Tag Archives: Abeta42

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.

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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.

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 more hydrophobic 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.

Baudelaire comes to Chemistry

Could an evil molecule be beautiful? In Les Fleurs du Mal, a collection of poems, Baudelaire argued that there was a certain beauty in evil. Well, if there ever was an evil molecule, it’s the Abeta42 peptide, the main component of the senile plaque of Alzheimer’s disease, a molecule whose effects I spent my entire professional career as a neurologist ineffectually fighting. And yet, in a recent paper on the way it forms the fibrils constituting the plaque I found the structure compellingly beautiful.

The papers are Proc. Natl. Acad. Sci. vol. 113 pp. 9398 – 9400, E4976 – E4984 ’16. People have been working on the structure of the amyloid fibril of Alzheimer’s for decades, consistently stymied by its insolubility. The authors solved it not by Xray crystallography, not by cryoEM, but by solid state NMR. They basically looked at the distance constraints between pairs of isotopically labeled atoms, and built their model that way. Actually they built a bouquet of models using computer aided energy minimization of the peptide backbone. Another independent study produced nearly the same set.

The root mean square deviation of backbone atoms of the 10 lowest energy models of the bouquets in the two studies was small (.89 and .71 Angstroms). Even better the model bouquets of the two papers resemble each other.

There are two chains of Abeta42, EACH shaped like a double horseshoe (similar to the letter S). The two S’s meet around a twofold axis. The interface between the two S’s is form by two noncontiguous areas on each monomer (#15 – #17) and (#34 – #37).

The hydrophilic amino terminal residues (#1 – #14) are poorly ordered, but amino acids #15 – #42 are arranged into 4 short beta strands (I only see 3 obvious ones) that stack up and down the fibril into parallel in register beta-sheets. Each stack of double horseshoes forms a thread and the two threads twist around each other to form a two stranded protofilament.

Glycines allow sharp turns at the corners of the horseshoes. Hydrogen bonds between amides link the two layers of the fibrils. Asparagine side chains form ladders of hydrogen bonds up and down the fibrils. Water isn’t present between the layers because the beta sheets are so close together (counterintuitively this decreases the entropy, because water molecules don’t have to align themselves just so to solvate the side chains).

Each of the horseshoes is stabilized by hydrophobic interactions among the hydrophobic side chains buried in the core. Charged residues are solvent exposed. The interface between the two horsehoes is a hydrophobic interface.

Many of the famlial mutations are on the outer edges of double S structure — they are K16N, A21G, D23N, E22A, E22K, E22G, E22Q.

The surface hydrophobic patch formed by V40 and A42 may explain the greater rate of secondary nucleation by Abeta42 vs. Abeta40.

The cryoEM structures we have of Abeta42 are different showing the phenomenon of amyloid polymorphism.

The PNAS paper used reombinant Abeta and prepared homogenous fibrils by repeated seeding of dissolved Abeta42 with preformed fibrils. The other study used chemically synthesized Abeta and got fibrils without seeding. Details of pH, peptide concentration, salt concentration differed, and yet the results are the same, making both structures more secure.

The new structure doesn’t immediately suggest the toxic mechanism of Abeta.

To indulge in a bit of teleology — the structure is so beautiful and so intricately designed, that the aBeta42 peptide has probably been evolutionarily optimized to perform an (as yet unknown) function in our bodies. Animals lacking Abeta42’s parent (the amyloid precursor protein) don’t form neuromuscular synapses correctly, but they are viable.