Tag Archives: alpha-Synuclein

Has the holy grail for Parkinson’s disease been found?

Will the horribly named SynuClean-D treat Parkinsonism?  Here is the structure described  verbally.  Start with pyridine.  In the 2 position put benzene with a nitrogroup in the meta position, position 3 on pyridine NO2, position 4 CF3, position 5 CN (is this trouble?) position 6 OH.  That’s it.  Being great chemists you can immediately see what it does.

Back up a bit.  One of the pathologic findings in parkinsonism in the 450,000 dopamine neurons we have in the pars compacta at birth, is the Lewy body, which is largely made of the alpha-synuclein protein.  This is thought to kill the neurons in some way (just which form of alpha-synuclein is the culprit is still under debate — the monomer, the tetramer etc. etc).  Even the actual conformation of the monomer is still under debate (intrinsically disordered) etc. etc.

The following paper [ Proc. Natl. Acad. Sci. vo. 115 pp. 10481 – 10486 ’18 ] claims that SynuClean-D inhibits alpha-synuclein aggregation, disrupts mature amyloid fibrils made from it, prevents fibril propagation and abolishes the degeneration of dopamine neurons in an animal model of Parkinsonism.  Wow ! ! !

Time for some replication — look at the disaster from Harvard Med School about cardiac stem cells, with 30+ papers retracted. https://www.nytimes.com/2018/10/15/health/piero-anversa-fraud-retractions.html.  Ghastly.

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Are the inclusions found in neurologic disease attempts at defense rather then the cause?

Thinking about pathologic changes in neurologic disease has been simplistic in the extreme.  Intially both senile plaques and neurofibrillary tangles were assumed to be causative for Alzheimer’s.  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 fascinating recent paper [ Neuron vol. 97 pp. 3 – 4, 108 – 124 ’18 ] http://www.cell.com/neuron/pdf/S0896-6273(17)31089-9.pdf gives strong evidence that some inclusions can be defensive rather than toxic.  It contains the following;

“In these studies, we found that formation of large inclusions was correlated with protection from a-synuclein toxicity”

The paper is likely to be a landmark because it ties two neurologic diseases (Parkinsonism and Alzheimer’s) together by showing that they may due to toxicity produced by single mechanism — inhibition of mitochondrial function.

Basically, the paper says that overproduction of alpha synuclein (the major component of the Lewy body inclusion of Parkinsonism) and tau (the major component of the neurofibrillary tangle of Alzheimer’s disease) produce death and destruction by interfering with mitochondria.  The mechanism is mislocalization of a protein called Drp1 which is important in mitochondrial function (it’s required for mitochondrial fission).

Actin isn’t just found in muscle, but is part of the cytoskeleton of every cell.  Alpha-synuclein is held to alter actin dynamics by binding to another protein called spectrin (which also binds to actin).  The net effect is to mislocalize Drp1 so it doesn’t bind to mitochondria where it is needed.  It isn’t clear to me from reading the paper, just where the Drp1 actually goes.

In any event overexpressing spectrin causes the alpha-synuclein to bind to it forming inclusions and protecting the cells.

There is a similar mechanism proposed for tau, and co-expressing alpha synuclein with Tau significantly enhances the toxicity of both models of tau toxicity which implies that they work by a common mechanism.

Grains of salt are required because the organism used for the model is the humble fruitfly (Drosophila).

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.

Kuru continues to inform

Neurologists of my generation were fascinated with Kuru, a disease of the (formerly) obscure Fore tribe of New Guinea. Who would have thought they would tell us a good deal about protein structure and dynamics?

It is a fascinating story including a Nobelist pedophile (Carleton Gajdusek) https://en.wikipedia.org/wiki/Daniel_Carleton_Gajdusek and another (future) Nobelist who I probably ate lunch with when we were both medical students in the same Medical Fraternity but don’t remember –https://en.wikipedia.org/wiki/Stanley_B._Prusiner

Kuru is a horrible neurodegeneration starting with incoordination, followed by dementia and death in a vegetative state in 4 months to 2 years. For the cognoscenti — the pathology is neuronal loss, astrocytosis, microglial proliferation, loss of myelinated fibers and the kuru plaque.

It is estimated that it killed 3,000 members of the 30,000 member tribe. The mode of transmission turned out to be ritual cannibalism (flesh of the dead was eaten by the living before burial). Once that stopped the disease disappeared.

It is a prion disease, e.g. a disease due to a protein (called PrP) we all have but in an abnormal conformation (called PrpSc). Like Vonnegut’s Ice-9 (https://en.wikipedia.org/wiki/Ice-nine) PrPSc causes normal PrP to assume its conformation, causing it to aggregate and form an insoluble mess. We still don’t know the structure of PrPSc (because it’s an insoluble mess). Even now, “the detailed structure of PrPSc remains unresolved” but ‘it seems to be’ very similar to amyloid [ Nature vol. 512 pp. 32 – 34 ’14]. Not only that, but we don’t know what PrP actually does, and mice with no PrP at all are normal [ Nature vol. 365 p. 386 ’93 ]. For much more on prions please see https://luysii.wordpress.com/2014/03/30/a-primer-on-prions/

Prusiner’s idea that prion diseases were due to a protein, with no DNA or RNA involved met with incredible resistance for several reasons. This was the era of DNA makes RNA makes protein, and Prisoner was asking us to believe that a protein could essentially reproduce without any DNA or RNA. This was also the era in which X-ray crystallography was showing us ‘the’ structure of proteins, and it was hard to accept that there could be more than one.

There are several other prion diseases of humans (all horrible) — mad cow disease, Jakob Creutzfeldt disease, Familial fatal insomnia, etc. etc. and others in animals. All involve the same protein PrP.

One can take brain homogenates for an infected animal, inoculate it into a normal animal and watch progressive formation of PrPSc insoluble aggregates and neurodegeneration. A huge research effort has gone into purifying these homogenates so the possibility of any DNA or RNA causing the problem is very low. There still is one hold out — Laura Manuelidis who would have been a classmate had I gone to Yale Med instead of Penn. n

Enter [ Nature vol. 522 pp. 423 – 424, 478 – 481 ’15 ] which continued to study the genetic makeup of the Fore tribe. In an excellent example of natural selection in action, a new variant of PrP appeared in the tribe. At amino acid #127, valine is substituted for glycine (G127V is how this sort of thing is notated). Don’t be confused if you’re somewhat conversant with the literature — we all have a polymorphism at amino acid #129 of the protein, which can be either methionine or valine. It is thought that people with one methionine and one valine on each gene at 129 were somewhat protected against prion disease (presumably it affects the binding between identical prion proteins required for conformational change to PrPSc.

What’s the big deal? Well, this work shows that mice with one copy of V127 are protected against kuru prions. The really impressive point is that the mice are also protected against variant Creutzfedlt disease prions. Mice with two copies of V127 are completely protected against all forms of human prion disease . So something about V/V at #127 prevents the conformation change to PrPSc. We don’t know what it is as the normal structure of the variant hasn’t been determined as yet.

This is quite exciting, and work is certain to go on to find short peptide sequences mimicking the conformation around #127 to see if they’ll also work against prion diseases.

This won’t be a huge advance for the population at large, as prion diseases, as classically known, are quite rare. Creutzfeldt disease hits 1 person out of a million each year.

There are far bigger fish to fry however. There is some evidence that the neurofibrillary tangles (tau protein) of Alzheimer’s disease and the Lewy bodies (alpha-Synuclein) of Parkinsonism, spread cell to cell by a ‘prionlike’ mechanism [ Nature vol.485 pp. 651 – 655 ’12, Neuron vol. 73 pp. 1204 – 1215 ’12 ]. Could this sort of thing be blocked by a small amino acid change in one of them (or better a small drug like peptide?).

Stay tuned.