Lactose intolerance and the proteins of the synaptic cleft

What does lactose intolerance have to do with the zillions of proteins happily infesting the synaptic cleft?  Only someone whose mind was warped into very abstract thinking by rooming with philosophy majors in college would see a connection.

The synaptic cleft is of immense theoretical interest to neuroscientists, drug chemists and pharmacologists, and of great practical interest to people affected by neurologic and psychiatric disease either in themselves or someone they know (e.g. just about everyone).

Almost exactly a year ago I wrote a post about a great paper on the proteins of the synaptic cleft by Thomas Sudhof.  You may read the post after the *****

Well Dr. Sudhof is back with another huge review of just how synapses are formed [ Neuron vol. 100 pp. 276 – 293 ’18 ], which covers very similar ground.

It is clear that he’s depressed by the state of the field.  Here are a few quotes

“I believe that we may need to pay more attention to technical details than customary because the pressures on investigators have increased the tendency to publish preliminary results, especially results obtained with new methods whose limitations are not yet clear.”

Translation: a lot of the stuff coming out is junk.

“Given the abundance of papers reporting non-validated protein interactions that cannot possibly be all correct, it seems that confidence in a possible protein-protein interaction requires either isolation of a stable complex or biophysical measurements of interactions using recombinant purified proteins.”

Translation:  Oy vey !

“Pre- or postsynaptic specializations are surprisingly easy to induce by diverse signals. This was first shown in pioneering studies demonstrating that polylysine beads induce formation of presynaptic nerve terminals in cultured neurons and in brain in vivo.” Obviously this means that you have to be very careful when you claim that a given protein or two causes a synapse to form, which researchers have not been.”

Translation not needed.

Then on to the meat of the review.  “An impressive number of candidate synaptic Cell Adhesion Molecules (CAMs) has been described (9 classes are given each with multiple members). For some of these CAMs, compelling data demonstrate their presence in synapses and suggest a functional role in synapses. Others, however, are less well documented. If one looks at the results in total, the overall impression is puzzlement: how do so many CAMs contribute to shaping a synapse?”

Then from 281 – 286 he goes into the various CAMs, showing the extent and variety of proteins found in the synaptic cleft.  Which ones are necessary and what are they doing?  Can they all be important.  There must be some redundancy as knockout of some doesn’t do much.

Here is where lactose tolerance/intolerance comes in to offer succor to the harried investigator.

Bluntly, they must be doing something, and something important,  or they wouldn’t be there.

People with lactose intolerance have nothing wrong with the gene which breaks down lactose.  Babies have no problem with breast milk.  The enzyme (lactase)  produced from the gene is quite normal in all of us.  However 10,000 years ago and earlier, cattle were not domesticated, so there was no dietary reason for a human weaned from the breast to make the enzyme.  Something turned off lactase production — from my reading, it’s not clear what.   The control region (lactase enhancer) for the lactase gene is 14,000 nucleotides upstream from the gene itself.  After domestication of cattle, so that people could digest milk their entire lives a mutation arose changing cytosine to thymine in the enhancer.  The farthest back the mutation has been found is 6.500 years. 3 other mutations are known, which keep the lactase gene expressed past weaning.  They arose independently.  All 4 spread in the population, because back then our ancestors were in a semi-starved state most of the time, and carriers had better nutrition.

How does this offer succor to Dr. Sudhof?  Simply this, here is a mechanism to turn off production of an enzyme our ancestors didn’t need past weaning.  Don’t you think this would be the case for all the proteins found in and around the synapse.  They must be doing something or they wouldn’t be there.  I realize that this is teleology writ large, but evolutionary adaptations make you think this way.

*****

The bouillabaisse of the synaptic cleft

The synaptic cleft is so small ( under 400 Angstroms — 40 nanoMeters ) that it can’t be seen with the light microscope ( the smallest wavelength of visible light 3,900 Angstroms — 390 nanoMeters).  This led to a bruising battle between Cajal and Golgi a just over a century ago over whether the brain was actually made of cells.  Even though Golgi’s work led to the delineation of single neurons he thought the brain was a continuous network.  They both won the Nobel in 1906.

Semifast forward to the mid 60s when I was in medical school.  We finally had the electron microscope, so we could see synapses. They showed up as a small CLEAR spaces (e.g. electrons passed through it easily leaving it white) between neurons.  Neurotransmitters were being discovered at the same time and the synapse was to be the analogy to vacuum tubes, which could pass electricity in just one direction (yes, the transistor although invented hadn’t been used to make anything resembling a computer — the Intel 4004 wasn’t until the 70s).  Of course now we know that information flows back and forth across the synapse, with endocannabinoids (e. g. natural marihuana) being the major retrograde neurotransmitter.

Since there didn’t seem to be anything in the synaptic cleft, neurotransmitters were thought to freely diffuse across it to being to receptors on the other (postsynaptic) side e.g. a free fly zone.

Fast forward to the present to a marvelous (and grueling to read because of the complexity of the subject not the way it’s written) review of just what is in the synaptic cleft [ Cell vol. 171 pp. 745 – 769 ’17 ] http://www.cell.com/cell/fulltext/S0092-8674(17)31246-1 (It is likely behind a paywall).  There are over 120 references, and rather than being just a catalogue, the single author Thomas Sudhof extensively discusseswhich experimental work is to be believed (not that Sudhof  is saying the work is fraudulent, but that it can’t be used to extrapolate to the living human brain).  The review is a staggering piece of work for one individual.

The stuff in the synaptic cleft is so diverse, and so intimately involved with itself and the membranes on either side what what is needed for comprehension is not a chemist but a sociologist.  Probably most of the molecules to be discussed are present in such small numbers that the law of mass action doesn’t apply, nor do binding constants which rely on large numbers of ligands and receptors. Not only that, but the binding constants haven’t been been determined for many of the players.

Now for some anatomic detail and numbers.  It is remarkably hard to find just how far laterally the synaptic cleft extends.  Molecular Biology of the Cell ed. 5 p. 1149 has a fairly typical picture with a size marker and it looks to be about 2 microns (20,000 Angstroms, 2,000 nanoMeters) — that’s 314,159,265 square Angstroms (3.14 square microns).  So let’s assume each protein takes up a square 50 Angstroms on a side (2,500 square Angstroms).  That’s room for 125,600 proteins on each side assuming extremely dense packing.  However the density of acetyl choline receptors at the neuromuscular junction is 8,700/square micron, a packing also thought to be extremely dense which would give only 26,100 such proteins in a similarly distributed CNS synapse. So the numbers are at least in the right ball park (meaning they’re within an order of magnitude e.g. within a power of 10) of being correct.

What’s the point?

When you see how many different proteins and different varieties of the same protein reside in the cleft, the numbers for  each individual element is likely to be small, meaning that you can’t use statistical mechanics but must use sociology instead.

The review focuses on the neurExins (I capitalize the E  to help me remember that they are prEsynaptic).  Why?  Because they are the best studied of all the players.  What a piece of work they are.  Humans have 3 genes for them. One of the 3 contains 1,477 amino acids, spread over 1,112,187 basepairs (1.1 megaBases) along with 74 exons.  This means that just over 1/10 of a percent of the gene is actually coding for for the amino acids making it up.  I think it takes energy for RNA polymerase II to stitch the ribonucleotides into the 1.1 megabase pre-mRNA, but I couldn’t (quickly) find out how much per ribonucleotide.  It seems quite wasteful of energy, unless there is some other function to the process which we haven’t figured out yet.

Most of the molecule resides in the synaptic cleft.  There are 6 LNS domains with 3 interspersed EGFlike repeats, a cysteine loop domain, a transmembrane region and a cytoplasmic sequence of 55 amino acids. There are 6 sites for alternative splicing, and because there are two promoters for each of the 3 genes, there is a shorter form (beta neurexin) with less extracellular stuff than the long form (alpha-neurexin).  When all is said and done there are over 1,000 possible variants of the 3 genes.

Unlike olfactory neurons which only express one or two of the nearly 1,000 olfactory receptors, neurons express mutiple isoforms of each, increasing the complexity.

The LNS regions of the neurexins are like immunoglobulins and fill at 60 x 60 x 60 Angstrom box.  Since the synaptic cleft is at most 400 Angstroms long, the alpha -neurexins (if extended) reach all the way across.

Here the neurexins bind to the neuroligins which are always postsynaptic — sorry no mnemonic.  They are simpler in structure, but they are the product of 4 genes, and only about 40 isoforms (due to alternative splicing) are possible. Neuroligns 1, 3 and 4 are found at excitatory synapses, neuroligin 2 is found at inhibitory synapses.  The intracleft part of the neuroligins resembles an important enzyme (acetylcholinesterase) but which is catalytically inactive.  This is where the neurexins.

This is complex enough, but Sudhof notes that the neurexins are hubs interacting with multiple classes of post-synaptic molecules, in addition to the neuroligins — dystroglycan, GABA[A] receptors, calsystenins, latrophilins (of which there are 4).   There are at least 50 post-synaptic cell adhesion molecules — “Few are well understood, although many are described.”

The neurexins have 3 major sites where other things bind, and all sites may be occupied at once.  Just to give you a taste of he complexity involved (before I go on to  larger issues).

The second LNS domain (LNS2)is found only in the alpha-neurexins, and binds to neuroexophilin (of which there are 4) and dystroglycan .

The 6th LNS domain (LNS6) binds to neuroligins, LRRTMs, GABA[A] receptors, cerebellins and latrophilins (of which there are 4)_

The juxtamembrane sequence of the neurexins binds to CA10, CA11 and C1ql.

The cerebellins (of which there are 4) bind to all the neurexins (of a particular splice variety) and interestingly to some postsynaptic glutamic acid receptors.  So there is a direct chain across the synapse from neurexin to cerebellin to ion channel (GLuD1, GLuD2).

There is far more to the review. But here is something I didn’t see there.  People have talked about proton wires — sites on proteins that allow protons to jump from one site to another, and move much faster than they would if they had to bump into everything in solution.  Remember that molecules are moving quite rapidly — water is moving at 590 meters a second at room temperature. Since the synaptic cleft is 40 nanoMeters (40 x 10^-9 meters, it should take only 40 * 10^-9 meters/ 590 meters/second   60 trillionths of a second (60 picoSeconds) to cross, assuming the synapse is a free fly zone — but it isn’t as the review exhaustively shows.

It it possible that the various neurotransmitters at the synapse (glutamic acid, gamma amino butyric acid, etc) bind to the various proteins crossing the cleft to get their target in the postsynaptic membrane (e.g. neurotransmitter wires).  I didn’t see any mention of neurotransmitter binding to  the various proteins in the review.  This may actually be an original idea.

I’d like to put more numbers on many of these things, but they are devilishly hard to find.  Both the neuroligins and neurexins are said to have stalks pushing them out from the membrane, but I can’t find how many amino acids they contain.  It can’t find how much energy it takes to copy the 1.1 megabase neurexin gene in to mRNA (or even how much energy it takes to add one ribonucleotide to an existing mRNA chain).

Another point– proteins have a finite lifetime.  How are they replenished?  We know that there is some synaptic protein synthesis — does the cell body send packages of mRNAs to the synapse to be translated there.  There are at least 50 different proteins mentioned in the review, and don’t forget the thousands of possible isoforms, each of which requires a separate mRNA.

Old Chinese saying — the mountains are high and the emperor is far away. Protein synthesis at the synaptic cleft is probably local.  How what gets made and when is an entirely different problem.

A large part of the review concerns mutations in all these proteins associated with neurologic disease (particularly autism).  This whole area has a long and checkered history.  A high degree of cynicism is needed before believing that any of these mutations are causative.  As a neurologist dealing with epilepsy I saw the whole idea of ion channel mutations causing epilepsy crash and burn — here’s a link — https://luysii.wordpress.com/2011/07/17/we’ve-found-the-mutation-causing-your-disease-not-so-fast-says-this-paper/

Once again, hats off to Dr. Sudhof for what must have been a tremendous amount of work

Baudelaire comes to Chemistry

Could an evil molecule be beautiful? In Les Fleurs du Mal, a collection of poems, Baudelaire argued that there was a certain beauty in evil. Well, if there ever was an evil molecule, it’s the Abeta42 peptide, the main component of the senile plaque of Alzheimer’s disease, a molecule whose effects I spent my entire professional career as a neurologist ineffectually fighting. And yet, in a recent paper on the way it forms the fibrils constituting the plaque I found the structure compellingly beautiful.

The papers are Proc. Natl. Acad. Sci. vol. 113 pp. 9398 – 9400, E4976 – E4984 ’16. People have been working on the structure of the amyloid fibril of Alzheimer’s for decades, consistently stymied by its insolubility. The authors solved it not by Xray crystallography, not by cryoEM, but by solid state NMR. They basically looked at the distance constraints between pairs of isotopically labeled atoms, and built their model that way. Actually they built a bouquet of models using computer aided energy minimization of the peptide backbone. Another independent study produced nearly the same set.

The root mean square deviation of backbone atoms of the 10 lowest energy models of the bouquets in the two studies was small (.89 and .71 Angstroms). Even better the model bouquets of the two papers resemble each other.

There are two chains of Abeta42, EACH shaped like a double horseshoe (similar to the letter S). The two S’s meet around a twofold axis. The interface between the two S’s is form by two noncontiguous areas on each monomer (#15 – #17) and (#34 – #37).

The hydrophilic amino terminal residues (#1 – #14) are poorly ordered, but amino acids #15 – #42 are arranged into 4 short beta strands (I only see 3 obvious ones) that stack up and down the fibril into parallel in register beta-sheets. Each stack of double horseshoes forms a thread and the two threads twist around each other to form a two stranded protofilament.

Glycines allow sharp turns at the corners of the horseshoes. Hydrogen bonds between amides link the two layers of the fibrils. Asparagine side chains form ladders of hydrogen bonds up and down the fibrils. Water isn’t present between the layers because the beta sheets are so close together (counterintuitively this decreases the entropy, because water molecules don’t have to align themselves just so to solvate the side chains).

Each of the horseshoes is stabilized by hydrophobic interactions among the hydrophobic side chains buried in the core. Charged residues are solvent exposed. The interface between the two horsehoes is a hydrophobic interface.

Many of the famlial mutations are on the outer edges of double S structure — they are K16N, A21G, D23N, E22A, E22K, E22G, E22Q.

The surface hydrophobic patch formed by V40 and A42 may explain the greater rate of secondary nucleation by Abeta42 vs. Abeta40.

The cryoEM structures we have of Abeta42 are different showing the phenomenon of amyloid polymorphism.

The PNAS paper used reombinant Abeta and prepared homogenous fibrils by repeated seeding of dissolved Abeta42 with preformed fibrils. The other study used chemically synthesized Abeta and got fibrils without seeding. Details of pH, peptide concentration, salt concentration differed, and yet the results are the same, making both structures more secure.

The new structure doesn’t immediately suggest the toxic mechanism of Abeta.

To indulge in a bit of teleology — the structure is so beautiful and so intricately designed, that the aBeta42 peptide has probably been evolutionarily optimized to perform an (as yet unknown) function in our bodies. Animals lacking Abeta42’s parent (the amyloid precursor protein) don’t form neuromuscular synapses correctly, but they are viable.

It ain’t the bricks, it’s the plan

Nothing better shows the utility (and the futility) of chemistry in biology than using it to explain the difference between man and chimpanzee. You’ve all heard that our proteins are only 2% different than the chimp, so we are 98% chimpanzee. The facts are correct, the interpretation wrong. We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

This is like saying Monticello and Independence Hall are just the same because they’re both made out of bricks. One could chemically identify Monticello bricks as coming from the Virginia piedmont, and Independence Hall bricks coming from the red clay of New Jersey, but the real difference between the buildings is the plan.

It’s not the proteins, but where and when and how much of them are made. The control for this (plan if you will) lies outside the genes for the proteins themselves, in the rest of the genome (remember only 2% of the genome codes for the amino acids making up our 20,000 or so protein genes). The control elements have as much right to be called genes, as the parts of the genome coding for amino acids. Granted, it’s easier to study genes coding for proteins, because we’ve identified them and know so much about them. It’s like the drunk looking for his keys under the lamppost because that’s where the light is.

We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

All the molecular biology you need to understand what follows is in the following post — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/

Briefly an enhancer is a stretch of DNA distinct from the DNA coding for a given protein, to which a variety of other proteins called transcription factors bind. The enhancer DNA and associated transcription factors, then loops over to the protein coding gene and ‘turns it on’ — e.g. causes a huge (megaDalton) enzyme called pol II to make an RNA copy of the gene (called mRNA) which is then translated into protein by another huge megaDalton machine called the ribosome. Complicated no? Well, it’s happening inside you right now.

The faces of chimps and people are quite different (but not so much so that they look frighteningly human). The cell paper studied cells which in embryos go to make up the bones and soft tissues of the face called Cranial Neural Crest Cells (CNCCs). How did they get them? Not from Planned Parenthood, rather they made iPSCs (induced Pluripotent Stem Cells — https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell) differentiate into CNCCs. Not only that but they studied both human and chimp CNCCs. So you must at least wonder how close to biologic reality this system actually is.

It’s rather technical, but they had several ways of seeing if a given enhancer was active or not. By active I mean engaged in turning on a given protein coding gene so more of that protein is made. For the cognoscenti, these methods included (1) p300 binding (2) chromatin accessibility,(3) H3K4Me1/K3K4me3 ratio, (4) H3K27Ac.

The genome is big — some 3,200,000,000 positions (nucleotides) linearly arranged along our chromosomes. Enhancers range in size from 50 to 1,500 nucleotides, and the study found a mere 14,500 enhancers in the CNCCs. More interestingly 13% of them were activated differentially in man and chimp CNCCs. This is probably why we look different than chimps. So although the proteins are the same, the timing of their synthesis is different.

At long last, molecular biology is beginning to study the plan rather than the bricks.

Chemistry has a great role in this and will continue to do so. For instance, enhancers can be sequenced to see how different enhancer DNA is between man and chimp. The answer is not much (again 2 or so nucleotides per hundred nucleotides of enhancer). The authors did find one new enhancer motif, not seen previously called the coordinator motif. But it was present in man in chimp. Chemistry can and should explain why changing so few nucleotides changes the proteins binding to a given enhancer sequence, and it will be important in designing proteins to take advantage of these changes.

So why is chemistry futile? Because as soon as you ask what an enhancer or a protein is for, you’ve left physicality entirely and entered the realm of ideas. Asking what something is for is an entirely different question than how something actually does what it is for.  The latter question  is answerable by chemistry and physics. The first question is unanswerable by them.  The Cartesian dualism of flesh and spirit is alive and well.

It’s interesting to see how quickly questions in biology lead to teleology.

Old dog does new(ly discovered) tricks

One of the evolutionarily oldest enzyme classes is aaRS (for amino acyl tRNA synthetase). Every cell has them including bacteria. Life as we know it wouldn’t exist without them. Briefly they load tRNA with the appropriate amino acid. If this Greek to you, look at the first 3 articles in https://luysii.wordpress.com/category/molecular-biology-survival-guide/.

Amino acyl tRNA syntheses are enzymes of exquisite specificity, having to correctly match up 20 amino acids to some 61 different types of tRNAs. Mistakes in the selection of the correct amino acid occurs every 1/10,000 to 1/100,000, and in the selection of the correct tRNA every 1/1,000,000. The lower tRNA error rate is due to the fact that tRNAs are much larger than amino acids, and so more contacts between enzyme and tRNA are possible.

As the tree of life was ascended from bacteria over billions of years, 13 new protein domains which have no obvious association with aminoacylation have been added to AARS genes. More importantly, the additions have been maintained over the course of evolution (with no change in the primary function of the synthetase). Some of the new domains are appended to each of several synthetases, while others are specific to a single synthetase. The fact that they’ve been retained implies they are doing something that natural selection wants (teleology inevitably raises its ugly head with any serious discussion of molecular biology or cellular physiology — it’s impossible to avoid).

[ Science vol.345 pp 328 – 332 ’14 ] looked at what mRNAs some 37 different AARS genes were transcribed into. Six different human tissues were studied this way. Amazingly, 79% of the 66 in-frame splice variants removed or disrupted the aaRS catalytic domain. . The AARS for histidine had 8 inframe splice variants all of which removed the catalytic domain. 60/70 variants losing the catalytic domain (they call these catalytic nulls) retained at least one of the 13 added domains in higher eukaryotes. Some of the transcripts were tissue specific (e.g. present in some of the 6 tissues but not all).

Recent work has shown roles for specific AARSs in a variety of pathways — blood vessel formation, inflammation, immune response, apoptosis, tumor formation, p53 signaling. The process of producing a completely different function for a molecule is called exaptation — to contrast it with adaptation.

Up to now, when a given protein was found to have enzymatic activity, the book on what that protein did was closed (with the exception of the small GTPases). End of story. Yet here we have cells spending the metabolic energy to make an enzymatically dead protein (aaRSs are big — the one for alanine has nearly 1,000 amino acids). Teleology screams — what is it used for? It must be used for something! This is exactly where chemistry is silent. It can explain the incredible selectivity and sensitivity of the enzyme but not what it is ‘for’. We have crossed the Cartesian dualism between flesh and spirit.

Could this sort of thing be the tip of the iceberg? We know that splice variants of many proteins are common. Could other enzymes whose function was essentially settled once substrates were found, be doing the same thing? We may have only 20,000 or so protein coding genes, but 40,000, 60,000, . . . or more protein products of them, each with a different biological function.

So aaRSs are very old molecular biological dogs, who’ve been doing new tricks all along. We just weren’t smart enough to see them (’till now).

Novels may have only 7 basic plots, but molecular biology continues to surprise and enthrall.