A new way to look at ALS (thank God)

It’s always good when a new way to look at a basically untreatable disease comes along.  We’ll know soon if looking at filamin A will be useful for Alzheimer’s disease.  Here’s another:  something we’ve known about for years (polyphosphate) may be important in Amyotrophic Lateral Sclerosis (ALS).   I used riluzole for ALS, but never saw any benefit.  It may have slowed the decline, but riluzole never stopped disease progression.

It is stated that 10% of ALS is familial, but I think this is an overstatement.  Even so mutations in a variety of proteins(superoxide dismutase 1 (SOD1) TDP43, C9orf72) do cause ALS, and studying them has taught us a lot about ALS.  There is plenty of work to do.  In 2016 a mere 160 mutations in the 153 amino acids of SOD1 had been found, but we still don’t know how they cause ALS despite hundreds of papers on the subject.  The proteins have allowed us to make mouse models of ALS, by putting in one or the other of mutated SOD1, TDP43, C9orf72 in motor neurons (or in whole animals)

Some real gumshoe work led to polyphosphate [ Neuron vol. 110 pp. 1603 – 1605 ’22 ].  Obviously in ALS, the motor neurons die, but recent work has shown that motor neurons are killed by neighboring astrocytes (containing any of the 3 the mutant proteins), when they are cultured together.   Normal astrocytes don’t do this.

So a lot of hard work found that it was polyphosphate in the supernatant fluid that was the killer.

So what is polyphosphate?  It’s been known for years, and is found in ALL cells — bacterial, plant, animal.  It also produced abiotically in volcanic exudates and deep sea steam vents.  No one knows what it does, so it has been called a molecular fossil.  Again teleology should inform biologic research (but it doesn’t).  Polyphosphate must be doing something useful or it wouldn’t be present in all living cells.

Chemically, polyphosphate is a chain of HUNDREDS to THOUSANDS of phosphate residues linked by high energy phosphoanhydride bonds.

Like this —

HO – PO2 – OH  + HO -PO2 -OH –>  HO – PO2 – 0 – PO2 – OH + H20

— the – O – in the middle is the phosphoanhydride bond

The authors treated motor neurons in culture with polyphosphate and found that it killed 40% of them.  So what?  Schmidt’s law of pharmacology, says that enough of anything will do anything,  So they looked at the spinal cords of patients dying of ALS and found that polyphosphate levels were higher than in neurologically normal controls.

So it’s open season on polyphosphate. Finding out what it does in normal cells, finding out how it kills motor neurons, finding out if decreasing its levels will help ALS (it does in cultures of motor neurons but that’s a long way from a living patient).  It’s an entirely new angle on an awful disease, with no useful treatment.  There is simply an enormous amount of work to be done.

Watch this space.

 

 

We now understand what amyloid actually is

Lately we have received an embarrassment of riches about amyloid and the diseases it causes.  I’ll start with the latest — the structure of TDP amyloid.

I must say it is a pleasure to get back to chemistry and away from the pandemic, however briefly.  So relax and prepare to enjoy some great chemistry and protein structure.

TDP43 (you don’t to know what the acronym stands for) is a protein which binds to RNA (among other things).  It also forms aggregates, and some 50 mutations are known producing FrontoTemporal  Dementia (FTD) and/or Amyotrophic Lateral Dementia (ALS).  I saw a case as a resident (before things were worked out) and knew something was screwy because while ALS is a horrible disease, patients are clear to the end (witness Stephen Hawking) and my patient was clearly dementing.

Mutations in TDP43 occur in 5% of familial ALS.  More to the point cytoplasmic aggregates of TDP43 occur in 95% of sporadic cases of ALS (no mutations), so neurologists have been fascinated with TDP43 for years.

Back before we knew much about the structure of amyloid, it was characterized by the dyes that would bind to it (Congo Red, thioflavin etc.) and birefringence (see below).  None of this is true for the aggregates of TDP43.

Well we now know what the structure of amyloid is.  You simply can’t do better than  Cell vol. 184 pp. 4857 – 4873 ’21 — but it might be behind a paywall.

So here’s the skinny about what amyloid actually is —

 

It is a significantly long polypeptide chain  flattening  out into a 4.8 Angstrom thick sheet, essentially living in 2 dimensions.  Thousands of sheets then pile on top of each other forming amyloid.  So amyloid is not a particular protein, but a type of conformation a protein can assume (like the alpha helices, beta pleated sheets etc. etc. ).

The structure also explained why planar molecules like Congo Red bind to amyloid (it slips between the sheets).   Or at least that’s what I thought.

 

Enter Nature vol. 601 pp. 29 – 30, 139 – 143 ’22 showing that some 79 amino acids of the 414 amino acids of TDP43 flatten out into single sheet in the aggregates, with the sheets piling on top of each other.  If that isn’t amyloid, what is?

 

Where are the beta strands producing birefringence if this is amyloid.  In fact where is the birefringence? (see below). The paper says that there are 10 beta strands in the 79 amino acids, but they are short with only two of them containing more than 3 amino acids (I guess they can see beta strands by measuring backbone angles a la Ramachandran plots).  The high number of glycine mediated turns prevents beta sheets from stacking next to each other precluding the crossBeta  structure (and birefringence).

 

Why doesn’t Congo Red bind?  My idea about how it binds to other amyloids (slipping between the sheets) clearly is incorrect.

 

There are all sorts of fascinating points about the amyloid of TDP43.  The filaments derived from patients are stable to heating to 65 C.   The structure of the TDP43 fibrils derived from patients with FTD/ALS are quite different in structure from synthetic filaments made from parts of TDP43, so possibly a lot of work will have to be done again.

 

Here is some more detail on amyloid structure:

 

So start with NH – CO – CHR.  NH  CO and C in the structure all lie in the same plane (the H and the side chain of the amino acid < R >  project out of the plane).

Here’s a bit of elaboration for those of you whose organic chemistry is a distant memory.  The carbon in the carbonyl bond (CO) has 3 bonding orbitals in one plane 120 degrees apart, with the 4th orbital perpendicular to the plane — this is called sp2 hybridization.  The nitrogen can also be hybridized to sp2.  This lets the pair of electrons above the plane roam around moving toward the carbon.  Why is this good?  Because any time you let electrons roam around you increase their entropy (S) and anything increasing entropy lowers their free energy (F)which is given by the formula F = H – TS where H is enthalpy (a measure of bond strength, and T is the absolute temperature in Kelvin.

 

So N and CO are in one plane, and so are the bonds from  N and C to the adacent atoms (C in both cases).

 

You can fit the plane atoms into a  rectangle 4.8 Angstroms high.  Well that’s one 2 dimensional rectangle, but the peptide bond between NH and CO in adjacent rectangles allows you to tack NH – CO – C s together while keeping them in a 3 dimensional parallelopiped 4.8 Angstroms high

 

Notice that in the rectangle the NH and CO bonds are projecting toward the top and bottom of the rectangle, which means that in each plane  NH – CO – CHR s, the NH and CO are pointing out of the 2 dimensional plane (and in opposite directions to boot). This is unlike protein structure in which the backbone NHs and COs hydrogen bond to each other.  There is nothing in this structure for them to bond to

 

What they do is hydrogen bond to another 3 dimensional parallelopiped (call it a sheet, but keep in mind that this is NOT the beta sheet you know about from the 3 dimensional structures of proteins we’ve had for years).

So thousands of sheets stacked together form the amyloid fibril.

 

Where does the 9 Angstrom reflection of cross beta (and birefringence) come from?  Consider the  [ NH – CHR – CO ]  backbone as it lies in the 4.8  thick plane (Having studied proteins structure since entering med school in ’62, I never thought such a thing would even be possible ! ).  It curves around like a snake lying flat.  Where are the side chains?  They are in the 4.8 thick plane, separating parts of the meandering backbone from each other — by an average of 9 Angstroms.

Here is an excellent picture of the Alzheimer culprit — the aBeta42 peptide as it forms the amyloid of the senile plaque

https://pubmed.ncbi.nlm.nih.gov/27355699/#&gid=article-figures&pid=figure-7-uid-6.

You can see the meandering backbone and the side chains keeping the backbone apart.

Then Nature [ vol. 598,  pp. 359 – 363 ’21] blows the field wide open, finding 19 different conformations of tau in clinically distinct diseases. Each clinical disease appears to be associated with a distinct polymorphism.  This is also true for the polymorphisms of alpha-synuclein, with distinct conformations being seen in each of Parkinsonism, multiple system atrophy and Lewy body dementia.

In none of the above diseases is there a mutation (change in amino acid sequence) in the protein.

Henry J. Heinz claimed to have 57 varieties of pickles in 1896, but Cell [ vol. 184 pp. 4857 – 4873 ’21  ] Page 4862 claims that 24 amyloid polymorphs of alpha-synuclein have been found and structurally characterized.  Recall that alpha-synuclein amyloid is the principal component of the Lewy body of Parkinsonism  and Lewy Body disese

How did they get the 24 different conformations?  They incubated the protein under different conditions (e.g. different salt concentrations, different alpha-synuclein concentrations, different salts).

Why is this incredibly good news? 

Because it moves us past amyloid itself, to the conditions which cause amyloid to form.  Certainly, removing amyloid or attacking it hasn’t resulted in any clinical benefit for the Alzheimer patient despite billions being spent by Big Pharma to do so.

We will start to study the ‘root causes’ of amyloid formation.   The amino acid sequence of each protein is identical despite the different conformations of the chain in the amyloid. Clearly the causes must be different for each of the different polymorphs of the protein.  This just has to be true.

What Cassava Sciences should do now

Apparently someone important didn’t like the way Cassava Sciences analyzed their data and their stock tanked again today..  Unfortunately all of this seems to be behind a paywall, and the someone important isn’t named.  I’d love a link if any reader knows of one — just put it in as a  comment below.

I’m not important, but I thought Cassava’s results were quite impressive.  They had enough cases and enough time for the results to be statistically significant

For one thing,  Cassava dealt with severely impaired people (see below) who would be expected to show greater neuronal dropout, senile plaques and neurofibrillary tangles, than recently diagnosed patients.   Neuronal loss is not reversible in man, despite hoards of papers showing the opposite in animals.

Since everything turns on ADAS-CoG, here is a link to a complete description along with some discussion — https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929311/

On a slide from Cassava’s presentation yesterday the ADAS-CoG average of the 50 patients on entry 9 months ago was 16.6.  With a perfect score of 70, it’s clear that these people were significantly impaired (please look at the test items to see how simple the tasks in ADAS-CoG actually are).    So an improvement of 3 points at 9 months  is significant, particularly since a drop of 5 points is expected each year — yes I’ve seen plenty of Alzheimer patients with ADAS-CoG scores of zero or close to it.

So an increase of 3 points in this group is about a16% improvement.

Here’s what Cassava should do now.  Their data should be re-examined as follows.  Split the ADAS-CoG scores into 3 groups: highest middle and lowest. Quartiles are usually used, but I don’t think 50 patients is enough to do this.  Then examine the median improvement in each of the three.  I’d use median rather than average as with small numbers in each group, a single outlier can seriously distort things — think of the survival of Stephen Hawking in a group of 12 ALS patients.

If the patients with the highest ADAS-CoG scores have the highest median improvement, there is no reason mildly impaired individuals should have a less than 16% improvement in their scores.  This means that a person with ADAS-CoG of 60 should achieve a perfect score of 70,  e.g. return to normal.

This would be incredibly useful for early Alzheimer’s disease.

There is a precedent for this.  Again it’s Parkinson’s disease.

As I mentioned in an earlier post, I was one of the first neurologists in the USA to use L-DOPA for Parkinsonism.  All patients improved, and I actually saw one or two wheelchair bound Parkinsonians walk again (without going to Lourdes).  They were far from normal, but ever so much better.

However,  treated mildly impaired Parkinsonians became indistinguishable from normal, to the extent that I wondered if I’d misdiagnosed them. These results were typical.   For a time, in the early 70s neurologists thought that we’d actually cured the disease.  It was a very heady time.  We were masters of the neurologic universe — schizophrenia was too much dopamine, Parkinsonism not enough. Bring on the next neurotransmitter, bring on the next disease.

We hadn’t cured anything of course, and the underlying loss of dopamine neurons in the substantia nigra continued.  Reality intruded for me with one such extremely normal appearing individual I’d diagnosed with Parkinsonism a few years earlier. He needed surgery, meaning that he couldn’t take anything by mouth for a while.  L-DOPA could only be given orally, and he looked quite Parkinsonian in a day or two.

If reanalysis of the existing data shows what I hope, Cassava Sciences should start another study in Alzheimer patients with ADAS-CoG scores of over 50.  If I’m right the results should be spectacular (and lead to immediate approval of the drug).

A little blue sky.  Sumafilam will then come to be known as intellectual Viagra, as all sorts of oldsters (such as yrs trly) will try to get it Alzheimer’s or no Alzheimer’s.

Book Review: Hawking Hawking

To this neurologist, Stephen Hawking’s greatest contribution wasn’t in physics. I ran a muscular dystrophy clinic for 15 years in the 70s and 80s. Few of my ALS patients had heard of Hawking back then. I made sure they did. Hawking did something for them, that I could never do as a physician — he gave them hope.

Which brings me to an excellent biography of Hawking by Charles Seife “Hawking Hawking” which tries to strip away the aura and myths that Hawking assiduously constructed and show the man underneath.

Even better, Seife is an excellent writer and has the mathematical and scientific  chops (Princeton math major, Yale masters in math) to explain the problems Hawking was wrestling with.

Hawking was smart.  One story tells it all (p. 328).  Apparently there were only 3 other physics majors at Oxford that year.  They were all given a set of 13 problems on electromagnetism and a week to do them.    One of the others (Derek Powney) tells the tale. “I discovered very rapidly that I couldn’t do any of them”.  So he teamed up with one of the others, and by the end of the week they’d done 1.5 problems.  The thrd student (working alone) solved one. 

At the end of the week “Stephen as always hadn’t even started”. He went to his room and came out 3 hours later. “Well, I’ve only had the time to do the first ten.”  “I think at that point we realized that it’s not just that we weren’t on the same street, we weren’t on the same planet.”

Have you ever had an experience like that?  I’ve had two.  The first occurred in grade school. I was a pretty good piano player, better than the rest of Dr, Rudnytsky’s students.  Then, someone told me that at age 3 his son would tell him what notes passing trains were whistling on, and that later on he’d sit behind a door listening to his father give lessons, and then come in afterwards and play by ear what the students had been playing.  The second occurred a within day or so of starting my freshman year in college. My roommate told me about a guy who thought he ought to know everybody in our class of 700+.  So he got out the freshman herald which had our pictures and names and a day later knew everyone in the class by name. 

The reason people of a scientific bent should read the book, is not the sociology, or the complicated sexuality of Hawking and his two wives, and god knows what else.  It is the excellent explanations of the problems in math and physics that Hawking faced and solved.  Even better, Seife puts them in context of the work done before Hawking was born.  

Two  examples

1. pp. 14 – 18 — a superb explanation of what Einstein did to create special relativity. 

2. pp. 240 – 245 an excellent description of the horizon problem, the flatness problem and how inflation solved it. 

Any really good book will teach you something.  People in physics, math and biology are consumed with the idea of information.  The book (pp. 131 – 134) explains why Hawking was so focused on the black hole information paradox.  It always seemed pretty arcane and superficial to me (on the order of how many angels could dance on the head of a pin).  

Wrong ! Wrong !

The black hole information paradox is at the coalface of ignorance in modern physics.  Why?  Because the two great theories we have in  (quantum mechanics and general relativity) disagree with what happens to the information contained in an object (such as an astronaut) swallowed by a black hole.  Relativity says it’s destroyed, while quantum mechanics says that’s impossible. 

So reconciling the two descriptions would lead to a deeper theory, and showing that one was wrong, would discredit a powerful theory. 

So even if you’re not interested in the sociology of the circles Hawking moved in or his sex life, there is a lot of well-explained physics and math to be learned for the general reader.  

The black hole information paradox resembles a similarly unresolved pair of phenomena in the world we live in, the Cartesian dualism between flesh and spirit.  It is writ large in biology.

Chemistry is great and can provide mechanistic explanations what we see, such as the example from the following old post, produced after the ***

It’s quite technical, but is an elegant explanation of how different cells make different amounts of two different forms of a muscle protein (beta actin and gamma actin ).  I never thought we’d have an explanation this good, but we do.  Well that’s the flesh and the physicality of the explanation.  Asking why different cells would want this, or what the function of all is puts you immediately in the world of spirit (ideas, which are inherently noncorporeal).  Physical chemistry and biochemistry are silent, and all the abstract explanations science gives us (the function, the why, the reason) is essentially teleological. 

*****

The last post “The death of the synonymous codon – II” puts you exactly at the nidus of the failure of chemical reductionism to bag the biggest prey of all, an understanding of the living cell and with it of life itself.  We know the chemistry of nucleotides, Watson-Crick base pairing, and enzyme kinetics quite well.  We understand why less transfer RNA for a particular codon would mean slower protein synthesis.  Chemists understand what a protein conformation is, although we can’t predict it 100% of the time from the amino acid sequence.  

Addendum 30 April ’21:  Called to task on the above  by a reader.  This statement is no longer true.  The material below the *** was bodily lifted from something I wrote 10 years ago.  Time and AI have marched on since then.

So we do understand exactly why the same amino acid sequence using different codons would result in slower synthesis of gamma actin than beta actin, and why the slower synthesis would allow a more leisurely exploration of conformational space allowing gamma actin to find a conformation which would be modified by linking it to another protein (ubiquitin) leading to its destruction.  Not bad.  Not bad at all.

Now ask yourself, why the cell would want to have less gamma actin around than beta actin.  There is no conceivable explanation for this in terms of chemistry.  A better understanding of protein structure won’t give it to you.  Certainly, beta and gamma actin differ slightly in amino acid sequence (4/375) so their structure won’t be exactly the same.  Studying this till the cows come home won’t answer the question, as it’s on an entirely different level than chemistry.

 

TDP43 and the anisosome

Neurologists have been interested in TDP43 (Tar Dna binding Protein of 43 kiloDaltons) for a long time. Mutants cause some cases of ALS (Amyotrophic Lateral Sclerosis — Lou Gehrig disease) and FTD (FrontoTemporal Dementia).  Some 50 different mutations in the protein have been found in cases of these two diseases.  Intracellular inclusions containing TDP are found in > 90% of sporadic ALS (no mutations) and 45% of FTD.

TDP43 contains 414 amino acids (as you might expect for a protein with a 43 kiloDalton mass).  There is an amino terminal ubiquitinlike fold, two RNA Recognition Motifs (RRMs) followed by a glycine rich low complexity sequence prion-like domain at the other (carboxy) end.  The disease causing mutations are found in the low complexity sequence. 

A  phase separated structure (the anisosome) never seen before involves  mutant TDP43 [ Science vol. 371 pp. 585, abb4309 pp. 1 –> 15 ’21 ].  It is a phase separated mass with liquid spherical shells and liquid cores.  The shells showed birefringence — evidence of a liquid crystal.  The cores show the HSP70 chaperone bound to TDP43 (which wasn’t binding RNA).

ATP is required to maintain the chaperone activity of HSP70. When ATP levels are reduced, the anisosome is converted into the protein aggregates seen in ALS and FTD.  So the anisosome is a protective mechanism. 

Biology is clearly leading chemistry around by the nose.  No chemist would ever have predicted something like this, or received a grant to mix all this stuff in a test tube not even thinking about stoichiometry and see what happened.  For more details on phase separation please see an old post — https://luysii.wordpress.com/2020/12/20/neuroscience-can-no-longer-ignore-phase-separation/

Here’s some stuff from that post to whet your appetite

Advances in cellular biology have largely 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.

 

Now is the winter of our discontent

One of the problems with being over 80 is that you watch your friends get sick.  In the past month, one classmate developed ALS and another has cardiac amyloidosis complete with implantable defibrillator.  The 40 year old daughter of a friend who we watched since infancy has serious breast cancer and is undergoing surgery radiation and chemo.  While I don’t have survivor’s guilt (yet), it isn’t fun.

Reading and thinking about molecular biology has been a form of psychotherapy for me (for why, see the reprint of an old post on this point at the end).

Consider ALS (Amyotrophic Lateral Sclerosis, Lou Gehrig disease).  What needs explaining is not why my classmate got it, but why we all don’t have it.  As you know human neurons don’t replace themselves (forget the work in animals — it doesn’t apply to us).  Just think what the neurons  which die in ALS have to do.  They have to send a single axon several feet (not nanoMeters, microMeters, milliMeters — but the better part of a meter) from their cell bodies in the spinal cord to the muscle the innervate (which could be in your foot).

Supplying the end of the axon with proteins and other molecules by simple diffusion would never work.  So molecular highways (called microtubules) inside the axon are constructed, along which trucks (molecular motors such as kinesin and dynein) drag cargos of proteins, and mRNAs to make more proteins.

We know a lot about microtubules, and Cell vol. 179 pp. 909 – 922 ’19 gives incredible detail about them (even better with lots of great pictures).  Start with the basic building block — the tubulin heterodimer — about 40 Angstroms wide and 80 Angstroms high.  The repeating unit of the microtubule is 960 Angstroms long, so 12 heterodimers are lined up end to end in each repeating unit — this is the protofilament of the microtubule, and our microtubules have 13 of them, so that’s 156 heterodimers per microtubule repeat length which is 960 Angstroms or 96 nanoMeters (96 billionths of a meter).  So a microtubule (or a bunch of microtubules extending a meter has 10^7 such repeats or about 1 billion heterodimers.  But the axon of a motor neuron has a bunch of microtubules in it (between 10 and 100), so the motor neuron firing to  the muscle moving my finger has probably made billions and billions of heterodimers.  Moreover it’s been doing this for 80 plus years.

This is why, what needs explaining is not ALS, but why we don’t all have it.

Here’s the old post

The Solace of Molecular Biology

Neurology is fascinating because it deals with illnesses affecting what makes us human. Unfortunately for nearly all of my medical career in neurology ’62 – ’00 neurologic therapy was lousy and death was no stranger. In a coverage group with 4 other neurologists taking weekend call (we covered our own practices during the week) about 1/4 of the patients seen on call weekend #1 had died by on call weekend #2 five weeks later.

Most of the deaths were in the elderly with strokes, tumors, cancer etc, but not all. I also ran a muscular dystrophy clinic and one of the hardest cases I saw was an infant with Werdnig Hoffman disease — similar to what Steven Hawking has, but much, much faster — she died at 1 year. Initially, I found the suffering of such patients and their families impossible to accept or understand, particularly when they affected the young, or even young adults in the graduate student age.

As noted earlier, I started med school in ’62, a time when the genetic code was first being cracked, and with the background then that many of you have presently understanding molecular biology as it was being unravelled wasn’t difficult. Usually when you know something you tend to regard it as simple or unimpressive. Not so the cell and life. The more you know, the more impressive it becomes.

Think of the 3.2 gigaBases of DNA in each cell. At 3 or so Angstroms aromatic ring thickness — this comes out to a meter or so stretched out — but it isn’t, rather compressed so it fits into a nucleus 5 – 10 millionths of a meter in diameter. Then since DNA is a helix with one complete turn every 10 bases, the genome in each cell contains 320,000,000 twists which must be unwound to copy it into RNA. The machinery which copies it into messenger RNA (RNA polymerase II) is huge — but the fun doesn’t stop there — in the eukaryotic cell to turn on a gene at the right time something called the mediator complex must bind to another site in the DNA and the RNA polymerase — the whole mess contains over 100 proteins and has a molecular mass of over 2 megaDaltons (with our friend carbon containing only 12 Daltons). This monster must somehow find and unwind just the right stretch of DNA in the extremely cramped confines of the nucleus. That’s just transcription of DNA into RNA. Translation of the messenger RNA (mRNA) into protein involves another monster — the ribosome. Most of our mRNA must be processed lopping out irrelevant pieces before it gets out to the cytoplasm — this calls for the spliceosome — a complex of over 100 proteins plus some RNAs — a completely different molecular machine with a mass in the megaDaltons. There’s tons more that we know now, equally complex.

So what.

Gradually I came to realize that what needs explaining is not the poor child dying of Werdnig Hoffman disease but that we exist at all and for fairly prolonged periods of time and in relatively good shape (like my father who was actively engaged in the law and a mortgage operation until 6 months before his death at age100). Such is the solace of molecular biology. It ain’t much, but it’s all I’ve got (the religious have a lot more). You guys have the chemical background and the intellectual horsepower to understand molecular biology — and even perhaps to extend it.

 

RIPK1

The innate immune system is intrinsically fascinating, dealing with invaders long before antibodies or cytotoxic cells are on the scene.  It is even more fascinating to a chemist because it works in part by forming amyloid inside the cell.  And you thought amyloid was bad.

The system becomes even more fascinating because blocking one part of it (RIPK1) may be a way to treat a variety of neurologic diseases (ALS, MS,Alzheimer’s, Parkinsonism) whose treatment could be improved to put it mildly.

One way to deal with an invader which has made it inside the cell, is for the cell to purposely die.  More and more it appears that many forms of cell death are elaborately programmed (like taking down a stage set).

Necroptosis is one such, distinct from the better known and studied apoptosis.   It is programmed and occurs when a cytokine such as tumor necrosis factor binds to its receptor, or when an invader binds to members of the innate immune system (TLR3, TLR4).

The system is insanely complicated.  Here is a taste from a superb review — unfortunately probably behind a paywall — https://www.pnas.org/content/116/20/9714 — PNAS vol. 116 pp. 9714 – 9722 ’19.

“RIPK1 is a multidomain protein comprising an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The intermediate domain of RIPK1 contains an RHIM [receptor interacting protein (rip) homotypic interaction motif] domain which is important for interacting with other RHIM-containing proteins such as RIPK3, TRIF, and ZBP1. The C-terminal DD mediates its recruitment by interacting with other DD-containing proteins, such as TNFR1 and FADD, and its homodimerization to promote the activation of the N-terminal kinase domain. In the case of TNF-α signaling, ligand-induced TNFR1 trimerization leads to the assembly of a large receptor-bound signaling complex, termed Complex I, which includes multiple adaptors (TRADD, TRAF2, and RIPK1), and E3 ubiquitin ligases (cIAP1/2, LUBAC complex).”

Got that?  Here’s a bit more

“RIPK1 is regulated by multiple posttranslational modifications, but one of the most critical regulatory mechanisms is via ubiquitination. The E3 ubiquitin ligases cIAP1/2 are recruited into Complex I with the help of TRAF2 to mediate RIPK1 K63 ubiquitination. K63 ubiquitination of RIPK1 by cIAP1/2 promotes the recruitment and activation of TAK1 kinase through the polyubiquitin binding adaptors TAB2/TAB3. K63 ubiquitination also facilitates the recruitment of the LUBAC complex, which in turn performs M1- type ubiquitination of RIPK1 and TNFR1. M1 ubiquitination of Complex I is important for the recruitment of the trimeric IκB kinase complex (IKK) through a polyubuiquitin-binding adaptor subunit IKKγ/NEMO . The activation of RIPK1 is inhibited by direct phosphorylation by TAK1, IKKα/β, MK2, and TBK1. cIAP1 was also found to mediate K48 ubiquitination of RIPK1, inhibiting its catalytic activity and promoting degradation.”

So why should you plow through all this?  Because inhibiting RIPK1 reduces oxygen/glucose deprivation induced cell death in neurons, and reduced infarct size in experimental middle cerebral artery occlusion.

RIPK1 is elevated in MS brain, and inhibition of it helps an animal model (EAE).  Mutations in optineurin, and TBK1 leading to familial ALS promote the onset of RIPK1 necroptosis

Inflammation is seen in a variety of neurologic diseases (Alzheimer’s, MS) and RIPK1 is elevated in them.

Inhibitors of RIPK1 are available and do get into the brain.  As of now two RIPK1 inhibitors have made it through phase I human safety trials.

So it’s time to try RIPK1 inhibitors in these diseases.  It is an entirely new approach to them.  Even if it works only in one disease it would be worth it.

Now a dose of cynicism.  Diseased cells have to die one way or another.  RIPK1 may help this along, but it tells us nothing about what caused RIPK1 to become activated.  It may be a biomarker of a diseased cell.  The animal models are suggestive (as they always are) but few of them have panned out when applied to man.

 

A pile of spent bullets — take II

I can tell you after being in neurology for 50 years that back in the day every microscopic inclusion found in neurologic disease was thought to be causative.  This was certainly true for the senile plaque of Alzheimer’s disease and the Lewy body of Parkinsonism.  Interestingly, the protein inclusions in ALS weren’t noticed for decades.

However there are 3 possible explanations for any microscopic change seen in any disease.  The first is that they are causative (the initial assumption).  The second is that they are a pile of spent bullets, which the neuron uses to defend itself against the real killer.  The third is they are tombstones, the final emanations of a dying cell, a marker for the cause of death rather than the cause itself.

An earlier post concerned work that implied that the visible aggregates of alpha-synuclein in Parkinson’s disease were protective rather than destructive — https://luysii.wordpress.com/2018/01/07/are-the-inclusions-found-in-neurologic-disease-attempts-at-defense-rather-then-the-cause/.

Comes now Proc. Natl. Acad. Sci. vol. 115 pp. 4661 – 4665 ’18 on Superoxide Dismutase 1 (SOD1) and ALS. Familial ALS is fortunately less common than the sporadic form (under 10% in my experience).  Mutations in SOD1 are found in the familial form.  The protein contains 153 amino acids, and as 6/16 160 different mutations in SOD1 have been found.  Since each codon can contain only 3 mutations from the wild type, this implies that, at a minimum,  53/153 codons of the protein have been mutated causing the disease.  Sadly, there is no general agreement on what the mutations actually do — impair SOD1 function, produce a new SOD1 function, cause SOD1 to bind to something else modifying that function etc. etc.  A search on Google Scholar for SOD1 and ALS produced 28,000 hits.

SOD1 exists as a soluble trimer of proteins or the fibrillar aggregate.   Knowing the structure of the trimer, the authors produced mutants which stabilized the trimer (Glycine 147 –> Proline) making aggregate formation less likely and two mutations (Asparagine 53 –> Isoleucine, and Aspartic acid 101 –> Isoleucine) which destabilized the trimer making aggregate formation more likely.  Then they threw the various mutant proteins at neuroblastoma cells and looked for toxicity.

The trimer stabilizing mutant  (Glycine 147 –> Proline) was toxic and the destabilizing mutants  (Asparagine 53 –> Isoleucine, and Aspartic acid 101 –> Isoleucine)  actually improved survival of the cells.  The trimer stabilizing mutant was actually more toxic to the cells than two naturally occurring SOD1 mutants which cause ALS in people (Alanine 4 –> Valine, Glycine 93 –> Alanine).  Clearly with these two something steric is going on.

So, in this experimental system at least, the aggregate is protective and what you can’t see (microscopically) is what kills cells.

Stephen Hawking R. I. P.

Stephen Hawking, brilliant mathematician and physicist has died.  Forget all that. He did something for my patients with motor neuron disease that I, as a neurologist, could not do.  He gave them hope.

What has chemistry done for them?  Quite a bit, but there’s so much left.

Chemistry, when successful, just becomes part of the wallpaper and ignored. All genome sequencing depends on what some chemist did.

For one spectacular example of what, without chemistry, would be impossible is Infantile Spinal Muscular Atrophy (Werdnig Hoffmann disease).  For the actual molecular biology behind it — please see — https://luysii.wordpress.com/2016/12/25/tidings-of-great-joy/.   Knowing the cause has led to not one but two specific therapies — an antisense oligonucleotide and a virus which infects neurons and actually changes the gene.

So knowing what the cause of a disease is should lead to a treatment, shouldn’t it?  Hold that thought.  Sometimes one form of motor neuron disease (amyotrophic lateral sclerosis or ALS) can be hereditary.  Find out what is being inherited to find how ALS is caused.

Well, the first protein in which a mutation is associated with familial ALS (FALS) was found exactly 25 years ago.  It is called superoxide dismutase (SOD1).  Over 150 mutations have been found in the protein associated with FALS, and yet despite literally thousands of papers on the subject we don’t know if the mutations cause a loss of function, a gain of function (and if so what that function is), an increased tendency to fold incorrectly, and on and on and on.  It’s a fascinating puzzle for the protein chemist and over the years my notes on the papers I’ve read about SOD1 have ballooned to some 25,000 words.

If you’re tired of working on SOD1, try a few of the other proteins in which mutations have been associated with FALS — Alsin, TAF15, Ubiquilin, Optineurin, TBK1 etc. etc.  The list is long.

Now it’s biology’s turn.  Motor neurons go from the spinal cord (mostly) and brain to produce muscle contraction.  Why should only this tiny (but crucial) minority of cells be affected.  The nerve fibers leave the spinal cord and travel to muscle in nerves which contain sensory nerve fibers making the same long trip, yet somehow these nerves are spared.

More than that, why should these mutations affect only these neurons, and that often after decades.  Also why should great athletes (Lou Gehrig, Ezzard Charles, etc. etc. ) get the disease.

One closing point.  Hawking shows why, in any disease median survival (when 50% of those afflicted die) is much a more meaningful statistic than average duration of survival.  Although he gave my patients great hope, they all died within a few years even as he mightily extended average survival.

 

We don’t understand amyloid very well

I must admit I was feeling pretty snarky about our understanding of amyloid and Alzheimer’s after the structure of Abeta42 was published.  In particular the structure explained why the alanine 42–> threonine 42 mutation was protective against Alzheimer’s disease while the alanine 42 –> valine 42 mutation increases the risk.  That’s all explained in the last post — https://luysii.wordpress.com/2017/10/12/abeta42-at-last/ — but a copy will appear at the end.

In that post I breathlessly hoped for the structure of aBeta40 which is known to be less toxic to neurons.  Well it’s here and it shows how little we understand about what does and what doesn’t form amyloid.  The structure appears in a paper about the amyloid formed by another protein (FUS) to be described later — Cell 171, 615–627, October 19, 2017 — figure 7 p. 624.

Now all Abeta40 lacks are the last 2 amino acids of Abeta42 — isoleucine at 41 and alanine at 42.  So solve the Schrodinger equation for it, and stack it up so it forms amyloid, or use your favorite molecular dynamics or other modeling tool.  Take a guess what it looks like.

Abeta42 is a dimer, a beta40 is a trimer, even though the first 40 amino acids of both are identical.

It gets worse. FUS (FUsed in Sarcoma) is a 526 amino acid protein which binds to RNA and is mostly found in the nucleus.  Neurologists are interested in it because over 50 mutations in have been found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).   FUS contains a low complexity domain (LCD) of 214 amino acids, 80% of which are one of 4 amino acids (glycine, serine, glutamine and tyrosine).  At high protein concentrations this domain of FUS forms long unbundled fibrils with the characteristic crossBeta structure of amyloid.  Only 57/214 of the LCD amino acids are part of the structured core of the amyloid — the rest are disordered.

Even worse the amino acids forming the amyloid core (#39 -#95) are NOT predicted by a variety of computational methods predicting amyloid formation (Agrescan, FISH, FOLDamyloid, Metamyl, PASTA 2.0).  The percentages of gly, ser, gln and tyr in the core forming region are pretty much the same as in the whole protein.  The core forming region has no repeats longer than 4 amino acids.

The same figure 7 has the structure of the amyloid formed by alpha-synuclein, which accumulates in the Lewy bodies of Parkinson’s disease.  It just has one peptide per layer of amyloid.

When you really understand something you can predict things, not just describe them as they are revealed.

 

Abeta42 at last

It’s easy to see why cryoEM got the latest chemistry Nobel.  It is telling us so much.  Particularly fascinating to me as a retired neurologist is the structure of the Abeta42 fibril reported in last Friday’s Science (vol. 358 pp. 116 – 119 ’17).

Caveats first.  The materials were prepared using an aqueous solution at low pH containing an organic cosolvent — so how physiologic could the structure actually be?  It probably is physiologic as the neurotoxicity of the fibrils to neurons in culture was the same as fibrils grown at neutral pH.  This still isn’t the same as fibrils grown in the messy concentrated chemical soup known as the cytoplasm.  Tending to confirm their findings is the fact that NMR and Xray diffraction on the crystals produced the same result.

The fibrils were unbranched and microns long (implying at least 2,000 layers of the beta sheets to be described).  The beta sheets stack in parallel and in register giving the classic crossBeta sheet structure.  They were made of two protofilaments winding around each other.  Each protofilament contains all 42 amino acids of Abeta42 and all of them form a completely flat beta sheet structure.

Feast your eyes on figure 2 p. 117.  In addition to showing the two beta sheets of the two protofilaments, it shows how they bind to each other.  Aspartic acid #1 of one sheet binds to lysine #28 of the other.  Otherwise the interface is quite hydrophobic.  Alanine2 of one sheet binds to alanine42 of the other, valine39 of one sheet binds to valine 39 of the other.  Most importantly isoLeucine 41 of one sheet binds to glycine38 of the other.

This is important since the difference between the less toxic Abeta40 and the toxic Abeta 42 are two hydrophobic amino acids Isoleucine 41 and Alanine 42.  This makes for a tighter, longer, more hydrophobic interface between the protofilaments stabilizing them.

That’s just a guess.  I can’t wait for work on Abeta40 to be reported at this resolution.

A few other points.  The beta sheet of each protomer is quite planar, but the planes of the two protomers are tilted by 10 degrees accounting for the helicity of the fibril. The fibril is a rhombus whose longest edge is about 70 Angstroms.

Even better the structure explains a mutation which is protective against Alzheimer’s.  This remains the strongest evidence (to me at least) that Abeta peptides are significantly involved in Alzheimer’s disease, therapeutic failures based on this idea notwithstanding.  The mutation is a change of alanine2 to threonine which can’t possibly snuggle up hydrophobically to isoleucine nearly as well as alanine did. This should significantly weaken the link between the two protofilaments and make fibril formation more difficult.

The Abeta structure of the paper also explains another mutation. This one increases the risk of Alzheimer’s disease (like many others which have been discovered).  It involves the same amino acid (alanine2) but this time it is changed to the morehydrophobic valine, probably resulting in a stronger hydrophobic interaction with isoLeucine41 (assuming that valine’s greater bulk doesn’t get in the way sterically).

Wonderful stuff to think and speculate about, now that we actually have some solid data to chew on.