibility that we are currently exploring.

(Totally sweet!)

The authors discuss the fact that their findings were highly spatially specific within the brain, involved a subset of cytokines and chemokines, and environmental enrichment did not seem to affect immune response in the periphery.

The immune response within the hippocampi of EE rats was markedly attenuated for a subset of cytokines and chemokines measured in our study. Importantly, not all measured immune molecules were blunted in the hippocampi of EE rats. Furthermore, the immune response was similar for each housing group in the parietal cortex as well as in the periphery. Within the hippocampus, however, EE rats had an attenuated response of interleukin-1b (IL-1b), the TNF family of genes, and several chemokines involved in the recruitment of leukocytes and monocytes. These families of genes indicate an altered hippocampal milieu in EE rats that may be less pro-inflammatory, more neuroprotective and less permeable to peripheral infiltrating immune cells.

There is a short discussion on the existing knowledge concerning IL-B and TNF-alpha in normal and pathological conditions, and how these findings are consistent with other findings involving environmental enrichment and cognition.

Tumor necrosis factor alpha (TNFa) is well characterized for its roles in inflammation and host defense, sepsis and, most intriguing for this study, apoptosis cascades (for review, see Hehlgans and Pfeffer, 2005). The observed attenuation after an immune challenge of TNFa and several associated genes in EE rats compared to HC controls indicates a potential enduring change in the hippocampal microenvironment of enriched rats, such that one mechanism by which EE may increase neuroprotection following insults to the CNS (Briones et al., 2011; Goldberg et al., 2011; Young et al., 1999) is via altered TNF tone and function, increasing the likelihood of cell survival by reducing apoptotic signaling. In addition to attenuated IL-1b and TNF responses, EE rats showed blunted responses for several chemokines known to influence the recruitment of circulating monocytes and leukocytes to the CNS.

Finally, the authors conclude how their findings add to the literature on environmental enrichment and brain function.

In summary, environmental enrichment is a relatively simple manipulation that results in robust beneficial outcomes for the brain. While previous studies have shown a role in post-insult rehabilitation for EE, our study provides evidence that enrichment need not follow the insult in order to be beneficial. Indeed, neuroinflammatory disease states might be attenuated or delayed in their onset in the face of ongoing EE. The translational reach of this manipulation remains to be explored, but in animal models of neuroinflammation, EE may provide a simple preventative measure for negative outcomes.

The bottom line is that a fuller rat life experience resulted in different neuroimmune profiles, findings with some consistency with previous observations that an enriched rat house resulted in improved behavioral manifestations of cognitive performance.  The qualities of these different neuroimmune profiles are also consistent with chemical profiles associated with positive outcomes in several conditions.

There is a deceivingly startling realization hidden in these finding, startling because it reveals the malleable nature of the seemingly different, but basic systems interacting and deceptive because it is so obvious.   How many of us have known someone who deteriorated upon entering a nursing home, or even retiring from working?  How many of us have kept their children inside for a week due to weather and watched their children go crazy after the already inferior indoor entertainment options are long exhausted?  Those changes in emotion, in behaviors and function, just like the findings from this study, are founded by chemistry.

But seeing evidence that relatively simple environmental modifications can rejigger the molecular atmosphere of the brain is still more than a little awe inspiring.   Knowing there is machinery underneath the hood is a little different than observing the cogs of cognition swell , shrink, or slow down; nothing less than a deeper understanding of the chemical basis of thought.  And that is pretty cool.

–          pD

Hello friends –

My son was a ‘gut kid’.  The irony is, for a while, because we were first time parents, we didn’t even know.  My son was flagged for evaluation for autism around a year of age and we met with the autism center people several times between his first and third birthdays, with his official diagnosis coming just after he turned three.  My wife came home from one of the early meetings convinced that his evaluators didn’t know the first thing about our son, autism, or anything else, and that in fact, they might be insane.

 ‘Do you know what those idiots asked me today?’

‘What?’

‘What his shits look like.  My kid can’t talk and they want to ask me about his diapers!’

‘Who fucking cares?

We wound up caring, a lot.  It turns out, this wasn’t a stupid question, it just seemed like one to us.   The answer to their question was that our son was having at least four or six very messy diapers a day, his stools were never firm logs that look like they came from an spherical filter, but always, always more liquid than solid, and frequently contained chunks of identifiable food.  But from our viewpoint, within the context of a child who was not speaking,  hurting himself, and never looked at anyone, the idea that we should be worrying about his shit was the stupidest thing we’d ever heard. 

But.  When we started paying attention, starting reading, and started meeting more people with children with autism, our incredulity waned.  We  tried GF/CF and probiotics.  We paid for lab tests to analyze the bacterial populations in his intestines. We experienced a life saving miracle with anti-fungal agents wherein my son essentially stopped hurting himself over the course of weeks after persistently banging his head dozens of times a day for six months.  For nearly a year we removed all complex carbohydrates from our son’s diet, an intervention that makes GFCF feel like a Sunday afternoon after college but before kids and autism.  We saw changes in our son based on how his GI tract was performing.  For our son, for us, we knew that by some mechanism, what got put in his mouth, and what happened along the way was tightly coupled with how our son felt and behaved.

This is why my vision with spots of rage when I see the ideas of GI and dietary involvement with autism mocked by pseudo-skeptics so rampantly on the Internet.  I cannot stand the thought of a single child continuing to suffer the way I watched my son suffer because they were told that there was no basis of GI interaction in autism.  That thought hurts.

Those biases stated, we are now, finally, starting to see research indicating that in some cases of autism, there are very real, non imaginary differences in GI function.

A few months ago, Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances was published [full, dense, but very cool paper available online].  Here is the abstract.

Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.

I’ll admit it.  From the outside, from the don’t-have-a-kid-with-autism-and-GI-problems perspective, that is some dense and seemingly bland stuff.  Essentially children with GI distress and children with GI distress and autism were compared and it was found that there were distinctly qualitative differences regarding the GI function in the groups.  This is validation of what a lot of us have been saying for a long time, that the GI problems our kids were experiencing weren’t coincidental to the autism, but somehow related.

For anyone who has been paying attention to the details of the autism-gut debate, these are dynamite findings.  These observations are the death knell for the overused, oversimplified notion that the GI connection to autism was a function of some kids having autism, some kids having GI distress, and that therefore, some kids with autism also have GI distress.  This research tells us that the reality is not so simple.

This study is the view from the microscope as opposed to the telescope, and took care not to study just anyone with an autism diagnosis, but those with an autism diagnosis and GI distress, problems so severe that invasive procedures to obtain tissue samples from the GI tract was warranted.   This is a critically important facet of the study design in my opinion, a lot of the negative findings in this arena have been epidemiological, and cast the widest possible net, capturing everyone with autism and comparing them with a sample of everyone else.  This is a great strength of the paper; for too long everyone has acknowledged the heterogeneous nature of autism, but few studies have tried to understand differences at a phenotype level.  This paper is different.

As evidence of the non-random population, the autism patient group had a regression incidence of over eighty percent, and nearly as many of the children in both groups were reported to have food allergies.

The details of the findings in the paper get deep pretty fast, but at a high level there were differences found in proteins involved with the digestion of carbohydrates and changes in bacterial populations between the groups, with some differences found with regard to specific locations in the intestine.  Based on these findings, the authors speculate that alterations in carbohydrate processing could result in abnormal bacterial populations by way of altered microbial food availability in parts of the gut.

Based on these findings, we propose a model whereby deficiencies in disaccharidases and hexose transporters alter the milieu of carbohydrates in the distal small intestine (ileum) and proximal large intestine (cecum), resulting in the supply of additional growth substrates for bacteria. These changes manifest in significant and specific compositional changes in the microbiota of AUT-GI children (see Figure 7A–C).

The authors discuss a potential feedback loop of effects of intestinal microbes and nutritional processing, and of the known and potential effects of altered bacterial populations.

Additionally, intestinal microbes can influence the expression of disaccharidases and transporters [59] through the influence of pathogen-associated molecular patterns (PAMPs) and butyrate (a byproduct of bacterial fermentation) on CDX2 expression and activity [60], [61], [62], [63]. In this regard, the observation that CDX2 was decreased in AUT-GI children with increased levels of Betaproteobacteria may be important.

Whatever the underlying mechanisms, reduced capacity for digestion and transport of carbohydrates can have profound effects. Within the intestine, malabsorbed carbohydrates can lead to osmotic diarrhea [64]; non-absorbed sugars may also serve as substrates for intestinal microflora that produce fatty acids and gases (methane, hydrogen, and carbon dioxide), promoting additional GI symptoms such as bloating and flatulence [65].

This is very similar to the underlying theory of the Specific Carbohydrate Diet, impaired carbohydrate digestion promotes bacterial imbalances in the intestine by altered food availability, leading to gastrointestinal distress.

Because of the varied nature of the protein imbalances found and absence of the common alleles associated with such conditions, the authors report that it is unlikely that the underlying cause of the imbalances is genetically based.

In our study, 93.3% of AUT-GI children had decreased mRNA levels for at least one of the three disaccharidases (SI, MGAM, or LCT). In addition, we found decreased levels of mRNA for two important hexose transporters, SGLT1 and GLUT2. Congenital defects in these enzymes and transporters are extremely rare [40], [41], and even the common variant for adult-type hypolactasia was not responsible for reduced LCT expression in AUT-GI children in this cohort. Therefore, it is unlikely that the combined deficiency of disaccharidases (maldigestion) and transporters (malabsorption) are indicative of a primary malabsorption resulting from multiple congenital or acquired defects in each of these genes.

There are a couple of ideas presented on what might be causing the altered disaccharide transporter levels, with food composition intake, immune or hormonal irregularities, and bacterial populations and their associated fermentation byproducts listed as possible candidates.  This study did not attempt to determine if any of these things were actually responsible, but an upcoming paper will also detail qualitative differences in expression of genes involved with inflammation in the autism group.

Regarding bacterial populations found, there were several differences identified by bacterial classification and location as well as some associations with onset of autistic behaviors and GI distress.

Pyrosequencing analysis of mucoepithelial bacteria revealed significant multicomponent dysbiosis in AUT-GI children, including decreased levels of Bacteroidetes, an increase in the Firmicute/Bacteroidete ratio, increased cumulative levels of Firmicutes and Proteobacteria, and increased levels of bacteria in the class Betaproteobacteria.

Stratification of AUT-GI children based on the timing of GI symptom development relative to autism onset revealed that the levels of Clostridiales and cumulative levels of Lachnospiraceae and Ruminococcaceae were significantly higher in AUT-GI children for whom GI symptoms developed before or at the same time as the onset of autism symptoms compared to AUT-GI children for whom GI symptoms developed after the onset of autism and compared to Control-GI children. However, we cannot discern whether changes in Clostridiales occurred before the onset of autism in this subgroup. We can only conclude that increased levels of Clostridiales members in biopsies taken after the development of both GI symptoms and autism are associated with the timing of GI onset relative to autism onset in this cohort. Although the reason for this association remains unclear, this finding may suggest that the timing of GI onset relative to autism is an important variable to consider in the design of future prospective studies investigating the microbiota of children with autism.

I am in love with the appreciation of the subtlety on display at the end, it may not be sufficient to simply categorize by GI and non GI autism, but also by the temporal relationship to onset of behavioral symptoms.  It makes for a messy outlook going forward, but one based on pragmatism as far as coming to valid conclusions.

As is appropriate, the authors end with an admission that we are still largely groping in the dusk about how the microbiome interacts with our tightly coupled systems, but give a variety of reasons to believe that what we do know makes system wide effects reasonable and a relationship with autism plausible.

Metabolic interactions between intestinal microflora and their hosts are only beginning to be understood. Nonetheless, there is already abundant evidence that microflora can have system-wide effects [76], [77], [78], [79], [80], [81], [82], [83] and influence immune responses, brain development and behavior [24], [25], [26], [84], [85].

No kidding!

It should be noted that this paper is a child of a 2010 IMFAR abstract, Intestinal Inflammation, Impaired Carbohydrate Metabolism and Transport, and Microbial Dysbiosis in Autism.  If I understand correctly, another paper is being prepared regarding the findings of intestinal inflammation that will be complimentary to Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances.  I’ll try to post something when it is published.

This study was small, with only twenty two participants, largely as a result of the difficulty in obtaining tissue specimens.  While this does give us cause for caution, it is important to note that this research does not exist in a vacuum, but rather, as a much larger set of research that tell us that the relationship between GI complaints and autism is more than the inceptions of DAN doctors.  Previously, Gastrointestinal abnormalities in children with autistic disorder, performed similar biochemistry and reported broadly consistent carbohydrate digestion problems, ‘Low intestinal carbohydrate digestive enzyme activity was reported in 21 children (58.3%), although there was no abnormality found in pancreatic function.’  Several other papers analyzing fecal samples have reported altered bacterial populations, including Low relative abundances of the mucolytic bacterium Akkermansia muciniphila and Bifidobacterium spp. in feces of children with autism, Gastrointestinal flora and gastrointestinal status in children with autism–comparisons to typical children and correlation with autism severity, Fecal lactoferrin and Clostridium spp. in stools of autistic children, and Pyrosequencing study of fecal microflora of autistic and control children, among others.

If the findings from this latest paper are spurious finding based on sample size problems, a lot of other studies are coincidentally finding the same type of thing in the wrong way.   Does anyone think that is likely?

I entered the autism world and online autism debate from a place of seeing with my own eyes the failures of a toddlers GI function and the difficult to overstate changes in that toddler alongside improvements in his GI health.  On one of the first autism blogs on which I participated I got into a discussion (argument?) with a blogger who I came to respect very much, but has since moved on.  I described the fact that my son had six or more diarrhea stools, a day, every day, for months on end, and that when we added dietary changes, probiotics, and later antifungal agents, the changes to his GI function were profound and impossible to misinterpret.  He told me something along the lines that humans were susceptible to illusions and sleight of hand, and I thought, ‘as if not knowing the difference between diarrhea and a log was along the lines of figuring out where the jack of spades went!’.   I couldn’t believe, could not fucking believe, someone would try to convince me that I had imagined my sons problems, and associated recovery.   This wasn’t a sugar pill study where I was asked if my child acted more or less hyperactive, this was a matter of asking myself, ‘How many diarrhea diapers did I change today?  Six?  Or Zero?’  [Repeat once a day for 180 days.]

I doubt this is necessary, but just in case, I will go on the record to state that it is easy, very easy, to tell the difference between a condition of chronic diarrhea and normal GI function.  There might not be a more simpleminded determination to make on Planet Earth or indeed, our perceptible universe.   This is a situation that is susceptible to placebo effects only in the most elaborate imaginations of people who have never experienced chronic GI problems.

From that time on, with nearly zero exceptions, I have become a little less shocked, but a little more saddened by the doublethink style skepticism applied to GI distress and autism in nearly every single conversation I have ever seen on the Internet.  I’ve put some time thinking toward this, why so many otherwise intelligent people house such extreme hostility on a relationship between GI function and autism.  I believe that the Wakefield / MMR autism debacle is at the heart of this disconnect; his ill fated and now retracted paper that launched a thousand Internet scribbles has seemingly forever tied GI complaints and autism to bad science.

It doesn’t have to be this way.  As a community, the vaccine wars and kissing cousin prevalence question has done a lot to fracture us, and very little to unite us.  That is a sad statement, and nothing makes it more unfortunate than the fact that it does not have to be this way.  Wakefield can be wrong about the MMR and there can still be very real differences in GI function in some cases of autism.  We can respectfully disagree about how well our existing prevalence studies inform us on the incidence of autism without also needing to accept a world view where every child with autism has raging bowel problems.

We should have the intellectual honesty to admit that there is nothing inherently dangerous about acknowledging what the data tells us; GI function seems to be abnormal in a subset of children with autism, and the underlying features of that GI distress are qualitatively different than what is found in ‘normal’ children.

–          pD

Hello friends –

Lately I’ve found myself reading papers and knowing and owning several of the references; tragically I can’t tell if I’m reading the right research and am onto something, or I am chasing phantoms and my web of pubmed alerts and reading interests are funneling my reference list into a narrowing echo chamber of sorts.   With that warning in mind, we can proceed to poking around several papers, only some of which mention autism per se.  Along the way, we will see evidence supporting the possibility of a biologically plausible mechanism of developmental programming of the neuroimmune environment, a sequence of events that may lead to impaired synaptic pruning in (some cases of?) autism.

By now, everyone has seen/read/heard about one form or another of the ‘a massive asteroid is going to destroy the world’ story.  One of the common survival strategies from an asteroid strike involves altering the path of the asteroid so that it misses the Earth.  The thoughtful analysis of this problem allows for the physics based reality of the problem, moving an asteroid out of an extinction based trajectory involves just a little work when the asteroid is ten thousand gazillion miles away, but a lot more work when it is only a gazillion miles away.  Upon careful evaluation living organisms display similar behavior, relatively minor disturbances in early life can alter the developmental trajectory, while that same disturbance later in life is unable to materially affect the organism beyond a transient effect.   The accumulated evidence that early life experiences can shape the adult outcome is nearly impossible to dispute with any remaining intellectual honesty, the question is instead, is how large is the effect in autism?

This analogy adequately symbolizes one of the more beautiful and terrifying concepts I’ve come across researching autism, that of ‘developmental programming’, which I blogged some about here, but essentially is the idea that there are critical timeframes during which environmental impacts can have long term persistent effects on a wide range of outcomes.  The most robustly replicated findings involve changes to metabolic profiles in response to abnormal prenatal nutritional environments, but there is also evidence of various other effects, including neurological, and reputable speculation, that autism, may in fact, be in part, a disorder of developmental programming.

Secondarily, there has long been speculation of problems in the removal of ‘excess’ synapses, i.e., ‘synaptic pruning’ in the autism population.   This culling of synapses begins in fetal life continuing throughout adolescence and the repeated observations of increased head circumference during infancy as a risk factor for autism has resulted in the idea that altered synaptic pruning maybe involved in autism.

In the last month or so several rather serendipitously themed papers have been published with tantalizing clues about some of the finer grained mechanisms of synaptic pruning, the possibility of impaired synaptic pruning in the autism population, and a known risk factor for autism that models a developmental programming event sequence that may tie them together.

First off, we have Synaptic pruning by microglia is necessary for normal brain development, (Paolicelli et all) with a very straightforward title, that has this dynamite in the abstract: (snipped for length)

These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.

This paper is short, but pretty cool, and very nice from a new territory perspective.  It also speaks directly towards one of the increasingly hilarious obfuscations you will sometimes see raised in online discussions about immunological findings in autism, namely, that we can’t know if the state of chronic inflammation in the CNS observed in autism is harmful or beneficial.   [hint: It might not be causative, but it isn’t beneficial.]

Here’s is a snippet from the Introduction:

Time-lapse imaging has shown that microglia processes are highly motile even in the uninjured brain and that they make frequent, but transient contact with synapses. This and other observations have led to the hypothesis that microglia monitor synaptic function and are involved in synapse maturation or elimination.  Moreover, neurons during this period up-regulate the expression of the chemokine fractalkine, Cx3cl1, whose receptor in the central nervous system is exclusively expressed by microglia and is essential for microglia migration. If, in fact, microglia are involved in scavenging synapses, then this activity is likely to be particularly important during synaptic maturation when synaptic turnover is highest.

Nice.  A time dependent participation by microglia in the critical process of optimization of neuron numbers, a process we are still very much groping our way in the dark towards untangling.  The researchers focused in on a particular molecular target, a chemical messenger of the immune system, fractalkine, and found that without fractalkine, the process of synaptic turnover was impaired.

A couple of tests were performed, first immunohistochemistry (i.e., exceedingly clever manipulation of antibodies to determine the presence or absence of proteins in very specific locations) which demonstrated that microglia were, in fact, ‘engulfing synaptic material’ in animals during periods of synaptic maturation.

Secondly, so called ‘knock out mice’ (i.e., genetically engineered mice constructed without the ability to make a specific protein, in this case, fractalkine) were used evaluate for changes in synaptic form and function based on a lack of fractalkine.  Changes in dendritic spine density were observed in the knock out mice group, with much higher densities in a very specific type of neuron during the second and third postnatal week of life.  The authors indicate this is a key timeframe in synaptic pruning, and state their findings are “suggesting a transient deficient synaptic pruning in Cx3cr1 knockout mice “.  The effect of not having fractalkine on spine density was time dependent as shown below.

Several other measurements were taken, including synaptic firing frequencies, which also implicated an increased surface area for synapses on dendritic spines, consistent with impaired pruning.  Time dependent effects on synaptic efficiency and seizure susceptibility were also found, which the led the authors to conclude that the findings were “consistent with a delay in brain circuit development at the whole animal level.”

For additional evidence of fractalkine participation in synaptic maintenance, we can look to the opposite direction, where researchers evaluating neuron loss in an Alzheimers model reported “Knockout of the microglial chemokine receptor Cx3cr1, which is critical in neuron-microglia communication, prevented neuron loss”.  Taken together, the conclusion that fractalkine processing is involved with neuron maintenance is highly likely, and correspondingly, highly unlikely to be a set of spurious findings.

There’s a couple paragraphs on potential mechanisms by which fractalkine could be interacting with microglia to achieve this effect, with the authors claiming that their data and other data generally supports a model wherein microglia were not effectively recruited to appropriate locations in the brain due to a lack of fractalkine, or, a ‘transient reduction in microglia surveillance.’

The conclusion is a good layman level wrap up that speaks toward the Interconnectedness of the brain and the immune system:

In conclusion, we show that microglia engulf and eliminate synapses during development. In mice lacking Cx3cr1, a chemokine receptor expressed by microglia in the brain, microglia numbers were transiently reduced in the developing brain and synaptic pruning was delayed. Deficient synaptic pruning resulted in an excess of dendritic spines and immature synapses and was associated with a persistence of electrophysiological and pharmacological hallmarks of immature brain circuitry. Genetic variation in Cx3cr1 along with environmental pathogens that impact microglia function may contribute to susceptibility to developmental disorders associated with altered synapse number. Understanding  microglia-mediated synaptic pruning is likely to lead to a better understanding of synaptic homeostasis and an appreciation of interactions between the brain and immune system

That’s all pretty cool, but there was precious little discussion of autism, except in the general sense of a ‘developmental disorder associated with altered synapse number’.   [But the references do speak to autism, the first reference provided, Dendritic Spines in Fragile X Mice displays a significant relationship to autism, and it describes how another flavor of knock out mice, this time designed to mimic Fragile-X, exhibit a ‘developmental delay in the downregulation of spine turnover and in the transition from immature to mature spine subtypes.’  Go figure!]

The other reason Paolicelli is of particular interest to the autism discussion is one of the major players in this study, the microglia (i.e., the resident immune cells of the CNS), have been found to be ‘chronically activated’ in the autism brain by direct  measurement in two studies (here, and here, [and by me, here]), and tons of other studies have shown indirect evidence of an ongoing state of immunological alertness in the autism brain.

Considering this is a brand new paper, I do not believe that there are any studies illuminating the results of a state of chronic activation of microglia on the process of synaptic pruning per se.  I will, however, go on the record that such an effect is very, very likely, and the logical leap is microscopically small that there will be some detrimental impact to such a state.  The inverse argument, a scenario wherein there could be a state of chronic microglial activation that does not interfere with microglia participation in the synaptic pruning requires logical acrobatics worthy of Cirque Du Soleil.  I am open to evidence, however.

So, from Paolicelli, we know that a ‘transient reduction in microglial surveillance’ induced by a reduction in the ability to production fractalkine can result in a condition ‘consistent with a delay in brain circuit development at the whole animal level’.

Next up, we have a paper that was all over the JerkNet in the days and weeks following its release, Neuron number and size in prefrontal cortex of children with autism.  This is a cool study, and likely a very important paper, but I must say that a lot of the online commentary exhibits an irrational exuberance towards one part of the findings.   Here is part of the abstract.

Children with autism had 67% more neurons in the PFC (mean, 1.94 billion; 95% CI, 1.57-2.31) compared with control children (1.16 billion; 95% CI, 0.90-1.42; P = .002), including 79% more in DL-PFC (1.57 billion; 95% CI, 1.20-1.94 in autism cases vs 0.88 billion; 95% CI, 0.66-1.10 in controls; P = .003) and 29% more in M-PFC (0.36 billion; 95% CI, 0.33-0.40 in autism cases vs 0.28 billion; 95% CI, 0.23-0.34 in controls; P = .009). Brain weight in the autistic cases differed from normative mean weight for age by a mean of 17.6% (95% CI, 10.2%-25.0%; P = .001), while brains in controls differed by a mean of 0.2% (95% CI, -8.7% to 9.1%; P = .96). Plots of counts by weight showed autistic children had both greater total prefrontal neuron counts and brain weight for age than control children.  [PFC == prefrontal cortex]

Essentially the authors used a variety of mechanisms to measure neuron number in a specific area of the brain, the prefrontal cortex, and found large variations (increases) in the autism group.   The prefrontal cortex is thought to be involved in ‘planning complex coginitive behaviors’, and ‘moderating correct social behavior’, among others, so this was a smart place to look.

The implicit hype on the internet is that this firmly indicates a ‘prenatal cause’ to autism, but if you read the paper, read what Courchense has said, and read recent literature, you know that the simplicity of this as a singular prenatal cause of autism is long broad strokes, and short on appreciation of the subtlety that textures reality.

A link @ LBRB sent me to the team at The Thinking Person’s Guide To Autism, who had a very nice transcription of a talk given by Courchesne at IMFAR 2011.  Here is a snipet that started my wheels turning.

What we see in autism is either an excess proliferation, producing an overabundance of neuron numbers, or the excess might be due to a reduced ability to undergo naturally occurring cell death. Or it could be both. We don’t know which and our data don’t speak to that, although our data do suggest that it’s probably both.

Finally, our evidence shows that across time, there’s a prolonged period of apoptosis, removal and remodeling of circuits. In order to get back to where neuron numbers are supposed to be, it takes a very long time for the autistic brain. In the normal developing brain, this takes just a few months. In autism, it’s a couple of decades.

[Note how well this fits within the model described by Paolicelli, i.e., “consistent with a delay in brain circuit development at the whole animal level”.  ]

I would highly recommend anyone who has read this far to go read the entire post @ TPGTA sometime.

As far as synaptic pruning goes, here is the associated segment of the paper:

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. A failure of subplate apoptosis might additionally indicate abnormal development of the subplate itself. The subplate plays a critical role in the maturation of layer 4 inhibitory functioning as well as in the early stages of thalamocortical and corticocortical connectivity development.inhibitory functioning and defects of functional and structural connectivity are characteristic of autism, but the causes have remained elusive.

Nearly half of the neurons in the area studied are expected to be removed through pruning, a process that extends well after birth.  That is something that you didn’t see referenced in too many places trumpeting this study as ‘proof’ that autism was caused by disturbances in the prenatal environment.  I’m not coming down on the prenatal environment as a critical timeframe for autism pathogensesis, just the difficult to defend underlying notion that this is the only time the environment should be evaluated, or the idea that if something is initiated prenatally other timeframes are therefore, unimportant.

So, I’d read that microglia were actively involved in proper synaptic pruning, contingent on utilization of fractalkine, and then read that impaired synaptic apoptotic mechanisms could be participating in autism, with a consequence of an over abundance of neurons.

Then, I got myself a copy of Microglia and Memory: Modulation by Early-Life Infection, which is another study in a growing body of evidence that immune challenges early in life can have unpredictable physiological consequences.  (This is another very cool paper with Staci Bilbo as an author, whom I think is seriously onto something.)  This study, in particular, focused on interactions microglia and formation of memories.   Here is the abstract:

The proinflammatory cytokine interleukin-1ß (IL-1ß) is critical for normal hippocampus (HP)-dependent cognition, whereas high levels can disrupt memory and are implicated in neurodegeneration. However, the cellular source of IL-1ß during learning has not been shown, and little is known about the risk factors leading to cytokine dysregulation within the HP. We have reported that neonatal bacterial infection in rats leads to marked HP-dependent memory deficits in adulthood. However, deficits are only observed if unmasked by a subsequent immune challenge [lipopolysaccharide (LPS)] around the time of learning. These data implicate a long-term change within the immune system that, upon activation with the “second hit,” LPS, acutely impacts the neural processes underlying memory. Indeed, inhibiting brain IL-1ß before the LPS challenge prevents memory impairment in neonatally infected (NI) rats. We aimed to determine the cellular source of IL-1ß during normal learning and thereby lend insight into the mechanism by which this cytokine is enduringly altered by early-life infection. We show for the first time that CD11b+ enriched cells are the source of IL-1ß during normal HP-dependent learning. CD11b+ cells from NI rats are functionally sensitized within the adult HP and produce exaggerated IL-1ß ex vivo compared with controls. However, an exaggerated IL-1ß response in vivo requires LPS before learning. Moreover, preventing microglial activation during learning prevents memory impairment in NI rats, even following an LPS challenge. Thus, early-life events can significantly modulate normal learning-dependent cytokine activity within the HP, via a specific, enduring impact on brain microglial function.

Briefly, the authors infected rats four days after birth with e-coli, and then challenged them with LPS in adulthood to simulate the immune system to evaluate if memory formation was affected.   As further evidence of an immune mediated effect, prevention of microglial activation in adulthood was sufficient to attenuate the effect.  Clearly the effect on memory formation was based on the immune system.  (Note:  Most of the studies I’ve read would indicate [i.e., educated guess] that a four day old rat is brain developmentally similar to the third trimester of a human fetus.)  While a terrifying and beautiful expression of developmental programming in its own right, there isn’t much to speak towards synaptic pruning in this paper, except maybe, potentially, one part of their findings.

In our study, CX3CL1 did not differ by group, whereas its receptor was decreased basally in NI rats, implicating a change at the level of microglia.

This is where things get either highly coincidental, or connected.  CX3CL1 is another name for fractalkine, i.e., animals that were infected in early life had decreased expression of the receptor for fractalkine compared to placebo animals, i.e., fractalkine is the same chemical messenger found to be integral in the process of synaptic pruning in Synaptic pruning by microglia is necessary for normal brain development!  From a functionality standpoint, having less receptor is very similar to having less fractalkine; as the animals in Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease tell us.

If, if synaptic apoptotic processes are impaired in autism, perhaps this is one mechanism of action. The timeline would involve a prenatal immune challenge, which causes a persistent decrease fractalkine receptor expression, which in turn, causes a consequent impairment in synaptic pruning through interference in microglial targeting.  There is near universal agreement that immune disturbances in utero are capable of altering developmental trajectory undesirably, and here, in an animal model, we have evidence that infections are capable of reducing availability of receptors of ligands known to play a critical role in synaptic pruning, the absence of which leads to conditions which are “consistent with a delay in brain circuit development at the whole animal level”. 

Only time, and more research, will tell if this is a pattern, a phantom, or a little of both.

–          pD


Hello friends –

One of the more beautiful and terrifying concepts I’ve come across in the last year or so is the idea of ‘developmental programming’, or sometimes fetal programming, or as I imagine it will eventually be recognized, the realization of subtle change is still change, and subtle change during critical timeframes can amplify into meaningful outcomes.  The underlying hypothesis is that environmental influences during early life, gestation, infancy, or even childhood, have the capacity to permanently influence physiological and behavioral state into adulthood.  The available evidence implicates the potential for developmental programming to be involved with an assortment of conditions that on the whole, you’d rather not have than have, including the spectrum sized set of disorders grouped as ‘metabolic syndrome’ that incorporates several risk factors for cardiovascular disorders, obesity, type II diabetes.  There is also less pronounced evidence for some autoimmune disorders, and perhaps, autism. 

Here is the most concise explanation of developmental programming I’ve seen so far, from Developmental Programming of Energy Balance and Its Hypothalamic Regulation

The concepts of nutritional programming, fetal programming, fetal origins of adult disease, developmental origins of health and disease, developmental induction, and developmental programming were all conceived to explain the same phenomenon: a detrimental environment during a critical period of development has persistent effects, whereas the same environmental stimulus outside that critical period induces only reversible changes.

I am absolutely in love with the importance of time dependent effects, a sort of combo pack of why the dose doesn’t always make the poison, and the importance of understanding subtle interactions in developing systems. 

The area of developmental programming that has a ton of research in the human field and animal models is the link between metabolic syndrome and a differently structured uterine and/or early postnatal environment.  A nice review from 2007, Developmental programming of obesity in mammals (full paper) has this:

Converging lines of evidence from epidemiological studies and animal models now indicate that the origins of obesity and related metabolic disorders lie not only in the interaction between genes and traditional adult risk factors, such as unbalanced diet and physical inactivity, but also in the interplay between genes and the embryonic, fetal and early postnatal environment. Whilst studies in man initially focused on the relationship between low birth weight and risk of adult obesity and metabolic syndrome, evidence is also growing to suggest that increased birth weight and/or adiposity at birth can also lead to increased risk for childhood and adult obesity. Hence, there appears to be increased risk of obesity at both ends of the birth weight spectrum.

And

Childhood and adult obesity are amongst the cardiovascular risk factors now considered to be ‘programmed’ by early life and, perhaps counter-intuitively, babies subjected either to early life nutritional deprivation or to an early environment over-rich in nutrients appear to be at risk. Supportive evidence includes the observation of a ‘U-shaped’ curve which relates birthweight to risk of adult obesity (Curhan et al. 1996).

[Check out that example of a hormetic dose curveTotally sweet!]

The list of papers supporting a link between abnormal gestational or birth parameters and subsequent obesity in the offspring is very, very voluminous.   The satellite level high view of the research starts with Dutch mothers during a time of famine, and the observations that these children were much more likely to be obese at nineteen in Obesity in young men after famine exposure in utero and early infancy.  Later, infants in England were found to have birth weight positively correspond to adult weight in Birth weight, weight at 1 y of age, and body composition in older men: findings from the Hertfordshire Cohort Study (full paper).  A study with twin pairs, Birth weight and body composition in young women: a prospective twin study  had similar findings, but with the additional coolness factor of being able to detect differences between genetically identical twins who happened to be born at different weights.  There are studies on infants that are born light but then ‘catch up’are consistently more likely to be obese, a review of which can be found in Rapid infancy weight gain and subsequent obesity: systematic reviews and hopeful suggestions.  Startlingly, Weight Gain in the First Week of Life and Overweight in Adulthood observed that formula fed babies who gained considerable weight during the first eight days after birth were more likely to be obese as adults, similar to other findings implicating formula fed babies with adult obesity.

Therearealsoconservativelya bazillionanimalmodelsthattellusthatthestudiesin humans are accurate.

Part of me hates the deterministic nature of these findings, it’s really just an extension of the fatalism of genetic assignment, but on the other hand, the data is the data.  I must admit, I am in love with the underlying evolutionary cleverness of the thrifty phenotype end of the U curve on display; a fetus or neonate that is deprived of nutrients, or perhaps, some types of nutrients, programs itself for an environment in which food is scarce, handling calories differently at a very fine grained metabolic level.  From a survival standpoint this modification is most definitely the smart move; all inbound indicators are signaling to the fetus that calorie acquisition is going to be tough on the outside, and as a result, the physiology is tweaked so that baby is ready to make the absolute most of any available nutrients.  If that child, however, is raised in a world with plentiful calories, if not always, beneficial calories, they tend to store fat more readily than a baby/child/adult that did not receive the same messages in utero.  Neat.

Like lots of things I seem to be running into, our observations of what is happening seem to be more advanced than our understanding of how it is happening.  The ideas of developmental programming have been around for a while, but we are still very much in the learning phase regarding mechanism of action, a very thorough review that I ran into can be found here:  Mechanisms of developmental programming of the metabolic syndrome and related disorders.   (full paper). 

Another example of programming a bit closer to home to the autism world has been in the news lately, namely the replication of findings that children who grow up around farm animals, or in some cases, pets, are less likely to suffer from allergies and /or asthma than children who grow up without that exposure.  These findings are also very robust, and appear to implicate similar critical developmental timeframes including the gestational environment, infancy, and toddlerhood. 

Here is an example of the kind of thing in this area,  Farming environment and prevalence of atopy at age 31: prospective birth cohort study in Finland

Cross-sectional studies have shown an association between the farming environment and a decreased risk of atopic sensitization, mainly related to contact with farm animals in the childhood. Objective Investigate the association of a farming environment, especially farm animal contact, during infancy, with atopic sensitization and allergic diseases at the age of 31. Methods In a prospective birth cohort study, 5509 subjects born in northern Finland in 1966 were followed up at the age of 31. Prenatal exposure to the farming environment was documented before or at birth. At age 31, information on health status and childhood exposure to pets was collected by a questionnaire and skin prick tests were performed. Results Being born to a family having farm animals decreased the risk of atopic sensitization [odds ratio (OR) 0.67; 95% confidence interval (CI) 0.56-0.80], atopic eczema ever (OR 0.77; 95% CI 0.66-0.91), doctor-diagnosed asthma ever (OR 0.74; 95% CI 0.55-1.00), allergic rhinitis at age 31 (OR 0.87; 95% CI 0.73-1.03) and allergic conjunctivitis (OR 0.86; 95% CI 0.72-1.02) at age 31. There was a suggestion that the reduced risk of allergic sensitization was particularly evident among the subjects whose mothers worked with farm animals during pregnancy, and that the reduced risk of the above diseases by farm animal exposure was largely explained by the reduced risk of atopy. Having cats and dogs in childhood revealed similar associations as farm animals with atopic sensitization. Conclusion and Clinical Relevance Contact with farm animals in early childhood reduces the risk of atopic sensitization, doctor-diagnosed asthma and allergic diseases at age 31.

That is one hell of a long running study and the findings are consistent with a wealth of similar studies across populations, including Exposure to environmental microorganisms and childhood asthma, and Effect of animal contact and microbial exposures on the prevalence of atopy and asthma in urban vs rural children in India.  These findings are part and parcel with the Hygiene Hypothesis, the idea that a relative reduction in ‘training’ of the immune system can lead to disturbances in normal immune system development and consequent development of autoimmune disorders.   (Here’s a nice review of the evidentiary backing for the Hygiene Hypothesis) From a clinical viewpoint, there are reasons to suspect this is a biologically plausible pathway; in Environmental exposure to endotoxin and its relation to asthma in school-age children the researchers reported an inverse relationship between the amount of endotoxin (i.e., a bacterial fingerprint that is recognized by the immune system) and the immune  response, stating, “Cytokine production by leukocytes (production of tumor necrosis factor alpha, interferon-gamma, interleukin-10, and interleukin-12) was inversely related to the endotoxin level in the bedding, indicating a marked down-regulation of immune responses in exposed children.”  We can also see immunomodulatory effects of farm or rural living in the cytokine profiles of breast milk between two populations, as reported in Immune regulatory cytokines in the milk of lactating women from farming and urban environments, which found much higher concentrations of TGF-Beta1, a critical immune modulator, in breastmilk and collustrum of ‘farm mothers’.  The concentration of TGF-Beta1 in breastmilk had already been implicated in infant development of atopic disease in Transforming growth factor-beta in breast milk: a potential regulator of atopic disease at an early age

The evidence supporting developmental programming in these instances is very problematic to overcome, clearly there are mechanisms by which the events of very early life can cause persistent changes to physiology into adulthood; be they changes ‘designed’ to be adaptive, or disturbed trajectories of usually tightly regulated systems that find inappropriate targets in an environment different than what our ancestors evolved in.  I’d note that none of what is above invalidates any findings of genetic involvement with cardiovascular problems, obesity, or asthma, but it should serve as a portrait of how genetic recipes are only part of the process. 

So, what about autism?  This is, admittedly, where things get a bit more speculative, there isn’t the same type of epidemiological evidence in the autism arena as what we see above.  Part of this discrepancy is an artifact of the fuzzy nature of autism, a bazillion different conditions each with their own personalized manifestation, a much more daunting set of variables to detangle compared with measuring BMI, triglyceride levels or asthma.  Those caveats in place, there is still room to discuss some potential examples wherein early life experiences might be participating in ‘programming’ some of what we see in autism. 

A nice review paper that speaks directly towards a developmental programming model that involves autism is Early life programming and neurodevelopmental disorders that includes as an author, Tom Insel, head of the National Institute of Mental Health, and generally, one of the good guys.   This is part of the abstract.

Although the hypothesized mechanisms have evolved, a central notion remains: early life is a period of unique sensitivity during which experience confers enduring effects. The mechanisms for these effects remain almost as much a mystery today as they were a century ago (Insel and Cuthbert 2009). Recent studies suggest that maternal diet can program offspring growth and metabolic pathways, altering lifelong susceptibility to diabetes and obesity. If maternal psychosocial experience has similar programming effects on the developing offspring, one might expect a comparable contribution to neurodevelopmental disorders, including affective disorders, schizophrenia, autism and eating disorders. Due to their early onset, prevalence and chronicity, some of these disorders, such as depression and schizophrenia, are among the highest causes of disability worldwide (World Health Organization, 2002). Consideration of the early life programming and transcriptional regulation in adult exposures supports a critical need to understand epigenetic mechanisms as a critical determinant in disease predisposition.

 

A concise explanation of the concept of developmental programming and the need for more finely detailed understandings of the likely epigenetic underpinnings.  Also included is a discussion of things like maternal stress during gestation, childhood environmental enrichment (or more specifically, ‘de-enriched’ or otherwise, terrible situations), and prenatal infection models.  Nice.  

What about specifics for the autism arena?  One environmental event that most everyone agrees can increase risk of an autism diagnosis is an immune challenge in the gestational period.  The animal models are robust and have been replicatedacross laboratories and epidemiological data supports an association.  A lot of groups have been studying the effects of maternal immune activation in animal models the past few years, what we can see are some striking parallel veins to what is observed in autism that involve the concept of developmental programming. 

One paper, with a title I love, is  Neonatal programming of innate immune function.  Here is a snipet of the abstract from the first paper:

There is now much evidence to suggest that perinatal challenges to an animal’s immune system will result in changes in adult rat behavior, physiology, and molecular pathways following a single inflammatory event during development caused by the bacterial endotoxin lipopolysaccharide (LPS). In particular, it is now apparent that neonatal LPS administration can influence the adult neuroimmune response to a second LPS challenge through hypothalamic-pituitary-adrenal axis modifications, some of which are caused by alterations in peripheral prostaglandin synthesis. These pronounced changes are accompanied by a variety of alterations in a number of disparate aspects of endocrine physiology, with significant implications for the health and well-being of the adult animal.

Another very cool, and very dense, paper with a salient title and content by the same group is  Early Life Activation of Toll-Like Receptor 4 Reprograms Neural Anti-Inflammatory Pathways (full paper) which reports that a single early life immune challenge results in persistently altered response to immune stimulants into adulthood, with differential responses in the CNS compared to the periphery.  Especially interesting in this paper is that the researchers have dug down a layer into the biochemical changes affected by early life immune challenge and found that alterations to HPA-Axis metabolites are responsible for the changes. 

Tinkering around with the HPA-Axis, an entangled neuroendicrine system that touches on stress response, immune function, mood, and more can have a lot of disparate effects.  It turns out, there is evidence that early life immune challenges can also modify behaviors in a way consistent with altered stress responses.

For example, the very recently published Peripheral immune challenge with viral mimic during early postnatal period robustly enhances anxiety-like behavior in young adult rats has a short, but to the point abstract:

Inflammatory factors associated with immune challenge during early brain development are now firmly implicated in the etiologies of schizophrenia, autism and mood disorders later in life. In rodent models, maternal injections of inflammagens have been used to induce behavioral, anatomical and biochemical changes in offspring that are congruent with those found in human diseases. Here, we studied whether inflammatory challenge during the early postnatal period can also elicit behavioral alterations in adults. At postnatal day 14, rats were intraperitoneally injected with a viral mimic, polyinosinic:polycytidylic acid (PIC). Two months later, these rats displayed remarkably robust and consistent anxiety-like behaviors as evaluated by the open field/defensive-withdrawal test. These results demonstrate that the window of vulnerability to inflammatory challenge in rodents extends into the postnatal period and offers a means to study the early sequelae of events surrounding immune challenge to the developing brain.

The methodology is very similar to what we see in a lot of animal models of early life immune activation, convince a young animals immune system that they are under microbial attack by mimicking either bacterial or viral invaders, and then measure behaviors, or physiology, later in life. This study could be seen as a complement to a much earlier (2005) paper, Early life immune challenge–effects on behavioural indices of adult rat fear and anxiety, which used a different immune stimulant (bacterial fingerprint/LPS versus viral fingerprint/Poly:IC), but which found generally consistent results.

There are more, for example, Early-Life Programming of Later-Life Brain and Behavior: A Critical Role for the Immune System (full paper), which reviews animal study evidence that early life immune challenges can have lifelong effects.  Here is part of the Introduction:

Thus, the purpose of this review is to: (1) summarize the evidence that infections occurring during the perinatal period can produce effects on brain and subsequent behavior that endure throughout an organism’s life span, and (2) discuss the potential role of cytokines and glia in these long-term changes. Cytokines are produced within the brain during normal brain development, but are expressed at much higher levels during the course of an immune response. In contrast to overt neural damage, we present data indicating that increased cytokine exposure during key periods of brain development may also act as a “vulnerability” factor for later-life pathology, by sensitizing the underlying neural substrates and altering the way that the brain responds to a subsequent immune challenge in adulthood. In turn, this altered immune response has significant and enduring consequences for behavior, including social, cognitive, and affective abilities. We discuss the evidence that one mechanism responsible for enduring cytokine changes is chronic activation of brain microglia, the primary immunocompetent cells of the CNS.

Check that out!  We have several papers showing, indeed, a ‘chronic activation of brain microglia’ in the autism population; one way, it seems, to achieve this, is ‘increased cytokine exposure during key periods of brain development’.  (Ouch!) 

Is developmental programming the mechanism by which gestational immune activation raises the risk of autism?  I don’t think we can answer that question with any authority yet, but the logical jumps to arrive at that conclusion are small, and  are supported by a great deal of evidence.  No doubt, we’ll be learning more about this in the years to come.

Ultimately, I think what all of this means is that, as usual, there is another layer of complexity thrown into the mix.  As far as autism goes, it seems likely that at least some of our children are manifesting behaviors consistent with autism as a result of things that happened to them very, very early in their life.  Figuring out if this is happening, how it is happening, and to which individuals, is a daunting, very difficult task; but at least we are approaching a level of knowledge to allow for such an endeavor.

This posting focused on the bad stuff, but the inverse is just as meaningful, having a ‘normal’ gestational period as far as nutrients go, programs you towards a more healthy weight, and being born to a mother exposed to a variety of microbial agents, as the overwhelming majority of mothers were for most of human existence, programs you away from asthma.  But from a broader standpoint, from a ‘every human on the planet’ view, I think we must begin to recognize that everyone is being programmed, in some ways for good, in others, for not so good.  Curiosity and thoughtful analysis is our way to illuminate the beautiful and dispassionate gears that propel the machinations of nature; developmental programming is one of the cogs in the natural world, hopefully, one day, we will acquire the wisdom to refine the program for our benefit, but in the meantime, it is still exciting to witness the discovery of the inner workings.

          pD

Hello friends –

I have decidedly mixed feelings on the genetic side of autism research; clearly genetics plays a part, but it does appear that autism has largely mirrored other complicated conditions in that what we thought we were getting when we cracked the genetic code has, for all practical purposes, failed to materialize.  To what extent our genetic makeup really plays a part in autism more than any other condition that is currently mystifying us, I don’t think we can say with much certainty; unless you want to count some.

To my mind, one particularly bright spot in the gene realm is the associations of the MET-C allele and an increased risk of an autism diagnosis.  At first glance, MET doesn’t seem like a big deal; lots of people have the MET-C mutation, in fact, nearly half of everyone has it.   But people with autism have it just a little more frequently, an observation that has been replicated many times.  But what is exciting is not only that the MET-C findings are robust, but they can also affect a lot of implicated systems in autism in biologically relevant ways.  From an ideological standpoint, the fissure in the autism community about research priorities regarding genetics versus environment, the MET-C studies are a superb example of just how much useful knowledge there is by starting at the genome and working upwards, and finding once we get there that the reality involves lots more than just genes.  There is something for everyone!

Getting to the big picture where we can appreciate the beautiful complexity takes a little bit of digging, but it’s worth the effort. 

Every now and again you’ll see a period piece about the forties, fifties or sixties, and you’ll get a glimpse of the female operator, someone who would take a call and literally connect two parties together; the gatekeeper. The operator’s actions were binary; either she connected the lines and the call went through, or she didn’t, and nothing happened.  Of course, one operator couldn’t connect you to any other phone, but participated in groupings of phones with some logical or functional structure.  Ultimately, the operators were the enabler of communication, physically putting two entities into contact to perform whatever business they had with each other. 

Within our bodies, tyrosine kinases  are enzymes responsible transferring phosphate to proteins; a chemical exchange critical towards a great number of cellular functions, and in a sense, the tyrosine kinases act as cellular operators, helping implement a physical swap of chemicals that ultimately set in motion a great number of processes.  Some very rudimentary cellular functions are initiated by the tyrosine kinases; for example, cell division, which is why mutated kinases can lead to the generation of tumors; i.e., the signaling for cell division gets turned on, and never gets turned off.  Inhibiting tyrosine kinases is the mechanism of action for some drugs that target cancer.  

The MET gene is responsible for creating the MET receptor tyrosine kinase.  This particular receptor is involved in lots of processes that are of great interest to autism; the MET receptor is expressed heavily during embryogenesis in the brain, has immune modulating capacities, and is associated with wound healing, and is particularly implicated in repair of the gastro-intestinal track. 

Kinases don’t just fire away, shuttling phosphates around any old time, they must be activated by a triggering molecule, or a ligand.  There is only one known ligand for the MET receptor; hepatocyte growth factor, or HGF (also sometimes referred to as HGF/SF, or hepatocyte growth factor/scatter factor).  We’ll get to why we bother worrying about HGF a little later on, but it is important to keep in mind that without HGF, the functions affected by the MET-C receptor, early brain development, immune modulating, and wound repair cannot be achieved. 

So what about autism, and why is it a beautiful illustration of complexity?  Walking our way through the MET findings in autism is a rewarding task; it is one of the few instances I’ve seen where the glimpses of relevance gleaned from straight genetic studies have been incrementally built upon to achieve a much grander understanding of autism.  This is the kind of thing that I think a lot of people who dismiss the utility of genetic studies are missing; genetics are only the first piece of the puzzle, it doesn’t only implicate genes, it tells us about the processes and the proteins disturbed in autism; and with that knowledge, we can perform targeted analysis for environmental participants.

The first clues about MET involvement with autism came in 2006, when A genetic variant that disrupts MET transcription is associated with autism (full paper) was published.  The abstract is longish, but here is a snipet:

MET signaling participates in neocortical and cerebellar growth and maturation, immune function, and gastrointestinal repair, consistent with reported medical complications in some children with autism. Here, we show genetic association (P = 0.0005) of a common C allele in the promoter region of the MET gene in 204 autism families. The allelic association at this MET variant was confirmed in a replication sample of 539 autism families (P = 0.001) and in the combined sample (P = 0.000005). Multiplex families, in which more than one child has autism, exhibited the strongest allelic association (P = 0.000007).

I appreciate the pleiotropic nature of what we are seeing here, a gene that is involved with brain growth and maturation, immune function, and GI repair.  The association in ‘multiplex’ (i.e., families with more than one child with autism) was very, very strong.  Even still, this was a pretty short paper, and it was all genetics.  Coolness factor:  3.

Neater studies were on the horizon shortly thereafter, a year later, some of the same group looked for expression of MET in post mortem brain tissue and found significantly decreased levels of MET protein in Disruption of cerebral cortex MET signaling in autism spectrum disorder

MET protein levels were significantly decreased in ASD cases compared with control subjects. This was accompanied in ASD brains by increased messenger RNA expression for proteins involved in regulating MET signaling activity. Analyses of coexpression of MET and HGF demonstrated a positive correlation in control subjects that was disrupted in ASD cases.

This is a nice follow up; lots of times a genetic study might suggest a hit, but we really don’t even know how such a genetic change might manifest physiologically, like having a jigsaw puzzle of solid black and finding two pieces that fit together.  In those instances, we can’t really go looking for different levels of the protein, so there you are.  In this case, the authors found an allele worth investigating, and then went looking to see if relevant proteins were altered in the population, and in the CNS no less!  Not only that, but they also looked at the initiating end of the process, the ligand, HGF, and found abnormalities.  Good stuff.  Unfortunately, I haven’t found myself a copy of this paper yet, but the fact that other proteins in the pathway were altered is another line of evidence that something is amiss.  I’ve begun to appreciate the fact that I have spent a long time under appreciating the interconnectedness of biological systems; you aren’t going to have a disturbance in one system without altering the way upstream, and downstream processes are working; so  the fact that we see other proteins, those related to MET functions, modified, makes beautiful sense.  Coolness factor: 5.

Likely because of the mixed findings of skewed proteins in the MET pathway (?), the next study in line is, Genetic Evidence Implicating Multiple Genes in the MET Receptor Tyrosine Kinase Pathway in Autism Spectrum Disorder (full paper available).  Here’s the abstract:

A functional promoter variant of the gene encoding the MET receptor tyrosine kinase alters SP1 and SUB1 transcription factor binding, and is associated with autism spectrum disorder (ASD). Recent analyses of postmortem cerebral cortex from ASD patients revealed altered expression of MET protein and three transcripts encoding proteins that regulate MET signaling, hepatocyte growth factor (HGF), urokinase plasminogen activator receptor (PLAUR) and plasminogen activator inhibitor-1 (SERPINE1). To address potential risk conferred by multiple genes in the MET signaling pathway, we screened all exons and 5 promoter regions for variants in the five genes encoding proteins that regulate MET expression and activity. Identified variants were genotyped in 664 families (2,712 individuals including 1,228 with ASD) and 312 unrelated controls. Replicating our initial findings, family-based association test (FBAT) analyses demonstrated that the MET promoter variant rs1858830 C allele was associated with ASD in 101 new families (P=0.033). Two other genes in the MET signaling pathway also may confer risk. A haplotype of the SERPINE1 gene exhibited significant association. In addition, the PLAUR promoter variant rs344781 T allele was associated with ASD by both FBAT (P=0.006) and case-control analyses (P=0.007). The PLAUR promoter rs344781 relative risk was 1.93 (95% Confidence Interval [CI]: 1.12−3.31) for genotype TT and 2.42 (95% CI: 1.38−4.25) for genotype CT compared to genotype CC. Gene-gene interaction analyses suggested a significant interaction between MET and PLAUR. These data further support our hypothesis that genetic susceptibility impacting multiple components of the MET signaling pathway contributes to ASD risk.

 

We’ve got two new genes added to the mix, PLAUR and SERPINE.  The juicy part here is that the authors didn’t look for these variants at random, but performed a targeted search; they knew that the proteins encoded by these genes interact with either MET receptor function or HGF, and they also had found altered expression of these genes in the CNS study.  From the Introduction:

The hepatocyte growth factor (HGF) gene encodes the activating ligand for the MET receptor. HGF is translated as an inactive precursor protein that requires cleavage for efficient binding to the MET receptor [Lokker et al 1992]. The activating cleavage of HGF is achieved most efficiently by the enzyme plasminogen activator (urokinase-type; uPA; gene symbol: PLAU) under conditions in which uPA binds to its receptor, the urokinase plasminogen activator receptor (uPAR; gene symbol: PLAUR). Activating cleavage of HGF can be suppressed by the plasminogen activator inhibitor-1 (PAI-1; gene symbol: SERPINE1). Together, these proteins regulate the activity of MET receptor tyrosine kinase signaling, and our recent microarray analyses of postmortem temporal lobe of individuals with ASD indicate that disrupted MET signaling may be common to ASD pathophysiology [Campbell et al 2007]. For example, we found that there is increased expression of the HGF, PLAUR and SERPINE1 transcripts in ASD in postmortem cerebral cortex. The observation of disrupted expression suggests a general dysfunction of MET signaling in the cerebral cortex of individuals with ASD.

The proteins encoded by PLAUR and SERPINE were also found increased in the expression study; a finding further supported by the genetic study here.  The really grand slice here is that the SERPINE protein suppresses cleavage of HGF; essentially another way MET function can be affected, from a disturbance upstream of HGF binding.   In other words, more SERPINE (possibly as a result of a ‘promoter allele’) would result in less MET receptor activation because the SERPINE interferes with the cleavage of HGF, and thus, another pathway to reduced MET activation.  In a finding that seems 20/20 with hindsight, a functional promoter of the SERPINE gene was found to increase autism risk; i.e., if you have more SERPINE, you get less functional HGF, and therefore less triggering of the MET receptor.  This is cool and begins a portrait of the complexity; it shows how the effect of reduced MET functionality can come from multiple drivers; the reduced MET allele, or, the promoter SERPINE allele, and what’s more, having both is an even bigger risk; the authors are describing a synergy of low penetrance genes.

From the discussion section of the paper:

Beyond genetic susceptibility, the functional integrity of the MET signaling system also is sensitive to environmental factors. This concept is supported by bioinformatics analyses that identified PLAUR, SERPINE1 and HGF as genes active in immune response regulation, sensitive to environmental exposures, and within chromosomal regions previously implicated in ASD linkage studies [Herbert et al 2006]. Moreover, a recent cell biological study shows that chemically diverse toxicants reduce the expression of MET in oligodendrocyte progenitor cells, a result that is interpreted as the convergence of toxicant effects on oxidative status and the MET-regulating Fyn/c-Cbl pathway

Here are links to the Hebert paper, Autism and environmental genomics, and the Li paper, Chemically Diverse Toxicants Converge on Fyn and c-Cbl to Disrupt Precursor Cell Function.  What is neat here is that we are starting to be able to see a pathway of genes, and resultant proteins, that can effect disparate systems.   I believe that there is a subset of acupuncture, acupressure that relies on more knuckles than needles, and while the science on accu* based therapies isn’t very good, it does occur to me that in a sense, our lattice work of HGF-PLAUR-SERPINE proteins that participate in the MET-C process are pressure points in a delicate system, push a little bit and things will bend down the line accordingly.  It also exemplifies why I am offended by highly negative attitudes on genetic studies held by people who believe in a non trivial, environmentally mediated increase in the rates of autism; we are approaching a nearly impossible to overturn reality that genes we know to be associated with autism are particularly sensitive to interference from environmental agents, and participate in immune function.  That is important information.  Coolness factor 8.  First glimpse of beauty factor: 10.

Next up we have Dynamic gene and protein expression patterns of the autism-associated Met receptor tyrosine kinase in the developing mouse forebrain (full paper). 

The establishment of appropriate neural circuitry depends upon the coordination of multiple developmental events across space and time. These events include proliferation, migration, differentiation, and survival – all of which can be mediated by hepatocyte growth factor (HGF) signaling through the Met receptor tyrosine kinase. We previously found a functional promoter variant of the MET gene to be associated with autism spectrum disorder, suggesting that forebrain circuits governing social and emotional function may be especially vulnerable to developmental disruptions in HGF/Met signaling. However, little is known about the spatiotemporal distribution of Met expression in the forebrain during the development of such circuits. To advance our understanding of the neurodevelopmental influences of Met activation, we employed complementary Western blotting, in situ hybridization and immunohistochemistry to comprehensively map Met transcript and protein expression throughout perinatal and postnatal development of the mouse forebrain. Our studies reveal complex and dynamic spatiotemporal patterns of expression during this period. Spatially, Met transcript is localized primarily to specific populations of projection neurons within the neocortex and in structures of the limbic system, including the amygdala, hippocampus and septum. Met protein appears to be principally located in axon tracts. Temporally, peak expression of transcript and protein occurs during the second postnatal week. This period is characterized by extensive neurite outgrowth and synaptogenesis, supporting a role for the receptor in these processes. Collectively, these data suggest that Met signaling may be necessary for the appropriate wiring of forebrain circuits with particular relevance to social and emotional dimensions of behavior.

Coooooool.   Here we touch on the complexity of brain formation, all the little things that need to go exactly right, and how MET might play a role in that incredibly complicated dance.  Even better, a mouse model is used to gain an understanding of where and when peak expression of MET proteins occur, a period of significant changes to neural structures and the formation of synapses, the physical structures that enable thought.   This is a dense paper, too dense to get deeply into blockquoting for this posting, but there are some parts that deserve notice, namely, documentation of spatially localized MET expression in brain areas associated with social behaviors and some fine grained information on the specific parts of synapse formation that utilize MET.    Coolness factor: 8.  Complexity Factor: 12.

Here is a paper that a lot of people that play skeptics on the Internet ought to hate, Distinct genetic risk based on association of MET in families with co-occurring autism and gastrointestinal conditions.  (full paper)

In the entire 214-family sample, the MET rs1858830 C allele was associated with both autism spectrum disorder and gastrointestinal conditions. Stratification by the presence of gastrointestinal conditions revealed that the MET C allele was associated with both autism spectrum disorder and gastrointestinal conditions in 118 families containing at least 1 child with co-occurring autism spectrum disorder and gastrointestinal conditions. In contrast, there was no association of the MET polymorphism with autism spectrum disorder in the 96 families lacking a child with co-occurring autism spectrum disorder and gastrointestinal conditions. chi(2) analyses of MET rs1858830 genotypes indicated over-representation of the C allele in individuals with co-occurring autism spectrum disorder and gastrointestinal conditions compared with non-autism spectrum disorder siblings, parents, and unrelated controls.

There is a lot of caution in this paper, but the nice part is that there are biologically plausible mechanisms by which a reduction in MET could snowball into problems in the gastro-intestinal track.

In the gastrointestinal system, MET signaling modulates intestinal epithelial cell proliferation, and thus acts as a critical factor in intestinal wound healing. For example, activation of MET signaling via application of exogenous hepatocyte growth factor has been shown to reduce the effects of experimentally induced colitis, inflammatory bowel disease, and diarrhea.

Pushing on the other end of the balloon, increasing MET signaling, has been shown to help GI problems; no less than evidence that a genetic change associated with autism has biologically plausible mechanisms by which GI problems would be more prevalent. In fact, unless our findings of MET alleles are in error, or our clinical findings of the effects of HGF are spurious, it is absolutely expected. There is also a section with the startlingly simple, and simultaneously great idea of why findings like these might be useful markers for phenotypic categorization in studies in the future; i.e., to discern the prevalence of GI problems in autism, it might, for example, make sense to design that study to take presence or absence of MET alleles into consideration.  Nice.  Coolness Factor: 7.  Insidiousness factor: 9.

Here’s another one that found associations with MET and social behavior, and GI disturbances again.  Association of MET with social and communication phenotypes in individuals with autism spectrum disorder

Autism is a complex neurodevelopmental disorder diagnosed by impairments in social interaction, communication, and behavioral flexibility. Autism is highly heritable, but it is not known whether a genetic risk factor contributes to all three core domains of the disorder or autism results from the confluence of multiple genetic risk factors for each domain. We and others reported previously association of variants in the gene encoding the MET receptor tyrosine kinase in five independent samples. We further described enriched association of the MET promoter variant rs1858830 C allele in families with co-occurring autism and gastrointestinal conditions. To test the contribution of this functional MET promoter variant to the domains of autism, we analyzed its association with quantitative scores derived from three instruments used to diagnose and describe autism phenotypes: the Autism Diagnostic Interview-Revised (ADI-R), the Autism Diagnostic Observation Schedule (ADOS), and both the parent and the teacher report forms of the Social Responsiveness Scale (SRS). In 748 individuals from 367 families, the transmission of the MET C allele from parent to child was consistently associated with both social and communication phenotypes of autism. Stratification by gastrointestinal conditions revealed a similar pattern of association with both social and communication phenotypes in 242 individuals with autism from 118 families with co-occurring gastrointestinal conditions, but a lack of association with any domain in 181 individuals from 96 families with ASD and no co-occurring gastrointestinal condition. These data indicate that the MET C allele influences at least two of the three domains of the autism triad.

Really sort of plain, but very nice to see the GI component validated in another data set.  Coolness factor 5.

Then a few months ago, Prenatal polycyclic aromatic hydrocarbon exposure leads to behavioral deficits and downregulation of receptor tyrosine kinase, MET was released, an uber cool showcase of the autism bigfoot, the often regaled, only very rarely documented, gene/environment interaction. 

Gene by environment interactions (G × E) are thought to underlie neurodevelopmental disorder, etiology, neurodegenerative disorders, including the multiple forms of autism spectrum disorder. However, there is limited biological information, indicating an interaction between specific genes and environmental components. The present study focuses on a major component of airborne pollutants, polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene [B(a)P], which negatively impacts cognitive development in children who have been exposed in utero. In our study, prenatal exposure of Cpr(lox/lox) timed-pregnant dams to B(a)P (0, 150, 300, and 600 μg/kg body weight via oral gavage) on embryonic day (E14-E17) consistent with our susceptibility-exposure paradigm was combined with the analysis of a replicated autism risk gene, the receptor tyrosine kinase, Met. The results demonstrate a dose-dependent increase in B(a)P metabolite generation in B(a)P-exposed Cpr(lox/lox) offspring. Additionally, a sustained persistence of hydroxy metabolites during the onset of synapse formation was noted, corresponding to the peak of Met expression. Prenatal B(a)P exposure also downregulated Met RNA and protein levels and dysregulated normal temporal patterns of expression during synaptogenesis. Consistent with these data, transcriptional cell-based assays demonstrated that B(a)P exposure directly reduces human MET promoter activity. Furthermore, a functional readout of in utero B(a)P exposure showed a robust reduction in novel object discrimination in B(a)P-exposed Cpr(lox/lox) offspring. These results confirm the notion that common pollutants, such as the PAH B(a)P, can have a direct negative impact on the regulated developmental expression of an autism risk gene with associated negative behavioral learning and memory outcomes.

Oh snap.  A common pollutant (well, common in the last few decades anyways), is shown to interact with MET in a dose dependent fashion to reduce protein expression in the brain during embryonic development and cause ‘a robust reduction in novel object discrimination’. (Ouch)  This is an example of just what we mentioned above, referenced Herbert, concerning the possibility of MET as a gene sensitive to ‘environmental exposures’.  Indeed.  From the discussions section:

The results from the present study demonstrate that the transcription and developmental expression patterns of a replicated ASD risk gene, MET, are highly sensitive to a common PAH pollutant. In utero exposure to B(a)P produces an oxidative milieu of B(a)P metabolites in offspring during a key postnatal period of synapse development, providing evidence that environmental exposure creates a sustained cerebral cortical burden that likely contributes to an increased oxidative load. Oxidative stressors in the form of metabolites would be expected to negatively impact gene expression (Kerzee and Ramos 2000) and, more specifically, receptor tyrosine kinase function, including Met (Li et al. 2007). These data suggest that B(a)P-induced exposure would impact the expression of key neurodevelopmental genes, including Met. Additionally, the predominance of the 3-OH and 9-OH metabolites places a sustained burden in the brain because of the potential for further oxidization to form B(a)P quinones (McCallister et al. 2008, Hood et al. 2000, Brown et al. 2007) which undergo redox cycling to generate reactive oxygen species (Kerzee and Ramos 2000, Bolton et al., 2000).

And

In conclusion, specific developmental events such as glutamatergic excitatory synapse formation and maturation may be particularly vulnerable to G x E effects that impact regulatory and signaling proteins involved in this process. While we do not suggest that the current study reflects specific defects related to a complex clinical condition such as the ASDs, current molecular, behavioral and functional imaging data are converging on the concept that the ASDs are a manifestation of altered local and long-distance cortical connectivity (Geschwind et al. 2007, Bill and Geschwind 2009, Geschwind and Levitt, 2007, Levitt and Campbell 2009). Also, Met and other related signaling components of this receptor tyrosine kinase pathway have been implicated in both syndromic and idiopathic disorders where the ASDs are diagnosed at a high rate. In combination with risk alleles in key genes, the in utero exposure to PAHs such as B(a)P, which results in both a reduction in absolute levels and the mistiming of peak Met expression, could drive the system toward a pathophysiological threshold that neither genetic risk nor environmental factors could produce individually. The present study focused on the neocortex, but given the highly restricted spatial and temporal expression of Met in mouse limbic circuits associated with social-emotional development and cognition (Judson et al. 2009), it is likely that perturbations occur throughout these key circuits, including in the hippocampus.

Really cool stuff; particularly the finding that developmental, in utero exposure was capable of driving abnormal protein expression well after birth. This is the best of both sides of the genetics versus environment conundrum; the kind of finding that sheds light on how environmental pollutants could be participating in increasing the number of children with autism by interacting with genetically susceptible children.  But what I love about this is that it is the death knell of the fairytale of a static rate, or near static rate of autism, just having the genes or the exposure isn’t enough; instead, the interaction of alleles and timed exposure ‘could drive the system toward a pathophysiolical threshold that neither genetic risk nor environmental factors could produce individually’.  I think there are some more findings coming from this group soon that might be exciting, or terrifying, depending on how you see it.  (or both).  Coolness factor: 99.   

So what have we learned and just how cool is it?

1)      The MET receptor enables some types of cellular signaling that have relevance to the autism community including synapse formation, immune modulation, and gastro intestinal function.  The ligand, or trigger of the MET receptor is HGF.

2)      Certain alleles of the MET gene that result in decreased expression are more common in children with autism than people without autism.

3)      Consistent with findings of increased prevalence of MET alleles, MET protein expression was found to be decreased in brain tissue from people with autism.  Other, related proteins, HGF, PLAUR, and SERPINE were also found to be disturbed.

4)      Following up on the differential findings of SERPINE and PLAUR, genetic studies found gene to gene interactions between the MET allele and alleles involved with production of SERPINE and PLAUR. Some of the proteins in question are known to be particularly vulnerable to environmental interference.

5)      Animal models tell us that MET is heavily expressed in many areas of the mammalian brain during prenatal and postnatal development, and we gain insight into the spatial and temporal expression of MET during the intricate dance of brain formation.

6)      Two studies add evidence that the one function of decreased MET expression, GI disturbances, are indeed found with greater consistency within children with autism and the MET allele.  This should be a relatively unsurprising finding considering what we know about MET and children with autism.

7)      Finally, a portrait of genetic / environmental interactions capable of disturbing physiology and behavior in ways consistent with findings in autism is rendered using an agent that is the product of the automobile age and already associated with decreased cognitive skills for groups with the highest gestational exposure.

It should be noted that this is just a slice of the MET papers out there in the autism realm; they all shared one or more authors, I picked them because they seem to show a nice progression of knowledge, and incremental approach towards learning more.   There is a lof more to learn, in particular, I think that the immune modulating effects of reduced expression would be an interesting subject, but one that will have to wait for another posting. 

–  pD

 

Hello friends –

Hot on the heels of Mitochondrial Dysfunction in Autism, another study on mitochondrial function in the autism population was just released, this time giving us insight into what is happening inside the gated community behind the blood brain barrier.  How potentially inconvenient.  Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism came out the other day; I’ve yet to receive a full copy (one has been promised to my real world email), but the abstract is juicy enough to warrant a small posting.

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

A few things immediately jump out at me.  Firstly, the Chauhan’s are authors of this paper, who have been around the autism / oxidative stress block since the get go, as authors of the very nice Oxidative stress in autism: increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin–the antioxidant proteins, a really nice paper that was one of the first I saw that broke the autism groups into classic and regressive phenotypes with findings of increased oxidative stress in the latter.

Secondly, one of the biggest concerns with Mitochondrial Dysfunction in Autism when it was released a few weeks ago was, whether or not the findings taken from lymphocytes, cells outside of the brain, could be reliably used as proxies for what is happening within the CNS.  Based on the findings in Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism it would seem that, at least in children, there is an increased frequency of mitochondrial problems in the brain.  Of course, if we acknowledge the reality of the interconnectedness of immune activation, oxidative stress, mitochondrial impairment and what we already know about the CNS in autism, these findings shouldn’t really be all that surprising.  None the less, it is nice to have some direct evidence of this.

Unfortunately, we still don’t know what is causing the problems with mitochondria function in the brain; it is possible, though exceedingly unlikely that all of the participants in this study also had a diagnosable electron chain disorder (I haven’t gotten a full copy of the paper yet).  I think it is possible that there is a feedback loop in place involving the immune response, oxidative stress, and mitochondria that for some reason our children’s physiology cannot shake loose from. 

The very small sample size of the children in this study, five, is an unfortunate reality for nearly all brain based studies in the autism world.  Though I’ve yet to read the full paper, my prediction is that it is liberally peppered with cautious language regarding interpreting the findings widely without further confirmation.  That is probably pretty good thinking.

But, if we look closely, and we taken notice of the where of mitochondrial problems in the autism group was observed, we may have evidence of participatory processes.  Specifically, Chauhan found decreased electron chain transport measurements in the cerebellum, frontal cortex, and temporal cortex.

In Group A, we observed significantly lower levels of complexes III and V in the cerebellum (p < 0.05), of complex I in the frontal cortex (p < 0.05), and of complexes II (p < 0.01), III (p < 0.01), and V (p < 0.05) in the temporal cortex of children with autism as compared to age-matched control subjects, while none of the five ETC complexes was affected in the parietal and occipital cortices in subjects with autism.

(my emphasis)

There have been a few other studies (that I know of) that have looked for brain region specific abnormalities that might be of interest to u.  Brain Region-Specific Changes in Oxidative Stress and Neurotrophin Levels in Autism Spectrum Disorders (ASD), which found increased markers of oxidative stress in the cerebellum:

Consistent with our earlier report, we found an increase in NT-3 levels in the cerebellar hemisphere in both autistic cases. We also detected an increase in NT-3 level in the dorsolateral prefrontal cortex (BA46) in the older autistic case and in the Wernicke’s area and cingulate gyrus in the younger case. These preliminary results reveal, for the first time, brain region-specific changes in oxidative stress marker 3-NT and neurotrophin-3 levels in ASD.

(My emphasis)

Interesting note: the ‘Wernicke’s area’ of the brain plays a large part in language skills, and in fact, damage to the Wernicke’s area can cause a type of aphasia. 

The number of studies that tie together oxidative stress and mitochondrial function are many and numerous to the point of cumbersomeness, I have a short list of them on a previous post about mitochondria function in autism, here

Two of the really nice neuroimmune studies in the autism realm, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism, and Immune Transcriptome Alterations In the Temporal Cortex of Subjects With Autism both provide evidence of an ongoing immune response in some of the specific areas of the CNS where Chauhan found impaired mitochondrial function, the cerebellum and the temporal cortex.

From Vargas:

We demonstrate an active neuroinflammatory process in the cerebral cortex, white matter, and notably

in cerebellum of autistic patients.

And

The neuroglial activation in the autism brain tissues was particularly striking in the cerebellum, and the changes were associated with upregulation of selective cytokines in this and other regions of the brain.

 

From Garbett:

 

Expression profiling of the superior temporal gyrus of six autistic subjects and matched controls revealed increased transcript levels of many immune system related genes. We also noticed changes in transcripts related to cell communication, differentiation, cell cycle regulation and chaperone systems.

 

Detangling if these findings are related, and if so, the direction of causality is for another series of studies to discern.  Calls towards the possibility that relationships like this are spurious are common, but I hate to invoke coincidences for no good reason other than coincidences do occur.  My suspicion is that the immune findings and impaired mitochondria findings are related, but a cautious suspicion is all that is warranted at this time.  I do believe that the relationship between immune activation and mitochondria function is being evaluated now; though I do not know if it is being addressed directly in the CNS, which would be ideal.

 

Curiously from my perspective, however, is the finding that young adults and adults with ASD in Chauhan did not exhibit decreased electron chain function.  The original microglia paper from Vargas, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism found extensive evidence of an ongoing immune response in the CNS of people with autism into adulthood.  From the standpoint of a theory wherein an immune response were driving the mitochondrial impairment due to increased oxidative stress, the findings in Chauhan of normal mitochondria function are contradictory to what was found in Vargas.  (?) 

 

A few other thoughts occurred to me as I considered the age differences found in Chauhan.  If mitochondrial dysfunction is part of the pathogenic force driving behaviors associated with autism, it is possible that a decrease as adulthood is reached conforms with a general improvement in adaptation many people seem to report.  Alternatively, if we are actually observing a true increase in the number of people with behaviors that can be classified as autistic, that is, the number of children with autism is a new phenomena, the age findings in Chauhan could be artifacts of different underlying causes of autism in the adults versus the children.  I’m a big believer in a wide range of physiological roads to the end point of autistic behaviors, so such a situation doesn’t really bother me conceptually, though it is very, very problematic to put to any kind of designed experiment. 

 

Lastly, for a while now I’ve been putting some thought towards something that’s really been bugging me about the neuroimmune findings in autism when put in context with other ‘classic’ neurological diseases that also exhibit a strong immune component; i.e., Alzheimer’s or Parkinson’s, both of which have strong immune findings as well, but are more strikingly degenerative in nature when compared to autism.  Generally you talk about a child with autism gradually getting better, or in some cases reaching a plateau; but very rarely (or never) is there the steady and unforgiving decrease in function that you see in diseases like Alzheimer’s.  I’m struggling with this reality and how our findings fit in.  I’m not sure how, or if, the age differences in Chauhan are meaningful towards this apparent paradox, but my pattern recognition unit sure is trying to tell me something, I just can’t tell if it’s sending me on (another) snipe hunt or not.

 

When the entire paper lands in my inbox, I may write another post about it.  I’m interested in seeing if any other blogs pick up on this paper or not and what their take on it is.  I’m still sort of in the dark on the machinations of the press cycle as it relates to autism news, but this paper doesn’t seem to have gotten the press release treatment that Mitochondrial Dysfunction in Autism did, even though its findings are just as interesting. 

 

          pD

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 1999Lee 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 2010Bitanihirwe 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.

(my emphasis)

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.

(my emphasis)

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.  

– pD


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