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.

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.

 

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.

 

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.

What reading the literature is like when things are barely understood

There is a very exciting paper to be described in a post to appear shortly. I ran a muscular dystrophy clinic for 15 years, and saw lots of Amyotrophic Lateral Sclerosis (ALS) — even though, strictly speaking it is not a muscular dystrophy. The muscular Dystrophy Association was founded by parents of weak children, before we could actually separate motor neuron disease from myopathy. In retirement, I’ve kept up an interest in ALS (particularly since all I could do for patients as a doc was — (drumroll) — basically nothing).

The fact that a fair amount of even sporadic ALS has a problem with a protein called C9ORF72 was particularly fascinating. All this came out less than five years ago (October 2011). Everything is far from clearcut even now.

That being the case, it might be of interest to look at the notes I accumulated as scientists began to explore what was wrong with C9ORF72, how the protein normally does whatever it does (we still don’t know really) and how the mutated product of the gene causes trouble (there are 3 main theories).

What you’ll see in what follows is the heat of scientific battle (warts and all), where things are far from clear. Enjoy. This is basically what used to be called a core-dump (back in the day when computer memory was made of metallic cores). Things are far from cut and dried even now so it might be of interest to see the many angles of attack on the problem, the confusion, the conflicting theories, as things became a bit more clear. It’s the scientific enterprise in action against a very horrible disease (trust me).

I’ll try and clear up the typos. I’ll also try to put the notes on the papers in semi-chronological order, but I make no guarantees. The notes may be incomprehensible, as they include only what I didn’t know rather than all the background needed to understand what’s in them .

First a bit of background — FTD stands for FrontoTemporal Dementia.

The #9p21 chromosomal region is another locus for ALS/FTD. It contains something called C9orf72, which contains a GGGGCC hexnucleotide repeat in the intron between noncoding exons 1a and 1b. Normal alleles contain less than 24 repeats (range 2 – 23). Those with ALS + FTD contain over 30 (actually they think the repeat length is much higher — 700 to 1,600 ! ! !). ORF probably stands for open reading frame.

The expansion is present in 12% of familial FTD and 22.5% of familial ALS — making it the most common genetic abnormality in both conditions. More importantly it is found in 21% of sporadic ALS and 29% of FTD in the Finnish population. Later they say it is the most common genetic cause of sporadic ALS (but only in 4%).

There are 3 possible mechanisms of toxicity
l. The RNA transcribed from the repeat acts as an RNA sponge, binding all sorts of RNAs it shouldn’t
2. Repeat Assoaicted Non-ATG translation (RAN translation) see later
3. Decreased expression of the mRNA for C9ORF72.

[ Science vol. 338 pp. 1282 – 1283 ’12 ] Now 40% of familial ALS, 21% of familial frontotemporal dementia, and 8% of sporadic ALS, 5% of sporadic frontotemporal dementia have expansions in C9orf72.

Not much is known about C9orf72 — it is conserved across species. It contains no previously known protein domains. The expansion leads to loss of one alternatively spliced C9ORF72 isoform (normally 3 isoforms are expressed), and to the formation of nuclear RNA foci (which appear to be composed mostly of the expansion). [ Neuron vol. 79 pp. 416 – 438 ’13 ] The function of C9ORF72 is unknown (8/13).

The current (12/12) thinking is that the repeats produce a glob of RNA which traps RNA binding proteins which have better things to do. The best analogy is myotonic dystrophy in which an expanded 3 nucleotide repeat sequesters muscleblind, an RNA binding protein involved in splicing.

The expansion is present in 46% of familial ALS in Finland and 21% of sporadic ALS there. But Finns are somewhat different genetically. The expansion is found in 1/3 of European ancestry familial ALS.

Interestingly some of the patients with FTD presented with nonfluent progressive aphasia.

[ Cell vol. 152 pp. 691 – 698 ’13, Neuron vol. 77 pp. 639 – 646 ’13 ] The protein aggregates of C9orf72 mutants contain TDP43 inclusions. But they also show additional p62 and ubiquilin positive pathology (with no TDP43 present). The abnormal proteins are due to translation of the expanded GGGGCC repeats (which should be nonCoding as they are in introns). This is an example of Repeat Associated Non-ATG translation (RAN). This was first shown for expanded CAG repeats, which can be translated in all 3 reading frames giving polyGlutamine, polyLysine and polySerine . A minimum of 58 CAG repeats was required for translation.

This work looked for translation of GGGGCC in all 3 reading frames (poly glycine-proline, poly glycine-alanine, polyglycine-arginine. They found that poly glycine-proline was found and in the protein inclusions which were p62 positive and TDP43 negative. Similar inclusions weren’t present in other neurodegenerative diseases, known to have nucleotide inclusions.

[ Proc. Natl. Acad. Sci. vol. 110 pp 7533 – 7534, 7778 – 7783 ’13 ] The expanded C9orf72 repeat is enough to cause neurodegeneration (mammalian neurons, and D. melanogaster). They placed either 3 or 30 copies of GGGCC into an epidermal growth factor vector between the start of transcription and the first ATG codon. The repeat can sequester the RNA binding protein Pur alpha (and other Pur family members). Interestingly, TDP43 didn’t bind to the repeat RNA, nor did hnRNP A2/B1 which binds to fragile X CGG repeat containing RNA. Overexpression of of Pur alpha is able to abort the neurogeneration in the mammalian neuonal cell line (Neuro-2a). So probably the excessive repeat number is acting as an RNA sponge.

Pur alpha is evolutionarily conserved. It controlls the cell cycle and differentiation. It is also a pomonent of the RNA transport granule. It interacts with Pur beta.

30 was as many repeats as they could manipulate experimentally — normals have 2 – 8 repeats, but patients with disease have from 100s to 1,000s of repeats, so the pathogenesis might be different.

[ Neuron vol. 80 pp. 257 – 258, 415 – 428 ’13 ] Expression of C9orf72’s mRNA in frontotemporal dementia/als (FTD/ALS) patients is reduced by 50%, and the expanded repeat and neighboring CgP islands are hypermethylated consistent with transcriptional silencing. Also the cytoplasmic aggregates staining positively for P62 appear to result from protein translation through the hexanucleotide repeat.

This work used induced pluripotent stem cells (iPSCs) derived from C9ALS/FTD patients. They show decreased C9orf72 mRNA, nuclear and cytoplasmic GGGGCC RNA foci, and expression of one RAN product (Gly Pro dipeptide). Neurons derived from the iPSCs also show enhanced sensitvity to glutamic acid excitotoxicity, and a transcriptional profile that ‘partially’ overlaps with transcriptional changes seen in iPSC neurons derived from mutant SOD1 ALS patients.

In addition, some 19 proteins were found which associate with the GGGGCC repeats in vitro. ADARB2 does this and participates in RNA editing.

ASOs (AntiSense OIigonucleotides ??) were used to suppress C9orf72 RNA expression. This led to reversal in many of the phenotypes of the iPSC neurons (suppression of glutamic acid toxicity, reduction in RNA foci formation). This implies that the GGGGCC repeats trigger toxicity through a gain of function mechanism. [ Proc. Natl. Acad. Sci. vol. 110 pp. E4530 – E4539 ’13 ] Nuclear RNA foci containing GGGCC in patient cells (wbc’s fibroblasts, glia, neurons) were ssen in patients with repeat expansion. The Foci weren’t present in sporadic ALS or ALS/FTD caused by other mutations (SOD1, TDP43, tau), Parkinsonism, or nonNeurological controls. Antisense oligonucleotides reduced the GGGGCC containing nuclear foci without alteraling overall C9orf72 RNA levels. SiNRAS didn’t work.

The Rx was applied to living mice and it was well tolerated.

[ Proc. Natl. Acad. Sci. vol. 110 pp E4968 – E4977 ’13 ] C9orf72 antisense transcripts are elevated in the brains of those with the expansion. Repeat expansion GGCCCC RNAs accumulate in nuclear foci in the brain. Sense and antisense foci accumulate in the blood and are potential biomarkers. RAN translation occurs in BOTH sense and antisense expansion transcripts — so all 6 proteins described above are made. The proteins accumulate in cytoplasmic aggregates in affected brain regions (e.g. frontal and motor cortex, spinal cord neurons).

[ Nature vol. 507 pp. 175 – 177, 195 – 200 ’14 ] C9orf72 has repeated hexanucleotide units (GGGGCC). Two or more G quartets stacked on top of one another form a G-quadruplex. In the expanded repeats of C9orf72 in ALS and frontotemporal dementia, stable quadruplexes form in DNA as well as the RNA transcribed from it.

Sequences which can form G-quadruplexes are conserved during evolution, so they presumably are doing something useful. They are found in transcriptional start sites. This work shows that G-quadruplex assembly in DNA increases transcriptional pauses in the expanded repeat (unsurprising). Also the G-quadruplexes in C9orf72 DNA promote the formation of stable R-loops — triple stranded structures that assemble when a newly form RNA transcript exiting RNA polymerase II invades the double helix and binds to one DNA strand, displacing the other. If the R-loops aren’t resolved, they can halt transcriptional elongation.

Not only that, but abortive GGGGCC containing RNAs accumulate in the spinal cord and motor cortex of patients with the expanded repeats. The RNAs are truncated in the GGGGCC region, and the amount is linearly proportional to the length of the hexanucleotide repeat. This explains how they could accumulate along with decreased level of full length C9orf72 mRNA (and presumably the protein made from it).

A ‘few dozen’ proteins binding the GGGGCC repeats have been found. One of them is nucleolin, involved in the formation of the ribosome within the nucleolus It is mislocalized to RNA foci in neurons of the motor cortex of patients with C9orf72 related disease. The lack of mature ribosomes results in the buildup of untranslated mRNA in the cytoplasm.

[ Science vol. 345 pp. 1118 – 1119, 1139 – 1145, 1192 – 1194 ’14 ] Normally the number of GGGGCC repeats in C9orf72 ranges from 2 to 23, with hundreds or even thousands of copies in the disease range. Possibilities
l. Interference with C9orf72 expression — e. g. loss of function
2. Sponging up RNA binding proteins by the transcript
3. Repeat associated non-ATG translation (RAN translation) in all reading frames (sense and antisense).

A series of stop codons in both the sense and antisense RNAs was engineered every 12 repeats, stopping formation of the dipeptide repeat proteins. The new RNAs still formed the G-quadruplexes, and both RNAs formed RNA foci when expressed in cultured neurons.

Putting them into Drosophila showed that the pure repeats able to form dipeptides causing degeneration in the fly eye, while the interrupted constructs (producing RNA only) did not. The same was true when expressed in the nervous systems of adult flies. Blocking translation of the RNA partially suppressed the phenotype.

There are 5 possible dipeptide products of RAN of GGGGCC (GA, GP, PA, GR, PR — G == Glycine, P == Proline, A == Alanine, R = Arginine). Then RNAs using alternate codons for the dipeptides were used (so GGGGCC wasn’t present). Expressing Glycine Arginine (GR) or Proline Arginine (PR) was toxic, Glycine Alanine showing ‘some’ toxicity later in life.

Some RNA binding proteins containing low complexity sequences (aka prion-like domains) — these are FUS, EWSR1, TAF14, hnRNPA2 — form polymeric assemblies, which incorporate into hydrogels in vitro. The assemblies are similar to RNA granules. Many of the RNA binding proteins associating with hydrogels hare serine arginine (SR) sequences. The SR domain proteins are regulated by phosphorylation on serine, also controlling the association with hydrogels. It is hypothesized that the GR and PR transcripts associate with hydrogels (or similar assemblies such as RNA granules), but are impervious to the regulatory action of the kinases (no serine to phosphorylate), so they might clog up the trafficking of SR domain containing RNA binding proteins moving in an out of the granules to transfer information throughout the cell.

[ Neuron vol 84 pp. 1213 – 1225 ’14 ] Proline Arginine dipeptides are neurotoxic. They form aggregates in nucleoli in experimental systems. Nuclear aggregates were also found in postmortem spinal cord from C9ORF72 ALS and ALS/FTD patients. Intronic GGGGCC transcripts are also toxic. Repeat associated non-ATG translation (RAN translation) is thought to depend on RNA hairpin structures using GC pairing.

[ Cell vol. 158 pp. 967 ’14 (abstract of something to appear in Science) ] Peptide translated from GGGGCC expansions containng arginines (Gly Arg and Pro Arg) are harmful — 3 other dipeptide repeats are harmless. The peptides bind to nucleoi and impede RNA biogenesis. Interestingly Ser-Arg repeats proteins (SR proteins) are important in RNA splicing. The GlyARG and PROARG repeat peptides alter splicing of the amino acid transporter EAAT2, similar to that seen in ALS. Interestingly, the peptides are readily taken up by cells in culture, translocating to the nucleus.

Also a small molecule has been developed which targets GGGGCC RNA expansions. It inhibits translation of the dipeptide repeat proteins from the expansions (see Science vol. 353 pp. 64 ****

GlyPro in CSF is a biomarker of ALS patients with the C9orf7s expansion.

The normal function of C9orf72 isn’t known. It is structurally related to DENN (Differentially Expressed in Normal and Neoplastic cells) proteins, which are GDP/GTP exchange factors for Rab GTPases.

At this point it isn’t known if the proteins generated by RAN are toxic. The protein inclusions are present in unaffected areas of the brain (lateral geniculate) as well as the vulnerable areas (cortex, hippocampus).

The initiation of RNA translation is thought to depend on RNA hairpin structures which use C:G complementary pairing. CAG (but not CAA) repeats undergo RAN translation. Protein aggregates occured only in brain intestes despite the fact that C9orf72 is expressed all over the body (but expression is highest in brain).

It is possible that antisense RNA could be formed from the opposite strand (e.g. CCCCGG) giving poly pro-ala, poly pro-gly and poly pro-arg.

[ Science vol. 1106 – 1112 ’15 ] Just expressing 66 GGGGCC repeats without an ATG start codon using an AdenoAssociated Virus (AAV) vector in mice was enough to produce neurodegeneration with RNA foci, inclusoins of poly QP, GA and GR and TDP43 pathology. There was cortical neuron and cerebellar Purkinje cell loss and gliosis.

[ Nature vol. 525 pp. 36 – 37, 56 – 61, 129 – 133 ’15 ] (GGGGCC)30 was expressed in the Drosophila eye. This leads to the rough eye trait and is easily scored, allowing you to look at the effect of other genes on it. Mutations activating RanGAP suppressed rough eyes. RanGAP binds to GGGGCC on the cytoplasmic face of the nuclear pore. Enhancing nuclear import or suppressing nuclear export of proteins also suppressed neurodegeneration. RanGAP physically interacts with the GGGGCC Hexanucleotide Repeat Expansion resulting in its mislocalization. The mislocalization is found in neurons derived from iPSCs from a patient with C9orf72 type ALS, and also in brain tissue from other patients with C9orf72 ALS.

Nuclear import is impaired due to HRE expression (fly and iPSC derived neurons). The defects can be ‘rescued’ by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. This may actually be a way to Rx ALS ! ! ! !

Another paper crossed (GGGGCC)58 flies with missing chromosomal segments. They found a variety of nuclear import factors whose inactivation worsened rough eye.

Expression of constructs of in GGGGCC)8, 28 and 58 lacking an AUG start codon in Drosophila was done. The constructs could only produce Repeat Associated NonAUG translation products (e.g. dipeptides). The dipeptides disrupt nuclear import of fluorescent test substrates and of normal nuclear proteins (notably TDP43). In addition RNA export from the nucleus is also compromised. The deleterious effects could be modified by 18 genetic regions (found by large scale unbiased genetic screening). THey coded for components of the nuclear pore complex, nuclear RNA export machinery and nuclear import.

Dipeptides produced from GGGGCC and GGGGCCn’s disrupt the nucleolus, so this may be an additional cause of repeat toxicity.

[ Neuron vol. 88 pp. 892 – 901 ’15 ] A mouse model containng the full human C9orf72 repeat which was either normal (15 repeats) or expanded (100 – 1,000 repeats) — using bacterial artificial chromosomes (BACs) — thes mice are called C9-BACexpanded. They show widespread RNA foci and RAN translated dipeptides. Nucleolin distribution was altered. However the mice showed normal behavior and there was no neurodegenration. This is surprising.

[ Nature vol. 535 p. 327’16 (abstr. of Sci. Transl. Med ’16) ] Mice with mutations diminishing or eliminating the function of C9ORF72 (unknown as of 8/13) developed autoimmune disease.

[ Science vol. 351 pp. 1324 – 1329 ’16 ] Two independent mouse lines lacking the ortholog of C9orf72 (3110043021Rik) in all tissues developed normally and aged without any motor neuron disease. Instead they developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophagelike cells. There was age related neuroInflammation similar to C9orf72 ALS but not sporadic ALS. There was no evidence of neurodegeneration however.

[ Neuron vol. 90 pp. 427 – 430, 531 -534, 535 – 550 ’16 ] BAC transgenic mice using patient derived gene constructs expressing (some of? all of?) C9ORF72 are reported.

A germline knockout develops blood abnormalities (splenomegaly, lymphadenopathy and premature death). The data conflict on which of the 5 products of RAN (Repeat Associated NonATG) translation are the most toxic (GP, GA, GR, PA, PA, PR).

In this study, mice with increased levels of repeats (up to 450) showed no evidence of motor neuron disease, and the brain was normal. They at least did have some trouble with cognition.

THe second study put in the full C9 gene with 5′ and 3′ flanking sequences. 4 lines of transgenics with repeats ranging from 37 to 500 were characterized. These mice did have peirpheral and central neurodegeneration, with motor deficits. There was a decrease in cortical neurons, Purkinje cells. This is the first time any transgenic has shown neurodegeneration. The deficits are reversible with antisense oligonucleotides. There was a disparity in disease expression between male and female mice.

RNA foci and DPR (DiPeptide Repeat) proteins don’t accumulate in the most affected brain regions.

[ Science vol. 353 pp. 647 – 648, 708 – 712 ’16 ] Spt4 is a highly conserved transcription elongation factor which regulates RNA polymerase II processivity (along with its binding partner Spt5). Spt4 is required to transcribe long trinucleotide repeats found in open reading frames, or in non protein coding regions of DNA templates (in S. cerevisiae). Mutations of Spt4 decrease synthesis of (and restored enzymatic activity to) expanded polyQ proteins (in yeast) without affecting genes lacking the excessive CAG repeats. It might also work in nonCAG repeats.

Targeting Spt4 (with antiSense oligonucleotides) reduces production of the C9orf72 expansion associated RNA and protein, and helps neurodegeneration in model systems. Repeat expansions are transcribed in both the sense and antisense directions. Yeast Spt4 (human homolog SUPT4H) is a small evolutionarily conserved zinc finger protein which forms a complex with Spt5, which then binds to RNA polymerase II regulating transcription elongation (pol II processivity).

DRB is a RNA polymerase II inhibitor. The complex of Spt4 and Spt5 homologs in man (SUPT4H, SUPT5H) is called DSIF (DRB Sensitivity Inducing Factor)

Depletion of Spt4 or its binding partner (Spt5 ) decreases the number of both sense and antisense repeat transcripts and RNA foci. One of the 6 RAN translation products (polyGlyPro) is substantially reduced by Spt4 depletion.

The study was in human c9ALS fibroblasts. However, side effects are certainly possible — in addition to decreasing the expression of C9ORF72, 95% depletion of SUPT4H1 altered (how?) the expression of another 300 genes. In mice deletion of both copies of SUPT4 is embryonic lethal, but deleting one produced no effects up to 18 months of age.

Don’t get your hopes up — but

Amyotrophic lateral sclerosis (ALS) is a God-awful disease, where patients progressively weaken and die because they aren’t strong enough to breathe, remaining mentally intact the entire time. A recent paper [ Science vol. 348 pp. 239 – 242 ‘ 15 ] showed that a drug already released by the FDA for treating hypertension — Wytensin (Guanabenz) was of benefit in a mouse model of the disease. So the drug is out there. If I were still in practice, I’d certainly give it a shot in my patients — off-label use be damned. Even better, enterprising organic chemists synthesized an analogue of Wytensin (Sephin1) which doesn’t lower blood pressure, but which still works in the mouse model.

Here’s why you shouldn’t get your hopes up too high. [ Nature vol. 4564 pp. 682 – 685 ’08 ] The work using SOD1 mutant mice (the mouse model of ALS mentioned above) is quite sloppy and nearly 12 drugs with benefit in mouse models have had no benefit in clinical trials. Minocycline which was effective in 4 studies in mice actually made things worse in a clinical trial of over 400 patients .

Now for a bit of background. Most cases of ALS aren’t familial, but a few are. One protein Superoxide Dismutase 1 (SOD1) was found to mutated in about 20% of familial ALS. It’s been studied out the gazoo, and some 140 different mutations have been found in its 153 amino acids in familial cases.

It’s hard to conceive of them all acting the same way, and literally thousands of papers have been written on the subject. It does seem clear that aggregated proteins occur in the dying neurons of ALS patients, but whether they are made mostly of SOD1 remains controversial (although it is present in the inclusions to some extent). Mature SOD1 is a 32 kiloDalton homodimeric metalloenzyme, in which each monomer contains Cu and Zn and one intrasubunit disulfide bond. It is one of the most abundant cellular proteins. It has a tendency to aggregate when overexposed.

The mouse results are impressive, as it improved established disease. In vivo, Sephin1 prevented the motor morphological and molecular defects of two unrelated protein misfolding diseases in mice (Charcot Marie Tooth 1B and ALS ! ! !). The mice had a mutant SOD1 (G93A). SOD1 mutants bind to Derlin1 on the the cytosolic side of the endoplasmic reticulum (ER) membrane blocking degradation of ER proteins causing ER stress. Very impressive ! ! ! !