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.

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.