Friday, November 11, 2011

Signal Transduction in Autism

EXECUTIVE SUMMARY: A study published this past summer analyzed tissue extracts from 20 donated brains, half (10) of which came from autistic donors. Half (5) of those people had histories of regression --- that is, they started out developing normally, speaking and everything, but then they lost some of the skills they had gained.

The brain tissue extracts were analyzed using a technique I describe in the main body of this post, that tests for the presence of a certain enzyme (protein kinase A, here) by giving it an opportunity to react with a sort of dummy peptide that can't really do anything except sit there and let the enzyme (and only that enzyme) act on it, and then introducing antibodies that will "tag" the altered peptides with an enzyme that will change a solution's color under certain conditions. This allowed the researchers to measure the relative activity of the enzyme across subjects or across brain regions; a similar measure, but using antibodies to the enzyme itself, rather than to its product, was used to measure the amount of enzyme present in each extract.

Using this method, the researchers found differences in protein kinase A activity and expression only in the frontal lobes, and only between the autism-with-regression subgroup of the autism group and both the controls and the rest of the autism group.

Protein kinase A is involved in intracellular signaling; it's one of the signal-boosting enzymes that helps the cell react quickly to changes in its environment. It modifies other proteins, affecting their activity. Some of its targets are proteins involved in neurotransmission (signaling between brain and nerve cells) and long-term potentiation (reinforcing those connections between neurons that are frequently used). It's this latter process that the study authors think may be disrupted in regressive autism.
SFARI News posted some time ago on a study published on August 31 in PLoS ONE, comparing the amount of a certain enzyme present in tissue extracts from different regions of the brain between deceased subjects with and without autism who had donated their brains to the National Institute of Child Heath and Development Brain and Tissue Bank for Developmental Disorders.

The enzyme in question is protein kinase A, which plays a hugely important role in the cell, helping mediate a process called signal transduction, through which the cell is able to react to its changing environment, or to signals from other cells. In signal transduction, a molecule from outside the cell (usually a hormone) attaches to a receptor outside the cell and causes the receptor to change shape, thus altering the part of the receptor that's inside the cell and triggering a chain reaction of changes in enzymatic activity within the cell.

Protein kinase A participates in one particular signaling pathway: the one involving a class of receptors called G proteins, which are actually clusters of several smaller proteins that split apart whenever something attaches to its extracellular binding site. The now-mobile subunits then go on to do other things in the cell, most importantly to activate* an enzyme responsible for turning adenosine monophosphate (AMP) into cyclic AMP, which works as a signaling molecule inside the cell.
(Here is a cartoon from Nature Publishing Group's Scitable website illustrating that splitting apart of the G protein after a signaling molecule binds to its associated receptor; I adapted the image somewhat to make it less busy)
(Cyclic AMP)

Cyclic AMP is part of a class of molecules called "second messengers," which are small molecules that can bind to, and either activate or inhibit, a wide range of enzymes. Also, the enzymes responsible for making these molecules are regulated by receptors on the surface of the cell, so that when a signaling molecule binds to the receptor, the enzyme gets switched on (in the case of adenylyl cyclase, which is what turns regular AMP into cAMP) and starts churning out second-messenger molecules, which then go on to tinker with their target enzymes. In this way --- by coupling receptor binding with synthesis of these second-messenger molecules --- the cell can amplify the signal it receives, allowing it to react more quickly to changes in its environment.(Cartoon showing signal transduction using cyclic AMP as a second messenger, taken from this community college's Anatomy & Physiology II webpage. You can see how a hormone binding to its receptor frees up the receptor-coupled G protein to exchange its GDP for GTP and then go off and --- depending on the hormone --- either activate or inhibit adenylyl cyclase, which either starts or stops churning out cAMP, which goes on to do lots of different things, like activating enzymes, telling the cell to secrete various things, opening ion channels, etc. The only thing I don't like about this cartoon is that it only shows one cAMP molecule as the output of all the running around happening in the cell membrane, when really cAMP is being continuously produced by every active adenylyl cyclase. So, what that looks like, relative to the amount of hormone coming to the cell from outside, is more like this other cartoon, down below) (See, look at the arrows coming out of that yellowish triangle. One arrow splits into five, then 25, then more than you can clearly see. This table from the Memorial University of Newfoundland's cell biology webpage lists the number of molecules affected by each step in a cAMP-dependent signaling pathway, from the one molecule changed when a single molecule of hormone binds to its receptor, to the 10,000 molecules changed by the time adenylyl cyclase starts producing cAMP).

Anyway, protein kinase A is one of the enzymes activated by cAMP binding to it, and it is also mostly a regulatory enzyme --- that is, it activates or deactivates other enzymes. Protein kinase A does that by transferring a phosphate group from ATP (a small molecule made up of a sugar, a nucleotide base and three phosphate groups) to certain amino acid residues on any of its target proteins.

What kinds of proteins does protein kinase A regulate? Well, that depends on what kind of cell all this is taking place in. Every cell in the body contains a complete human genome; the differences between cell types are differences in which genes are expressed --- i.e., which proteins are present. So each cell type is going to have a different mix of proteins whose activity needs to be coordinated.

Some of its targets are proteins expressed in almost every cell type: these include a histone, one of a large family of proteins whose function is to condense chromosomal DNA that is not actively being transcribed or replicated; transcription factors (most notably, from the CREB family); a metabolic enzyme involved in storing energy for later use; ion channels; and other kinases (enzymes that alter the activity of other proteins by transferring phosphate groups onto them from ATP).

Although protein kinase A performs specialized functions in just about every cell type, I'm only going to talk about what it does in the brain, since that is the cell type relevant to this post. There, in addition to the stuff mentioned above, protein kinase A 1) helps regulate the synthesis of a common precursor to a variety of neurotransmitters, 2) helps form synapses by guiding the specialized proteins that allow the membranous sacs that deliver neurotransmitters from one neuron to the next toward the tip of the developing axon, and 3) with another protein kinase, regulates the ion-channel activity of the NMDA receptor, which is involved in strengthening the more frequently-used conntections between neurons. There may be more, but this is what I've been able to find.

For all that background information, the experiment I'm going to describe is actually pretty simple: like I said above, the researchers took tissue samples from five different regions of donated brains from autistic and non-autistic subjects, homogenized them (basically, ran them through a blender) and tested each sample for protein kinase A activity. The test they used is called the ELISA (for Enzyme-Linked ImmunoSorbent Assay --- see why people would rather call it Eliza?), which is a plastic plate covered with small circular wells (0.7 cm across by 1 cm deep) with, in this case, short peptides containing either serine or threonine (the two amino acids to which protein kinase A can attach a phosphate group), anchored to the bottom. (ELISA is most often used to test for the presence of antibodies --- that's how HIV testing is done --- so for that, the thing stuck to the bottom of the well would be the antigen to which whatever antibody you're testing for responds). They added their brain tissue extracts one by one to each well, along with a small amount of ATP dissolved in water (for the protein kinase to "borrow" phosphate groups from), then waited an hour and a half before emptying out the wells (the substrates, which were permanently affixed to the bottoms of the wells, would stay, along with, presumably, any phosphate groups that had been attached to them during the previous 90 minutes) and introducing an antibody specifically designed to bond with the phosphorylated form of the substrate peptide. Next, they washed the wells out thoroughly (to weed out everything that was not chemically bonded to the fixed substrates) and added a second antibody, chosen for its ability to bind to the first antibody, and which was also attached to an enzyme known for producing dramatic color changes as a side effect of its interaction with certain organic molecules. (A solution containing the molecule in question was also added, so that the wells in which the greatest proportion of the well-bottom peptides had been phosphorylated, and thus had the whole antibody rigmarole sticking off of them, would have the deepest color. There is even a way to measure color --- a device that can measure the degree to which something absorbs light at a given wavelength --- so that you don't have to rely on just your eyes to tell you whether this well or that one is a darker shade of yellow).

They used a somewhat similar technique, called Western blotting, to compare the amount of active protein kinase A between groups for each brain region. They injected their tissue samples from each of the different brain regions into a polyacrylamide gel, and ran an electric current through the gel to get the proteins to move through it. Since the gel resists having things move through it, different size proteins will travel through it at different rates. After a while, most of the proteins will separate themselves into bands along the gel, by size. Once this happened, the researchers transferred the proteins to a nitrocellulose membrane, and added antibodies specific to the catalytic (active) subunit of protein kinase A. Just like with the ELISA, there was also a secondary antibody coupled to a color-producing enzyme.

One thing that's a bit unusual in this study is that the researchers divided their brains from autistic donors into two groups, based on the developmental history of the donors. They had a "regressive autism" group, whose members started out developing typically but then lost some of the skills they'd acquired: speech was the most common skill that was lost, but some of the donors in this category also lost social skills and interest in social interaction. There was also a "non-regressive autism" group, whose members were delayed in language and social development from birth.

Subtyping autism is an increasingly popular thing for researchers to do, since "autism" is such a broad, flexible category that encompasses people with a very wide range of developmental and medical histories. It makes sense that researchers would want to subdivide this large, diverse group further to make sure they're comparing apples to apples when they look at different studies of "the autistic brain" or "the autistic immune system" or whatever.

The thing that's strange about subtyping in this study is that the number of brains being looked at is already so small. Each big group (autism, both regressive and not, and controls) had samples from ten people in it, and the researchers couldn't always get a sample from every point of interest on every brain, so sometimes the number of samples in a given category (brain region + donor neurotype) was less than ten; the smallest n for any category was 7. But that means that, with subtyping, the biggest n possible for either autism subgroup is 5, which looks more like a case study than a comparison across populations. But then, histological studies of donated brains always have to deal with smaller sample sizes, since there isn't exactly a superabundance of donated brains, and I guess if you have big differences among your subjects, you might as well sort them into subcategories, even if your subcategories are tiny.

At some point in this post I should probably mention the results of this study I've gone to such lengths to describe. The authors only found differences in protein kinase A activity in one region --- the frontal cortex --- and this difference was largest between one subgroup of the autistic group --- the autism-with-regression subgroup --- and both the non-regressive autism subgroup and the control group. The regressive autism subgroup had maybe a little less than half the PKA activity of the controls and the non-regressive autism subgroup (those two groups did not differ). Taken as a whole, the autism group had about 35% less PKA activity in the frontal-lobe samples than the control group.

The results were similar for the Western blot; the only region that showed any differences in PKA expression was the frontal lobe, and again, it was only the regressive autism subgroup that differed. Tissue extracts from that group had siginificantly less PKA in them than extracts from either the control group or the non-regressive autism subgroup; the unified autism group did not differ from the control group.

The researchers also looked for a correlation between their measure of PKA activity and various possible confounding factors, like how long each donor had been dead, the age of the donors when they died, whether they had any history of seizures, and what medications they were taking; they didn't find any relationship between any of these things and either outcome variable. Their measure of PKA expression also involved measuring how much of another protein was present in each tissue extract, both because that protein is about the same size as PKA, and thus cannot be separated from it using electrophoresis, and also to have a protein whose expression is not expected to vary across groups with which to compare relative amounts of the protein that is expected to vary.

Here is a picture of the Western blot showing both PKA (top row) and the other protein, a structural protein called beta-actin (bottom row), from all tissue samples:(Figure 2A, in Ji et al., 2011 - samples from autistic donors are on the left, and subdivided into non-regressive and regressive subtypes. Controls are on the right. You can see that, in the bottom row, the blobs are all approximately the same size, indicating expression of beta-actin is more or less the same across groups. You can also see that the blobs in the top row are a lot thinner - one space has nothing at all in it - in the regressive autism group than they are in either the non-regressive autism group or the control group. It looks like PKA expression is a bit more variable within the control group than beta-actin is, though.)

So, for a couple of reasons --- the extreme smallness of sample size, and also the degree of variation in PKA expression within the control group --- I am a bit skeptical as to whether this finding will hold up. It definitely needs to be tested a few more times, with bigger donor pools.

Leaving that aside, though --- what are the implications of this finding, should it be substantiated? The study authors refer to earlier literature that describes a role for cAMP signaling pathways in both brain development (obviously germane to a study about developmental disability) and long-term memory formation and learning (relevant to the question of how people can lose skills they once had). But it's not clear yet exactly what that role is; if you search for "protein kinase a brain" on BioNOT (a database of negative experimental results), you find an article claiming to find no difference in PKA activity between tissue samples taken from donors with Alzheimer's disease and those taken from healthy donors. So that complicates things a bit, as Alzheimer's is, even more than regressive autism, characterized by a loss of learned skills and memories.

Ji, L., Chauhan, V., Flory, M., & Chauhan, A. (2011). Brain Region–Specific Decrease in the Activity and Expression of Protein Kinase A in the Frontal Cortex of Regressive Autism PLoS ONE, 6 (8) DOI: 10.1371/journal.pone.0023751

*What does it mean to activate an enzyme? Well, an enzyme is a kind of protein, and like all proteins, it has a range of three-dimensional configurations** it can assume, and only some of these possible shapes leave the binding site for the molecule the enzyme acts upon freely accessible. So when an enzyme is in one of those arrangements, and molecules of its particular substrate can just drift along and come into contact with the binding site(s), that's when the enzyme can be considered active. Binding of a phosphate group or some other small molecule at a different binding site will usually trigger a shape change; that is how enzymes can be activated or deactivated by other enzymes.

**I have this idea that proteins are called proteins just because of this shape-changing ability they have, in which they resemble the mythical Proteus.


Lindsay said...

Well, that's depressing ... I got five spam comments on this post, and three of them are for porn.

How's that song go again?

Clay said...

I would comment, except the whole thing is so far over my head... :-P

Lindsay said...

@Clay - oh, rats. My goal with these posts is to make them intelligible to non-scientists.

Signal transduction might be one of the hardest things to write clearly about, though --- all those different steps where molecule X does something to molecule Y, which causes Z to happen ...

What could I do differently to make posts like this more readable?

I know that keeping it shorter helps; that's why I do the Executive Summaries. (I want to go into detail, too, though, because that's something I can provide that regular media coverage can't. So, while I know longer posts are harder for the reader to digest, I need the length if I want to spell out exactly how an experiment was done, or what a particular molecule does and why scientists care about it). But maybe I should be less shy about splitting posts up, having a separate post for each thing I want to talk about. Like, maybe instead of having one monster post about this study, maybe I should've written one short post on what signal transduction is, one on cAMP and protein kinase A, and THEN one about this particular experiment?

Apoptosis said...


Thanks for providing these useful tips over here. Signal transduction mechanisms that regulate the development and structural plasticity of excitatory synapses, these functional and structural changes of synapses, collectively referred to as synaptic plasticity...

That Hairy Canadian said...

I think that you did an excellent job of explaining the processes. I'd give it an A+.

You took a dry and somewhat confusing subject and brought it to life perfectly. Your choice of graphics worked well. Although I found your cross references and links were spot on, they may have been confusing to readers more used to a non-technical style of writing.

I don't know whether breaking it up into smaller posts would be a good idea. If I were to suggest any alterations I would try to break up some of the longer sentences. That would be a small shame since they are written so well, but could improve the over-all readability.

I'm going to be sharing the link to this post often. I hope it doesn't disappear.

Anonymous said...

I didnt understand the concluding part of your article, could you please explain it more?

Clay said...

@That Hairy Canadian - FINALLY! An intelligent comment on this post. (You have no idea how many spam/porn spam comments have had to be deleted, some in Russian, some in Chinese.)

That Hairy Canadian said...

@Clay - Thanks for the compliment, I hope I continue to deserve such praise. I wasn't trying to be smart, so much as trying to be constructive. Anything I had to say pales in comparison to Lindsay's insighful knowledge on the subject at hand.

BTW - How do you know it wasn't me who posted all of those previous spam comments? ;)

Clay said...

@That Hairy Canadian: Some of those spam comments were in Russian, some in Chinese. Many/most had links to what I assume were porn sites.

Anonymous said...

Dear Lindsay,

This article looks to be fascinating!

I'm a relatively young sapling - 24, with Asperger's, best analogised as positive/negatively-charged ions controlling water uptake, etc - and am in no way qualified to answer any specific charges articulated here, specifically Long-Term Potentiation on which I wish to hone.

I hope, in time, to draw upon the experimental data, if, at the very least, in raising my vigilance in not falling sucker (if they can be apprehended and, if it holds, represented through mental states - as an internal cognitive cavity - and/or related phenomena?) to precisely these chronic incidences that result in cognitive decline.

As much as I wish to conjecture here, my inexperience is vast. I want to link with it very much.

This is, by all accounts, a sloppy surmise from me. Please pardon my indiscretions and, hopefully, you'll see (in time) that with your intent, I may find the combination that unlocks those, provisionally and philosophically.

Kindest regards,

(Yours) urgently,

Richard Taylor