passionless Droning about autism

Archive for the ‘Phenotypes’ Category

Hello Friends – There are (at least) two big
classifications of microglia findings in autism, an altered
morphology (i.e., shape and function, or ‘activated’ versus
‘quiescent’), and an increased number (i.e., more), with both
parameters varying with each other and spatially.  In other
words, disparate parts of the brain have different numbers of
microglia in them, and the functional profile of those microglia
also varies from one area to another. 
[Note: There is ongoing
discussion
regarding the appropriate definition of
‘activation’ of microglia, with evidence of (at least) four states
of microglial morphology.] Recently I saw a discussion on the SFARI
site about the fancy in
vivo study of microglia numbers in high functioning males with
autism
.  (I believe I am growing
increasingly jaded, as it occurs to me that radio tracing against
[11C](R)-PK11195) to
show microglial activation is a fancy trick, but one leaving us
open to detecting other stuff too.)  In any case, the findings
are not especially unexpected by now, well not to me anyways, but a
comment at the SFARI site really got me thinking about the chain of
events that could lead to different spatial and
morphological characteristics of microglia.  Perhaps we could
gain insight into the question of what the microglia are doing by
trying to understanding how they got there. Do we have any
biologically plausible models that might educate us on how a
different morphology and distribution of microglia could be
achieved? 
A while ago I got a copy of a few
articles that don’t have autism in them per se, but they kept
coming to the forefront of my mind when I thought about that
question.  The first is Distribution
of microglia in the postnatal murine nigrostriatal
system
,
which had a disease focus on
Parkinson’s, but what really grabbed my attention is what they
learned about the developmental pathway microglia took to populate,
and then depopulate the substantia nigra (SN), a little wedge of
brain involved with motor skills, reward seeking, and addiction.

Interestingly, the SN
has been shown to contain more microglia than
adjacent 
structures. We have analysed
changes in microglia numbers and in microglial morphology in the
postnatal murine nigrostriatal system at various stages ranging
from postnatal day 0 (P0) up to 24 months of age. We
clearly show that the microglia numbers in the SN and in the
striatum dramatically increase from P0 to P15
and
significantly decrease in both areas in 18-month-old and
24-month-old animals.

[Note: There seems to be
some variance in the appropriate ‘rat-to-human-age’ approximations;
especially when trying to do something as
expeditionary as comparing brain development.  We should
extrapolate only with caution.] The part that makes me grin is that
it illustrates our nascent understanding of the process of
microglial colonization into the CNS, the hows,
whens
, wheres, and whys are still
shrouded in mystery. The broadest outlines tell us that microglial
penetration into the brain is a long running, dynamic process; the
microglia are slow infiltrators, gaining access into parts of the
brain in concert with a swath of proliferating and inhibitory
factors, all at a time of once in a lifetime neurodevelopmental
modifications. Regulation
of postnatal forebrain amoeboid microglial cell proliferation and
development by the transcription factor Runx1
paints a
beautiful portrait of functionality.  Runx1 is a chemical
messenger that participates in phenotyopic determination of blood
cell progenitors into mature cells.  The researchers observed
spatial, time dependent expression of Runx1 in the developing
forebrain, and differential levels following injury.

Here we show that the mouse
transcription factor Runx1, a key regulator of myeloid
cell proliferation and differentiation, is expressed in forebrain
amoeboid microglia during the first two postnatal weeks
.
Runx1 expression is then downregulated in ramified microglia. Runx1
inhibits mouse amoeboid microglia proliferation and promotes
progression to the ramified state. We show further that
Runx1 expression is upregulated in microglia following nerve injury
in the adult mouse nervous system.
These findings provide
insight into the regulation of postnatal microglia activation and
maturation to the ramified state and have implications for
microglia biology in the developing and injured brain.

It doesn’t really tell us much about a
persistent change in microglia per se, but it does render a picture
of proliferation and differentiation as an easily
disrupted
symphony.  When we think about the
developing brain, I won’t pretend to have more than a lightyear
close guess at what microglia might be doing
differently between amoeboid and ramified
morphologies in this locale, at this time, but I
very highly doubt there isn’t
a functional impact on microenvironment neurodevelopment; our
developing brains are using opportunities like the Indians used the
buffalo, no waste, no excess, and because balance is important,
everything is important. Moving back to the
question of the plausibility of a pathway to the autism state,
luckily (or unluckily?) the literature is veritably littered with
insults that perturb microglial development, leading to
 persistent changes to microglial morphology, ultimately
percolating up to behavioral changes. Prenatal stress is a bad, bad
thing, and here is a study that finds that extreme mice stress can
persistently alter the mice activation profile of mice microglia.
Prenatal
stress increases the expression of proinflammatory cytokines and
exacerbates the inflammatory response to LPS in the hippocampal
formation of adult male mice
, was just published, and
comes wrapped up with a double hit, and
different resting and stimulated neuroimmune environments.

Under basal conditions,
prenatally stressed animals showed increased expression of
interleukin 1ß and tumor necrosis factor-a (TNF-a) in the
hippocampus and an increased percentage of microglia cells with
reactive morphology in CA1 compared to non-stressed males.
Furthermore, prenatally stressed mice showed increased TNF-a
immunoreactivity in CA1 and increased number of Iba-1
immunoreactive microglia and GFAP-immunoreactive astrocytes in the
dentate gyrus after LPS administration. In contrast, LPS did not
induce such changes in non-stressed animals. These
findings indicate that prenatal stress induces a basal
proinflammatory status in the hippocampal formation during
adulthood that results in an enhanced activation of microglia and
astrocytes in response to a proinflammatory
insult.

Note: I have not read this
paper so I do not know if a qualitative number of microglia, or
just more immune-targeted microglia were found, but likely the
latter. A similar, full free paper, Prenatal
stress causes alterations in the morphology of microglia and the
inflammatory response of the hippocampus of adult female
mice
, found broadly similar results; perturbed resting
and stimulated states in the treatment group.

Prenatal stress, per se,
increased IL1ß mRNA levels in the hippocampus, increased the total
number of Iba1-immunoreactive microglial cells and increased the
proportion of microglial cells with large somas and retracted
cellular processes. In addition, prenatally stressed and
non-stressed animals showed different responses to peripheral
inflammation induced by systemic administration of LPS.
LPS induced a significant increase in mRNA levels of IL-6,
TNF-a and IP10 in the hippocampus of prenatally stressed mice but
not of non-stressed animals.
 

Going back to my
ramblings on glial priming
, it seems that here we have an
example of a type of cross system priming (sweet!), where
disturbing the stress response system changed the immune system;
such is the way of the polyamorous chemical families interacting in
our brain.  It also occurs to me that given the
delicate nature of the developing brain, and the
crazy important
tasks going on in there, we might want to think very
carefully before we ‘induced a significant increase in
mRNA levels of IL-6, TNF-a and IP10 in the hippocampus‘

on subgroups who might be environmentally predisposed to react with
exaggerated vigor.  But what do I know? Of course, the
prenatal immune challenge arena holds a ton of studies on
persistent microglial function, and ‘consequences’.  There are
way too many to list, but a quick overview of some very recent ones
would include: Enduring
consequences of early-life infection on glial and neural cell
genesis within cognitive regions of the brain
, an early
life real infection model with e coli that
concludes, “Taken together, we have provided evidence that
systemic infection with E. coli early in life has significant,
enduring consequences for brain development and subsequent adult
function
.”  (Staci Bilbo!)  This paper was sort
of a quinella, as it showed both changes in immune responsiveness
into adulthood; it also demonstrated the ability
of an immune insult to alter
the developmental trajectory of the
microglia, i.e., E. coli increased the number of newborn
microglia within the hippocampus and PAR compared to controls. The
total number of microglia was also significantly increased in E.
coli-treated pups, with a concomitant decrease in total
proliferation.
Neonatal
lipopolysaccharide exposure induces long-lasting learning
impairment, less anxiety-like response and hippocampal injury in
adult rats
very directly blasted rats with some LPS
immune activation action, and includes, ”Neonatal LPS
exposure also resulted in sustained inflammatory responses in the
P71 rat hippocampus, as indicated by an increased number of
activated microglia and elevation of interleukin-1ß content in the
rat hippocampus.”  
(Sound familiar?) Interleukin-1
receptor antagonist ameliorates neonatal lipopolysaccharide-induced
long-lasting hyperalgesia in the adult rats
took the
extra step of adding a set of animals that got inhibited
inflammatory responses.  Results are increasingly
unsurprising.

Neonatal
administration of an IL-1 receptor antagonist (0.1mg/kg)
significantly attenuated long-lasting hyperalgesia induced by LPS
and reduced the number of activated microglia in the adult rat
brain. These data reveal that neonatal intracerebral LPS
exposure results in long-lasting hyperalgesia and an elevated
number of activated microglia in later life. This effect is similar
to that induced by IL-1ß and can be prevented by an IL-1 receptor
antagonist

I love how (once again) we
can see how interrupting the immune response can have an effect.
Environmental impacts outside of the immune
activation realm may also find a place within the ‘big tent’ of
microglial agitation with consequent developmental impacts. 
The people who made the first big neuroimmune / autism splash at
Johns Hopkins later came out with Neuroinflammation
and behavioral abnormalities after neonatal terbutaline treatment
in rats: implications for autism
, which found that an
agent used to prevent labor in some situations could
produced a robust increase in microglial activation on PN
30 in the cerebral cortex”
in treatment animals. 
The drug in question, terbutaline, has been weakly associated with
increased incidence of autism, i.e., Prenatal
exposure to ß2-adrenergic receptor agonists and risk of autism
spectrum disorders
, and beta2-adrenergic
receptor activation and genetic polymorphisms in autism: data from
dizygotic twins
. And now, in 2013, Beta-adrenergic
receptor activation primes microglia cytokine production
,
displays another example of cross system
priming.

To determine
if ß-AR stimulation is sufficient to prime microglia, rats were
intra-cerebroventricularly administered isoproterenol (ß-AR
agonist) or vehicle and 24h later hippocampal microglia were placed
in culture with media or LPS. Prior isoproterenol treatment
significantly enhanced IL-1ß and IL-6, but not TNF-a production
following LPS stimulation. These data suggest that central
ß-AR stimulation is sufficient to prime microglia cytokine
responses.

In other words, they gave
the rats a drug in the class of terbutline, and subsequently
observed an increased microglia responsiveness in cultured
cells.  What a crazy coincidence. Detecting total
populations
of microglia in adulthood, either regionally
or in the brain as a whole is a little more difficult, the little
buggers are a lot easier to detect when we light them up with neon
green tracers that stick to proteins expressed at ‘activation’
time, and it just doesn’t look like the question has been asked too
many times.  I did, however, find something that has a sort of
chip shot on this analysis, Prenatal
stress alters microglial development and distribution in postnatal
rat brain
, which looked at regional microglia populations
and phenotypes at two time periods following prenatal stress
events.

Prenatal
stress consisting of 20 min of forced swimming occurred on
embryonic days 10–20. On postnatal days 1 and 10, stressed and
control pups were killed. Microglia were identified using Griffonia
 simplicifolia lectin and quantified in the whole encephalon.
In addition, plasma corticosterone was measured in dams at
embryonic day 20, and in pups on postnatal days 1 and 10.
At postnatal day 1, there was an increase in number of
ramified microglia in the parietal, entorhinal and frontal
cortices, septum, basal ganglia, thalamus, medulla oblongata and
internal capsule in the stressed pups as compared to controls, but
also there was a reduction of amoeboid microglia and the total
number of microglia in the corpus callosum.
By postnatal
day 10, there were no differences in the morphologic type or the
distribution of microglia between the prenatal stress and control
groups, except in the corpus callosum; where prenatal stress
decreased the number of ramified microglia. The stress procedure
was effective in producing plasma rise in corticosterone levels of
pregnant rats at embryonic day 20 when compared to same age
controls. Prenatal stress reduced the number of immature
microglia and promoted an accelerated microglial differentiation
into a ramified form.

They did a lot
of clever stuff at analysis time, taking samples from several
locations after birth and ten days later, and
also did some fine grained classification of the
shape of the microglia.  They include spatial and temporal
mappings of four microglial developmental profiles.  It looks
as if prental stress was able to alter the developmental speed of
microglia from one morphology to another in different parts of the
brain.  There was as small section in the discussion that
speculated on what such changes might mean for neurodevelopment.

Given that during the
early postnatal period occur numerous brain developmental processes
(e.g. neurogenesis, myelination, synaptogenesis, astrogliogenesis,
neuronal cell death and blood–brain barrier maturation) [6, 19, 22,
25, 36, 52] it is possible that altered microglial
development induced by in utero stress may affect other
developmental processes either changing microenvironment molecular
constitution or triggering earlier inflammatory changes secondary
to the blood–brain barrier opening induced by prenatal
stress
.  Although punctual, the altered microglial
development might alter extensively the other
neurodevelopmental processes
ensuing perdurable
structural changes
; for example it is possible that the
change in the distribution pattern of microglia in the prenatal
stress group may render vulnerable some neuroanatomic
regions due to the reduction of neurotrophic factors
,
such as the corpus callosum where there is a continuous axonal
growth

No kidding! [There is also some very
interesting notes regarding microglial participation in purkinje
cell death that deserves and entire post. . .] This should be the
point that any rational observer must accept that we several lines
of evidence that early life experiences can persistently alter
microglial function with plausible mechanisms that could affect
neurodevelopment.  Our data concerning total population
numbers in adulthood is a lot more difficult to come by, but I
think this will probably be getting looked at soon enough. Of
course, in any particular individual it is difficult (or
impossible?) to know how they may have arrived at a state of
increased microglial activation, but at the same time, it is not as
if we have no clue on possible pathways to this destination; our
short list of environmental factors includes immune insult, stress,
and chemical agents. If the question is, ‘what are the microglia
doing in the autism population?’, one plausible answer is ‘their
phenotype was persistently altered by an early life event through a
developmental programming model’. As I was mulling all of this
over, two things happened.  First, a maternal CRP
study
came out, and found a pretty strong relationship
between direct measurements of mommy inflammation with increased
risk of baby autism.  The nice part is that they had a
gigantic data set (1.2M births!) to work with thanks to a few
decades of single payer medicine.  (Very
nice!
)

For maternal CRP
levels in the highest quintile, compared with the lowest quintile,
there was a significant, 43% elevated
risk.
This finding suggests that
maternal inflammation may have a significant role in autism, with
possible implications for identifying preventive strategies and
pathogenic mechanisms in autism and other neurodevelopmental
disorders.

Just after that paper came out, I
made some Fred Flintstone style beef ribs.  I ‘primed’ the
meat with a Moroccan inspired spice rub overnight, then
slow, slow, slow cooked them with a
low, low, low heat all day
long
, and blasted away with a date glaze under the
broiler just before go time and they were caveman style
primal fucking awesome
.  The key to arriving there
was the slow cooking. The rib preparation
process got me thinking about our population wide experiment
in replacing infection with inflammation
where we have
traded in death by pathogens or other once fatal ailments in
exchange for a longer life frequently plagued by conditions
associated with higher inflammation.  Our analysis on long
term alterations to microglial proliferation and morphology is
largely comprised of studying acute insults
(sound familiar?), i.e., injection of purified bacterial cell
components known to trigger a robust immune response, ten sessions
of mouse based pregnant forced swimming, or exposure to chemicals
with rare and particular exposure routes in humans.  Mostly I
think this is due to the black swan nature of the developmental programming
model
alongside the very new idea that microglia are
doing jobs other than responding to infections; our models are
crude because of our relative ignorance.  What will we find
when our filters are appropriately powered to detect for chronic,
but subtle insults? It occurs to me that there may be a ribs model
of altered microglial colonization of the fetal brain; it seems
clear that proliferation and differentiation of microglia can
clearly be changed by powerful inputs, but the
chemical messengers that impact that change are closely related (or
the same) as the measurement points in the maternal CRP study.
Could a slow cooking of slightly higher but not acutely
increased
maternal inflammation be participating in the
genesis of autism (in some children) through altering the migration
and proliferation of microglia into the neonatal brain?  Could
the same chemical messengers of inflammation be subtly
priming
the microglia to respond with increased vigor to
insults later in life?  Has our replacement of infection with
inflammation included an unanticipated effect that alters the
developmental pathway of the very cells that help shape our
children’s brains? I don’t think we are (quite) clever enough to
answer these types of questions yet, but we are at least starting
to generate the right kind of data to inform us on how to get
started.  I don’t know what we will find, but the initial data
doesn’t look very good.  In the meantime, I am recommending
you go get some ribs and let them cook all day long.      

pD

Advertisements

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 –

A while ago I saw a completely fascinating Nova  called Ghost In Your Genes concerning the nascent field of epigenetics, the study of the relative expression of genes, which is a bit different than the presence or absence of genetic differences.   I’d recommend this program to anyone interested in learning.

In a general sense, our genes are simply blueprints for the production of proteins; the traditional model of genetic research involves structural changes in the genetic blueprints, so that we might understand that a person with a particular mutation might produce more, or less, of a particular protein than someone without that mutation.  As protein gradients are altered, physiological effects accumulate, and we can begin to associate genetic differences with identifiable classifications.

But.  It turns out, structural differences in the DNA aren’t the only way to affect the production of genes.  Genes can also be regulated by a variety of factors, and these changes in regulation, in turn, are measured as expression of genes, essentially a measure of which genes are active, or inactive, and to what extent.

From Wikipedia:

In biology, epigenetics is the study of inherited changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: επί– over, above) genetics. These changes may remain through cell divisions for the remainder of the cell’s life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism;[1] instead, non-genetic factors cause the organism’s genes to behave (or “express themselves”) differently.[2]

For another interesting write up, see this one by PZ Meyers; it gets technical quickly, but is a nice read.  Specific mechanisms aside, it is sufficient for our purposes that epigenetics is the study of how a variety of non structural changes can affect how our genes operate.

An analogy might of a series of car engines, sitting at idle.   All of them power a car, but at a structural level there are differences, most models are roughly generating the same amount of force at idle, but, for example, the Prius engine is generating much lower force than Porsche engine.   At a very general level, we might consider physical mutations of our genome and protein generation capacities to be equivalent to the difference between the Porshce and the Prius at idle.  But there are other means to affect the energy being put out by the engine, the accelerator, tweaking the cylinders, or a variety of other means. This is a big shift and weights heavily on the ‘genetic and environment interaction’ theme that gets a lot play in the autism realm.  Despite a lot of studies, and spectrums of dollars there have been very few findings involving autism genes that do anything but confer a very limited risk of a diagnosis.  Furthermore, a lot of the studies are finding that seemingly very common mutations are implicated, but with very delicate effects.  An example of this might be the MET genes, that have several neat papers (here, here, here), but the specific MET-C allele associated with autism is still very common, found in nearly fifty percent of everyone.  None the less, it is just a little more prevalent in the autism cohort, but the impact is very subtle, and likely dependent on the presence of a variety of other genes (or expression patterns), or other factors.  Excepting known genetic  conditions that confer great risk, but can be responsible for only a fraction of our autism, mutations such as Fragile-X or Rhett Syndrome, the vast majority of genetic findings impart small increases in risk.

But once we start looking at the wide array of different genetic expression in autism, it becomes clear that which genes you use, and to what extent, might be as important as which genes you are born with.  By way of example, a very cool paper out recently, “Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain ” that is available in full online and I would recommend to anyone interested in how analyzing epigenetic changes and the accompanying differential gene expression can teach us more about autism.  A lot of the press around this study involves the hope that eventually this kind of finding might lead a treatment opportunities, something I personally consider to be a long term goal that still faces significant technical hurdles; but it does gives us insight into the nature of autism, and the usefulness of the half truth ‘differently wired’ argument concerning autism treatments.

– pD

Note: Updated link to PZ.

I have yet to get my hands on a copy of Plasma cytokine profiles in Fragile X subjects: Is there a role for cytokines in the pathogenesis?, but plan on doing so relatively soon.  Even still, the abstract looks pretty good:

BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder with a broad spectrum of involvement and a strong association with autism. Altered immune responses have been described in autism and there is potential that in children with FXS and autism, an abnormal immune response may play a role. OBJECTIVES: To delineate specific patterns of cytokine/chemokine profiles in individuals with FXS with and without autism and to compare them with typical developing controls. METHODS: Age matched male subjects were recruited through the M.I.N.D. Institute and included: 19 typically developing controls, 64 subjects with FXS without autism and 40 subjects with FXS and autism. Autism diagnosis was confirmed with ADOS, ADI-R and DSM IV criteria. Plasma was isolated and cytokine and chemokine production was assessed by Luminex multiplex analysis. RESULTS: Preliminary observations indicate significant differences in plasma protein levels of a number of cytokines, including IL-1alpha, and the chemokines; RANTES and IP-10, between the FXS group and the typical developing controls (p<0.01). In addition, significant differences were observed between the FXS group with autism and the FXS without autism for IL-6, Eotaxin, MCP-1 (p<0.04). CONCLUSIONS: In this study, the first of its kind, we report a significantly altered cytokine profile in FXS. The characterization of an immunological profile in FXS with and without autism may help to elucidate if an abnormal immune response may play a role and help to identify mechanisms important in the etiology of autism both with and without FXS.

I love the MIND guys.  Anyways, the quick glance gives me some ideas:

  • The big one here would seem to be that we seem to have additional evidence that immune dysregulation has very specific ties to autism; a pattern not just of immune dysregulation, but indeed, the beginings of an immunological signature, one so precise that given nothing but blood samples, we could beat Vegas odds in selecting which child has Fragile-X, and which child has Fragile-X and autism.   This is also more evidence that an abberant immunological response might be playing a causative role in autistic behavior genesis; just having Fragile-X isn’t enough for you to have this profile, you also have to have autism. 
  • Some of the players there we know about already; IL-6 was found in increased levels in the CNS by  Vargas, and Li , and was found to be generated at higher levels in several studies including Ashwood, Enstrom  , Jyonouchi, and Jyonouchi.  It also has great deal of support for a place in seizure generation.  MCP-1 was also found in Vargas, Eotaxin is also chemotaxic, though I haven’t read anything about it yet.  

 

That’s the abstract view, the whole paper should get here soon.


Enter your email address to subscribe to this blog and receive notifications of new posts by email.

Join 36 other followers