Tag Archives: ALS

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.

 

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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 ! ! ! !