Cholesin

You wouldn’t think that there was anything more to be said about cholesterol metabolism after decades of work by med school classmate Mike Brown and a host other researchers.  But there is.

The body can synthesize cholesterol starting from scratch and Mike found out how this is inhibited when cholesterol levels get too high.  Here is a brief summary of how this happens from a recent paper [ Cell vol. 187 pp. 1685 – 1700 ’24 ]

“Cholesterol biosynthesis and uptake are tightly regu-lated through a negative feedback mechanism that senses the cellular cholesterol levels. When cells are deficient in cholesterol, SREBP2, along with its escort protein SREBP cleavage-acti- vating protein (SCAP), is transported in coat protein complex II (COPII) vesicles from the endoplasmic reticulum (ER) to the Golgi apparatus. In the Golgi, SREBP2 is sequentially cleaved by site-1 and site-2 proteases. The N-terminal domain of SREBP2, released by this cleavage, travels to the nucleus, where it func- tions as a transcription factor to enhance the expression of genes involved in cholesterol synthesis and uptake. Conversely, when cellular cholesterol levels rise, cholesterol molecules bind to SCAP, triggering its interaction with insulin-induced gene (INSIG). This interaction retains SREBP in the ER and prevents the subsequent activation of SREBP and the expression of genes involved in cholesterol metabolism”.

 

Well now you can see why this took decades to figure out.

However a recently discovered protein cholesin cuts off cholesterol synthesis when you eat and absorb cholesterol, which is much more proactive as it doesn’t wait for cholesterol levels to increase.   Cholesin is secreted into the blood by the gut when cholesterol is absorbed (secretion into the blood is what makes it a hormone).   Human cholesin contains 195 amino acids and works its magic by binding to a G Protein Coupled Receptor (GPCR) called GPCR146 which shuts off signaling by protein kinase A (PKA). This prevents  SREPB2 from turn on cholesterol synthesis (primarily in the liver).

So obviously GPCR146 and cholesin do a biochemical dance together.  Amazingly, dance is more than a metaphor, and the two proteins are coded (and entwined) on opposite strands of the same genetic locus of chromosome #7 with the code for GPCR146 on one strand inside the code for cholesin on the other.

I find this both bizarre and fantastic.  The discoveries of molecular biology never cease to amaze (me at least, and you too if your molecular biological soul isn’t completely dead).

The viruses in our brains

PNMA2 (ParaNeoplastic antigen MA2) is a protein initially found as the target of the immune response (autoantibodies) producing a nasty dementing neurologic disease (Paraneoplastic encephalitis).  The PNMA2 protein is exclusively expressed in neurons which implies that neurons are using it for something.   This is teleological thinking, usually looked down on, but always needed in molecular biology and cellular physiology.

What PNMA2 does is amazing.  It forms icosahedral viral capsids which are released from cells (in culture) as nonEnveloped capsids.  It isn’t clear if this normally happens in our  brains.    Probably it doesn’t, and when the capsid somehow gets out of the producing cell or neuron immunological hell breaks loose and autoimmune encephalitis is the result.

PNMA2 is derived from one of the long terminal repeat retrotransposons (LTR retrotransposons), viral remnants that make up 8% of the human genome (https://en.wikipedia.org/wiki/LTR_retrotransposon). This explains why it makes particles that look like viruses.  Such particles can contain RNA, so big pharma is interested in them as a way of delivering mRNA drugs.

Totally off topic but yesterday I read a paper about E. Coli DNA gyrase, an amazing enzyme which untangles DNA ( Science vol. 384 pp. 227 – 232 ’24 ).

Here is what it does.   If you’ve got some venetian blinds in your home twist it 20 or so times (keeping the ends fixed, and you have the DNA double helix, with two strands winding around each other.  Now to read or copy a single strand, you must grab both strands where you want this to happen  and pull them apart keeping the ends of the venetian blind fixed.  This immediately increases the coiling elsewhere. Since there are only 10 nucleotides/turn of the double helix, copying a gene for a 100 amino acid protein means you are removing 33 twists from the separated strands (and producing new ones elsewhere).   The cords of the venetian blind quickly become a tangled mess when this happens.  This is where DNA gyrase comes in.  It cuts both strands of the DNA double helix, holding on to the cut ends, and slides an intact double helix of the twisted DNA through the cut.   Sounds fantastic doesn’t it?  Hard to see how evolution could come up with something like this but it did.

The paper contains the following passage toward the end

“A second model based on a sign-inversion reaction wassuggested to describe introduction of (–)SC by this enzyme (28). This model proposed that the enzyme binds to a positive crossover followedby a DNA strand passage through a DNA double-strand break that results in a sign inversion.”

(28) is 28. P. O. Brown, N. R. Cozzarelli,Science206, 1081–1083 (1979).

The paper is 45 years old and has now been shown to be correct.  N. R.  Cozzarelli is my late good friend and Princeton classmate Nick, and it is very nice to see him honored here.

A few words about Nick.  Although Princeton was full of rich kids, they still had the brains to take in someone like Nick whose father was an immigrant shoemaker in Jersey City.  Nick worked his way through Princeton waiting on tables in commons (where all Freshmen ate).  I can still see the time that some rich preppie jerk gave him a hard time about the service.

Nick got his PhD at Harvard and later became a professor at Berkeley where he did his great work.  Nick later edited the Proceedings of the National Academy of Sciences (USA) for 10 years before his very untimely death over 20 years ago from Burkitt’s lymphoma.  R. I. P. Nick.

Why me, O Lord, Why me?

It was very hard for my multiple sclerosis (MS) patients to understand why they were singled out for MS, given the publicity given to theories of viral causation, popular at least since I started getting seriously interested in neurology as a 3rd year medical student in 1964.  Herpes simplex (fever blisters) was a popular culprit, but we all know lots of people who’ve had them without coming down with MS.

The best explanation I could give them was of my med school classmate Marty, a Jewish kid from Pittsburgh.  Graduating in 1966 at the height of American involvement in Vietnam, all my classmates entered the service within a few years.  Marty was sent to Vietnam  as a GMO (General Medical Officer).  He was quickly sent back stateside as he developed a severe anemia.  Why?

Well malaria was endemic in Vietnam, and anyone going over received an antiMalarial as prophylaxis.  The malarial parasite does its damage by infecting red blood cells.  The antiMalarial drug he received inhibited a red cell enzyme Glucose 6 Phosphate Dehydrogenase (G6PD), essentially starving the parasites.  Marty had a partial deficiency of this enzyme.  Such deficiencies are relatively common in areas endemic for Malaria, as it is protective, just as the sickle cell trait is protective against Malaria in Africa.  A variety of other mutations in different red cell proteins arising in endemic areas are also protective (example Thalassemia in Greece, etc. etc.)

So if Marty had never been sent to Vietnam he would never have become anemic.  I’d tell my patients that they had some biochemical difference (totally unknown back then) that made them susceptible to complications of infection with a common organism.  Not very satisfying, but it was the best I could do.

In the case of another virus Epstein Barr Virus (EBV) which causes infectious mononucleosis, this explanation (50+ years later) turned out to be exactly correct.    Not only that it shows the extreme subtlety of what ‘causation’ in medicine actually means.

Not only must the unlucky people getting MS after EBV infection be different biochemically, they must be infected with a particular variant of EBV (not all EBV is the same, just as not all people or SARS-CoV-2 are the same).

That’s the view from 30,000 feet.  You can stop here but the full explanation is unsparingly technical.  It is to be found in Cell vol. 186 pp. 5675 – 5676, 5708 – 5718 ’23 ]

Here goes.

Long term control of Epstein Barr Virus is mediated by cytotoxic T lymphocytes which recognize parts of EBV proteins.  One such protein is EBNA1, and antibodies to amino acids #386 – #405 of EBNA1 cross react with amino acids #370 – #389 of a human protein called GlialCAM (for Glial Cell Adhesion Molecule) which is important in maintaining the myelin sheath around axons in the brain (MS is basically a destructive immune attack on myelin).

Such an antibody is called autoreactive, in the sense that it is reacting to a normal human protein.  Cells producing autoreactive antibodies (autoreactive cells) are normally eliminated by cytotoxic natural killer cells.  In the case of EBV specific T cells they are eliminated by natural killer cells expressing proteins NKG2C and NKG2D.  They target the autoreactive GlialCAM specific autoreactive B cells.  Some people have deletion of the gene coding for NKG2C rendering them more susceptible to MS after EBV infection.

But wait, there’s more. There are many EBV variants and some of them upregulate another human protein HLA-E by containing another protein (LMP1) which stabilizes HLA-E.  HLA-E blunts the natural killer cell attack on autoreactive GlialCAM cells.

So it’s a delicate dance of unfortunate events ‘causing’ MS.  A mild genetic defect in the human, and a genetic variant in the virus, both of which must occur for causation.

Who knew that medical causation could be so subtle.

Why studying the cell is like the blind men and the elephant

I’ve been reading about stress granules for over 20 years.  My notes on them contain over 60,000 characters and have my summaries of the information in over 50 papers.  They go by a lot of names — processing body, P body etc.  They are an example of phase separation in the cell, similar to other better known players such as the nucleolus.  They are formed by the cellular response to a variety of stresses — starvation, lack of oxygen, reactive oxygen species, changes in cellular pH, problems with mRNA translation into protein, etc. etc.

One protein called G3BP1 is constantly found in them, but like a lot of phase separated bodies their composition isn’t fixed and they contain lots of different proteins and RNAs which come and go from the body.  Heraclitus would have loved the stress granule, you never step into (study) the same stress granule twice. They may be a new form of matter — https://luysii.wordpress.com/2015/12/06/a-new-form-of-matter/.  They are fascinating to the chemist as their composition (stoichiometry) isn’t constant — https://luysii.wordpress.com/2022/07/20/bye-bye-stoichiometry-2/  and chemists, particularly physical chemists have spent a lot of time studying them.

Most of their components (mostly proteins and RNAs) are characterized by having multiple areas which can bind to other areas.  Protein sequences able to bind to RNA are common in them, as are areas of proteins which never settle down to a single structure, and areas of proteins with a very simple amino acid composition (low complexity domains).  So phase separated bodies are a fascinating field of study for cell biologists, molecular biologists, protein chemists, physical chemists, physicists and (not so much) organic chemists, all busily studying away about their chemical properties.

Into this mix comes a completely different way of looking at stress granules, e.g. as molecular plugs for holes arising in the cellular membranes found in lysosomes and endosomes.  This is an entirely new (and very important) function for stress granules, which hadn’t even been considered (until now).  Here, their physicality rather than their chemical nature is what matters, allowing another set of blind men to study the stress granule elephant in a completely different way.  For details please see Nature vol. 923 pp. 919 – 920, 1062 – 1069 ’23.  Given the subject matter, I find it fascinating that one Alex S. Holehouse is of the authors of 919 – 920

Res Ipsa Loquitur

The first sequencing of the human genome was ‘completed’  in April 2003 taking 13 years and costing over a billion dollars.

It really wasn’t complete, and even by 2022 10 megaBases (of the 3.2 gigaBases) hadn’t been sequenced

Science vol. 382 1 December p. 980 ’23 The UK biobank just released the WHOLE genome sequences of 500,000 people.

Imagine what we’d know if our understanding of the results had similarly accelerated.

 

 

Phase separation strikes again

Phase separated droplets of protein, RNA and God knows what else have gotten everyone’s attention.  For background see after the ***.

Neurologists know that phase separated TDP43 droplets are involved in a variety of neurologic diseases.  For background see after the &&&

Now it’s everyone’s concern because a recent paper [ PNAS vol. 120 e230355120 ’23 ] shows that phase separation is involved in the construction of the pandemic virus SARS-CoV-2.

Cellular Nucleic Acid Binding Protein (CNBP) is part of the interferon generated response to RNA virus infections of our cells.  In response to infection CNBP is phosphorylated and translocates from the cytoplasm to the nucleus where it turns on the interferon beta genes.

SARS-CoV-2 evades detection by our RNA sensing pathways (a story in itself), so CNBP is retained in the cytoplasm.

CNBP has another trick up its sleeve, binding to the 3′ and 5′ long terminal repeats (LTRs) of the viral genome, competing with the viral nucleocapsid protein.  This prevents the two from liquid liquid phase separation (LLPS — another name for macromolecular phase separation) which is critical for viral replication.   Did you know this? I didn’t.  The paper gives 4 references in journals I don’t read all appearing in the last few years.

Cells and animals lacking CNBP have higher SARS-CoV-2 viral loads after infection.

****

Bye bye stoichiometry

I’m republishing this old post from 2018, to refresh my memory (and yours) about liquid liquid phase separation before writing a new post on one of the most interesting papers I’ve read in recent years.  The field has exploded since this was written.

Until recently, developments in physics basically followed earlier work by mathematicians Think relativity following Riemannian geometry by 40 years.  However in the past few decades, physicists have developed mathematical concepts before the mathematicians — think mirror symmetry which came out of string theory — https://en.wikipedia.org/wiki/Mirror_symmetry_(string_theory). You may skip the following paragraph, but here is what it meant to mathematics — from a description of a 400+ page book by Amherst College’s own David A. Cox

Mirror symmetry began when theoretical physicists made some astonishing predictions about rational curves on quintic hypersurfaces in four-dimensional projective space. Understanding the mathematics behind these predictions has been a substantial challenge. This book is the first completely comprehensive monograph on mirror symmetry, covering the original observations by the physicists through the most recent progress made to date. Subjects discussed include toric varieties, Hodge theory, Kahler geometry, moduli of stable maps, Calabi-Yau manifolds, quantum cohomology, Gromov-Witten invariants, and the mirror theorem. This title features: numerous examples worked out in detail; an appendix on mathematical physics; an exposition of the algebraic theory of Gromov-Witten invariants and quantum cohomology; and, a proof of the mirror theorem for the quintic threefold.

Similarly, advances in cellular biology have come from chemistry.  Think DNA and protein structure, enzyme analysis.  However, cell biology is now beginning to return the favor and instruct chemistry by giving it new objects to study. Think phase transitions in the cell, liquid liquid phase separation, liquid droplets, and many other names (the field is in flux) as chemists begin to explore them.  Unlike most chemical objects, they are big, or they wouldn’t have been visible microscopically, so they contain many, many more molecules than chemists are used to dealing with.

These objects do not have any sort of definite stiochiometry and are made of RNA and the proteins which bind them (and sometimes DNA).  They go by any number of names (processing bodies, stress granules, nuclear speckles, Cajal bodies, Promyelocytic leukemia bodies, germline P granules.  Recent work has shown that DNA may be compacted similarly using the linker histone [ PNAS vol.  115 pp.11964 – 11969 ’18 ]

The objects are defined essentially by looking at them.  By golly they look like liquid drops, and they fuse and separate just like drops of water.  Once this is done they are analyzed chemically to see what’s in them.  I don’t think theory can predict them now, and they were never predicted a priori as far as I know.

No chemist in their right mind would have made them to study.  For one thing they contain tens to hundreds of different molecules.  Imagine trying to get a grant to see what would happen if you threw that many different RNAs and proteins together in varying concentrations.  Physicists have worked for years on phase transitions (but usually with a single molecule — think water).  So have chemists — think crystallization.

Proteins move in and out of these bodies in seconds.  Proteins found in them do have low complexity of amino acids (mostly made of only a few of the 20), and unlike enzymes, their sequences are intrinsically disordered, so forget the key and lock and induced fit concepts for enzymes.

Are they a new form of matter?  Is there any limit to how big they can be?  Are the pathologic precipitates of neurologic disease (neurofibrillary tangles, senile plaques, Lewy bodies) similar.  There certainly are plenty of distinct proteins in the senile plaque, but they don’t look like liquid droplets.

It’s a fascinating field to study.  Although made of organic molecules, there seems to be little for the organic chemist to say, since the interactions aren’t covalent.  Time for physical chemists and polymer chemists to step up to the plate.

Neurologic Velcro

Neurons must in some way respond to mechanical stimuli or you wouldn’t have a sense of touch.  A remarkable ion channel called piezo2 in the axon membrane opens in response to stretch of the axon causing a nerve impulse to fire.  They are huge proteins with anywhere from 2,100 to 4,700 amino acids.  Three come together to form an ion channel.  Each monomer contains 35 or so transmembrane segments, arranged in an arc.  (If I could ever figure out how to get an image from a paper or captured on the web into a WordPress document, I’ll update this post, and I’m going to work on it this coming weekend. ) In the meantime content yourself with figure 1 from this https://www.nature.com/articles/s41586-019-1505-8.  The 3 arcs are called blades and are made mostly of alpha helices.   The complex is huge, fitting into a circle of diameter 270 Angstroms.   The structure distorts the membrane, indenting it so the ion is channel formed by the junction of the 3 monomers is closed.  Stretch the membrane and the indentation disappears opening the channel, ions flow inside the axon and a nerve impulse is fired.

Enter Neuron vol. 111 pp. 3211 – 3229 ’23 which has fantastic pictures of 3 types of mechanically sensitive sense organs — the Pacinian corpuscle, the Meissner corpuscle and the lanceolate complex found around hair follicles.  Each picks up a different type of mechanical stimulus, yet they all use the same ion channel — piezo2.

The answer was totally unexpected — each axon has hundreds to thousands of small (1 micron) projections (containing piezo2) looking exactly like velcro (see figure 8 p. 3225) and these are tacked to the nonneuronal cells of the sense organ by adherens junctions, so that when any part of the sense organ is moved piezo2 is stretched and the axon fires.  Again apologies — I hope to get these figures in here over the weekend.

So the specificity of the sense organ for the type of mechanical stimulus doesn’t lie in the neuron’s axon which is the same in all 3, but in the non-neuronal cells it is attached to.

AlphaFold fails the Glass Eye test

It’s been 3 years since AlphaFold blew away the competition in the Critical Assessment of Protein Structure (CASP) coming up with the 3 dimensional structure of proteins given just the amino acid sequence of the protein.  The structure was known to the people at CASP, but not to the contestants.

I wrote a post far back in the mists of time (2009 to be exact — https://luysii.wordpress.com/2009/11/29/time-for-the-glass-eye-test-to-be-inserted-into-casp/) saying that this wasn’t fair, as (1) the contestants knew that the protein had a single structure and (2) some proteins didn’t have a structure in all their parts but had intrinsically disordered region, or were completely disordered (like alpha-Synuclein, the main protein component of the Lewy body found in Parkinson’s disease.

I argued that contestants should be given a protein with an intrinsically disordered region (IDR) to see if they came up with a structure that wasn’t there, similar to the glass eye test inflicted on hapless medical students.

What is the glass eye test all about.  Here is part of the old post.

One of the hardest things for a fledgling doc to learn, is how to do a decent physical examination (PE). Whole courses are given on the subject. One of the hardest parts of the PE is looking into the back of the eye with an ophthalmoscope. This is important (particularly in neurology) because it’s the only place in the body where you can actually see a nerve (the optic nerve) along with arteries and veins (everything else you do in the PE results in just inference about nerves, rather than direct observation). If the optic nerve appears swollen then you know that pressure inside the head is elevated (always serious).

The temptation to fudge is ever present. This is why the patient with the glass eye is so valuable. If one is present and the unlucky med student attempts to say that they saw the nerve, it’s time for a lecture (hopefully not too sadistically). It didn’t happen to me, but the basic lesson is to report what you see honestly, whether you understand it or not — there’s probably someone smarter or with more experience who will (that’s what I heard when I reported a bunch of findings that just didn’t make sense).

Well AlphaFold just got the glass eye test and failed [Proc. Natl. Acad. Sci. vol. 120 e2304302120 ’23 ] It assigned structures (with high confidence to boot) to 15% of intrinsically disordered regions (IDRs).  It is known that some IDRs will fold to a single structure on binding to a target (this is called conditional folding) and here AlphaFold ‘often’ predicts the structure of these states.  Of interested is that mutations causing human disease are 5 times more common in conditionally folded IDRs than IDRs in general.

Even more interesting is that 80% of bacterial IDRs conditionally fold, but only 20% of eukaryotic IDRs do.

In defense of Cassava Sciences — Part I — models

Cassava Sciences has been under attack  since 8/21 reaching a crescendo recently — https://www.science.org/content/article/co-developer-cassava-s-potential-alzheimer-s-drug-cited-egregious-misconduct and https://www.science.org/content/blog-post/saga-cassava.  Both contain demands that their current double blind placebo controlled studies on Simufilam, their Alzheimer drug be stopped.

Full disclosure.  I do not own any Cassava stock and have not received anything from them for writing about them (aside from a free meal from Lindsay Burns 11/21 at the CTAD (Clinical Trials in Alzheimer’s Disease) in Boston.   There was and is no quid pro quo about anything I’ve written.

I do know Derek Lowe and have spent several pleasant afternoons discussing organic chemistry and drug development with him at my cousin’s New Year’s Day bash, before COVID put a stop to the affair.

I’ve known Lindsay since she was a teenager, when I was practicing neurology in Montana.  This was primarily through her parents who were sheep ranchers and good friends of my wife and me.

The basic position of the two articles above, is that some of the protein electrophoreses backing up Cassava’s model of Simufilam were either fudged or missing.

For details about Cassava’s model and the science behind it please see — https://luysii.wordpress.com/2023/04/23/the-science-behind-cassava-sciences-sava-the-latest-as-of-19-april-23/

But now it’s time to talk about models of disease causation and how important and reliable they are.  I’ll start with an impeccable model of Alzheimer’s disease and the exquisite chemistry, biochemistry and genetics backing it up.   It’s technical, but so are the protein electrophoreses which have been criticized.

Basically the model says that the accumulation of Abeta peptides, the major component of the senile plaque, causes Alzheimer’s disease, and removing them should help the disease.

The following is pretty long and if you want to skip the details for the rest of the argument — fast forward to *****

First (and probably the best evidence) is the mutation that protects against Alzheimer’s disease.  As most of you know, the aBeta peptide (39 to 42 amino acids) is part of a much larger protein the Amyloid Precursor Protein (APP) which contains 639 to 770  amino acids.  This means that enzymes must  cut it out.  Such enzymes (called proteases) are finicky, cutting only between certain amino acids.  In what follows A673T stands for the 673rd position which normally has amino acid Alanine (A) there.  Instead there is amino acid Threonine (T).   The enzyme cleaving at 673  is Beta Secretase 1 (BACE1).

       [ Nature vol. 487 pp. 153 ’12 ] A mutation in APP protects against Alzheimer’s disease.   First the genome sequence APP of 1,795 Icelanders  were studied to look for low frequency variants.  They found a mutation A673T adjacent to the site that is cleaved by beta secretase 1 (BACE1) which doesn’t vary — it’s gamma secretase which cleaves at variable sites leading to Abeta40, Abeta42 formation.  The mutation is at position 2 in Abeta.  The mutation results in a 40% reduction in the formation of amyloidogenic peptides  in vitro (293T cells transfected with variant and normal APP). Amazingly, a different variant at 673 (A673V — V stands for the amino acid Valine) — increases Abeta formation.    Because BACE1 can’t cleave APP containing the A673T mutation, alternative processing of APP at another site the alpha site (which is within aBeta preventing formation of the full 39 – 43 amino acid peptide).

So if you can’t make the full aBeta peptide you don’t get Alzheimer’s (or have less chance of getting it).

Then there are the mutations in the part of APP which code for the aBeta peptide which increase the risk of Alzheimer’s.  They cause the different forms of familial Alzheimer’s disease.   Now that we know the actual structure of the aBeta amyloid fiber, we can understand how they do this.  This is further evidence that the aBeta peptide is involved in the causation of Alzheimer’s disease.

You’ll need some protein chemistry chops to understand the following

Recall that in amyloid fibrils the peptide backbone is flat as a flounder (well in a box 4.8 Angstroms high) with the amino acid side chains confined to this plane.  The backbone winds around in this plane like a snake.  The area in the leftmost loop is particularly crowded with bulky side chains of glutamic acid (single letter E) at position 22 and aspartic acid (single letter D) at position 23 crowding each other.  If that wasn’t enough, at the physiologic pH of 7 both acids are ionized, hence negatively charged.  Putting two negative charges next to each other costs energy and makes the sheet making up the fibril less stable.

To make an amyloid fiber just stack 1000 or more of these flounder aBeta peptides  on top of each other.

The marvelous paper (the source for much of this) Cell vol. 184 pp. 4857 – 4873 ’21 notes that there are 3 types of amyloid — pathological, artificial, and functional, and that the pathological amyloids are the most stable.

In 2007 there were 7 mutations associated with familial Alzheimer’s disease (10 years later there were 11). Here are 5 of them.

Glutamic Acid at 22 to Glycine (Arctic)

Glutamic Acid at 22 to Glutamine (Dutch)

Glutamic Acid at 22 to Lysine (Italian)

Aspartic Acid at 23 to Asparagine (Iowa)

Alanine at 21 to Glycine (Flemish)

All of them lower the energy of the amyloid fiber  making them more stable

Here’s why

Glutamic Acid at 22 to Glycine (Arctic) — glycine is the smallest amino acid (side chain hydrogen) so this relieves crowding.  It also removes a negatively charged amino acid next to the aspartic acid (which is also negatively charged).  Putting two negative charges next to each other costs energy — because like charges repel. Both effects lower the energy of the amyloid fiber

Glutamic Acid at 22 to Glutamine (Dutch) — really no change in crowding, but it removes a negative charge next to the negatively charged Aspartic acid

Glutamic Acid at 22 to Lysine (Italian)– no change in crowding, but the lysine is positively charged at physiologic pH, so we have a positive charge next to the negatively charged Aspartic acid, lowering the energy because like charges attract.

Aspartic Acid at 23 to Asparagine (Iowa) –really no change in crowding, but it removes a negative charge next to the negatively charged Glutamic acid next door

Alanine at 21 to Glycine (Flemish) — no change in charge, but a reduction in crowding as alanine has a methyl group and glycine a hydrogen.

As a chemist, I find this immensely satisfying.  The structure explains why the mutations in the 42 amino acid aBeta peptide are where they are, and the chemistry explains why the mutations are what they are.

****

It doesn’t get any better than this, yet therapy based on the model (monoclonal antibodies to remove the aBeta peptide) have produced minimal benefit (a less than 30% decrease in the rate of decline) and serious side effects namely brain hemorrhage, because the aBeta peptides don’t confine themselves to the senile plaque but can be found in the walls of blood vessels.  This is called cerebral amyloid angiopathy (CAA).

What about wildly successful therapies which were found without ‘benefit’ of any theory, with theoreticians scrambling to explain them post hoc.

Here are 3:

l. Lithium for mania– the story is particularly fascinating — https://www.nature.com/articles/d41586-019-02480-0. We still don’t know how it works but theories abound, despite the original discovery reaching the ripe old age of 70.

2. Ketamine analogs for depression.  For decades we were taught that antidepressants take a few weeks to work, and this was true of the drugs we had (tricyclics, selective serotonin reuptake inhibitors).  Hell, I taught it myself back in the day at Montana State University. This was until ketamine analogs (esketamine) lifted depression within hours.  We are still trying to figure out how this works, and what it tells us about depression  (which is almost certainly a lot).

3. Wegovy, Ozempic for weight loss.  I’ve been reading about glucagon like peptide (GLP) for years, because it is a peptide neurotransmitter. People have been studying it for years.   Only when  GLP agonists used to treat diabetes produced weight loss did theorists scramble to figure out why.

As an old med school friend, warped by his experiences at the University of Chicago used to say.  “That’s how it works in practice, but how does it work in theory?”

So here we have a superb model for Alzheimer’s disease which hasn’t produced a terribly useful therapy, and three incredibly useful therapies for which we presently have no satisfying model.

So dumping on Cassava and wishing to stop an ongoing study because the model underneath it is based on flawed data just doesn’t make sense.

In particular, it doesn’t make sense given the data Cassava has already released, but that’s for part II.

The brain just got a lot more complicated

People have been studying the locus coeruleus (LC) for at least 60 years.  For one thing it is easy to see on dissection of the brain (it’s blue — think cerulean blue).  For another it’s contains the largest collection of neurons using norepinephrine as a neurotransmitter (about which much more later) whose effects on arousal, mood, addiction and psychiatric disease have long been known.  So anything altering LC activity is likely to be important clinically.

Neuroscience has largely concentrated on just a few neurotransmitters — glutamic acid, the major excitatory neurotransmitter in brain which opens ion channels causing neurons to fire and gamma amino butyric acid (GABA) the major inhibitory neurotransmitter which shuts ion channels inhibiting neurons from firing.  Then there are the volume neurotransmitters (dopamine, serotonin, norepinephrine, histamine, acetyl choline) about which much more later.

Lastly, there are the peptide neurotransmitters, of which the brain’s own opiates (the enkephalins) are the best known and studied.  There are tons of them —  over 100 are known — https://en.wikipedia.org/wiki/Neuropeptide#:~:text=There%20are%20over%20100%20known,molecules%20in%20the%20nervous%20system.

Technology has marched on and it is now possible to isolate a single neuron and study the messenger RNA (mRNA) it is making.  Not to leave anyone behind, we assume that if the cell is making mRNA coding for a protein, the ribosome will grab the mRNA and make the protein.  This is a major advance, since you don’t have to test for each of the 20,000 different proteins the genome codes for (and we don’t have tests for all of them). What distinguishes the wildly different cell types in our body is the collection of proteins they make, and the way they organize the membranes of the cell.  Each cell type has a different collection of proteins.

Enter Proc. Natl. Acad. Sci. vol. 120 e2222095120 ’23 — which used single cell RNA sequencing (scRNA-seq) on neurons in the rodent locus coeruleus (which has only 3,000 neurons, unlike man which has 50,000.

Based on the RNA found, they were able to study LC neurons making norepinephrine (as judged by presence of mRNAs for the enzymes making it).   The staggering finding (which the authors don’t make much of) is that, as a group,  LC neurons using norepinphrine express mRNAs for 19 different neurotransmitters and 30 neuropeptide receptors.   It is hard for me to find out the maximum or mean number of neurotransmitters and receptors expressed in and on a single neuron (I have written the authors on this point), but we do know that expression of norepinephrine and the neuropeptide galanin is common in this group.

But forget that.  It is reasonable to assume that if a neuron expends the metabolic energy to transcribe a gene for a neurotransmitter receptor into mRNA,  makes the protein corresponding to the mRNA and inserts it into the neuronal membrane — that it will respond to the neurotransmitter.

To respond to a neurotransmitter/neuropeptide a neuron must have a receptor, just as to respond to Ni Hao you must have the language receptor (understand the language) for Chinese.

We’re not in the Kansas of glutamic acid, GABA, norepinephrine, dopamine, serotonin, histamine, acetyl choline any more.  The complexity of 3o different neurotransmitters/neuropeptide producing effects on the 3,000 cells of the locus coeruleus is staggering.   Almost certainly someone is doing a similar study on the cerebral cortex.

Life becomes even more complex if a given norepinephrine neuron expresses multiple receptors (as I think they do — we’ll see what the author says).

Now a bit about why norepinephrine neurons are so important.

The locus coeruleus sends fibers all over the brain, releasing norepinephrine everywhere, and not just at synapses.  This is called volume neurotransmission. Most places on the axons of a LC neuron showing synaptic vesicles (where norepinephrine is found), don’t have a dendrite or any sort synaptic specialization next to them.  So the LC innervates the whole brain, in the same way that our brain innervates our muscles.  Stimulate the LC of a rat and the brain is flooded with norepinephrine and the animal wakes up.  Think of it as the sprinkler system of an office building.

This means that the length of axons of neurons acting by volume neurotransmission (this includes dopamine, serotonin, acetyl choline and histamine) must be enormous.  Here’s one reference for dopamine — “Individual neurons of the pars compacta (which uses dopamine as a neurotransmitter) are calculated to give rise to 4.5 meters of axons once all the branches are summed”  — [ Neuron vol. 96 p. 651 ’17 ].”   That’s just one cell doing all that.

I can’t find an actual source, so it may be a neuroscience urban myth, that every neuron is very close (within a few neuronal cell body diameters) to an axon of a volume transmitting neuron.  If anyone knows a source please write a comment.