Faked Beta-Amyloid Data. What Does It Mean?

Written by Derek Lowe on August 2, 2022. Posted in Current News

Late last week came this report in Science about doctored images in a series of very influential papers on amyloid and Alzheimer’s disease.

That’s attracted a lot of interest, as well it should, and as a longtime observer of the field (and onetime researcher in it), I wanted to offer my own opinions on the controversy.

First off, I’ve noticed a lot of takes along the lines of “OMG, because of this fraud we’ve been wasting our time on Alzheimer’s research since 2006”. That’s not really the case, as I’ll explain.

But don’t get your hopes up: from one point of view, the main inaccuracy in that statement is that we’ve been actually been wasting our time in Alzheimer’s research for even longer than that. So that’s not very comforting, either.

We’ll start with some background and history, the better to appreciate the current furor in context. Those of you who know the field can skip ahead to later sections as marked, but your admission ticket is valid for the entire length of the ride if you want to get on here.

The Amyloid Hypothesis

An association between Alzheimer’s disease and amyloid protein in the brain has been around since. . .well, ever since the early 1900s, when Alois Alzheimer (and Oskar Fischer, independently) recognized some odd features in the brains of people who had died with memory loss and dementia. There were dark plaques among the neurons, which stained in a way that suggested they were some sort of amyloid protein.

The association of these plaques with dying neurons made a compelling case that they were involved in the disease, although it was recognized at the same time that there were “neurofibrillary tangles” that were also present as a sign of pathology. A classic AD plaque has a dense core of precipitated protein, surrounded by a less dense halo of abnormal protein around it, and also surrounded by a lot of rather unhealthy-looking extensions of nearby neurons.

Activated astrocytes and microglia are present as well, suggesting that some degenerative process has been taking place for some time and that the usual repair mechanisms have not been up to the job.

In the mid-1980s, the main protein in the plaques was conclusively identified as what became known as beta-amyloid, a fairly short (36 to 42 amino acid) piece that showed a profound tendency to aggregate into insoluble masses. Pure “A-beta” is not a lot of fun to work with or even to synthesize; it really does gum things up alarmingly. A lot of proteins can do that to some degree, with various types of amyloid high on that list.

The same protein was found in the (similar) lesions that develop in the brain tissue of Down’s syndrome patients, who often show memory loss and AD-like symptoms even earlier than usual. By the early 1990s, the “amyloid cascade hypothesis” of Alzheimer’s was the hot topic in the field.

Beta-amyloid had been found to be a cleavage product from inside the sequence of a much larger species (APP, or amyloid precursor protein), and the cascade hypothesis was that excess or inappropriately processed beta-amyloid was in fact the causative agent of plaque formation, which in turn was the cause of Alzheimer’s, with all the other neuropathology (tangles and so on) downstream of this central event.

And for the last thirty years, this has been the reigning idea in the field, although there are others (such as tau protein, which is more involved with the neurofibrillary tangles). It has not been a smooth ride, though.

Before getting to that part, please keep in mind that there’s a lot of support for the amyloid hypothesis itself, and I say that as someone who has been increasingly skeptical of the whole thing. For example, mutations in APP that lead to easier amyloid cleavage also lead to earlier development of Alzheimer’s symptoms, and that’s pretty damn strong evidence.

There are human families around the world (in Sweden, Holland, Mexico, Colombia and more) with “hereditary early onset Alzheimer’s” of this sort, and these things almost always map back to mutations in amyloid processing. If you engineer excess beta-amyloid in cells in culture, you see toxic effects, and the same goes for engineering that in living animals. Now, none of those mice or whatever develop syndromes quite like human Alzheimer’s, to be sure – we’re the only animal that does, interestingly, but excess beta-amyloid is always trouble.

But along with these strong signals there have always been plenty of mysteries, too: no one’s quite sure of all the functions of the APP protein, for starters, and that goes double for whatever the normal functions of beta-amyloid might be. Meanwhile, although there does seem to be a correlation between amyloid plaques and dementia, there are people who show significant amyloid pathology on examination after death who did not show real signs of Alzheimer’s.

Progress has been slowed by the longstanding problem of only being able to see the plaques post-mortem (brain tissue biopsies are not a popular technique) – there are now imaging agents that give a general picture in a less invasive manner, but they have not helped settle the debates. The results of the clinical trials of the last twenty years or so have only added to these problems.

Putting It to the Test

Ever since the 1990s, researchers and clinicians have been spending uncountable hours (and uncountable dollars) trying to turn the amyloid hypothesis into a treatment for Alzheimer’s. I would not like to count the number of such attempts, nor even to try to list all of the variations. There have been all sorts of treat-the-symptoms approaches, for sure, but also a number of direct shots on goal.

The enzymes that cleave beta-amyloid out of the APP protein (beta-secretase and gamma-secretase) have been targeted for inhibition, naturally. Small molecules have been sought that would slow down amyloid aggregation or even to promote its clearance. Most famously, antibodies have been produced against various forms of beta-amyloid itself, in attempts to interrupt their toxicity and cause them to be cleared by the immune system.

Every single one of these interventions has failed in the clinic. Every last damn one. If you look for the best outcome of all, actual reversal of Alzheimer’s symptoms, you never see it. No one has, and given the level of neuronal damage, it’s quite possible that no one ever will, unfortunately. What about just slowing down the inexorable progress that the disease seems to show in so many patients?

No luck there, either. Compared to control patients, none of these therapies have shown meaningful effects on the rate of decline. One of the gamma-secretase inhibitor trials actually seemed to speed it up, for reasons yet unknown.

The antibody trials have been the most disconcerting. Some of them have actually shown real reductions in amyloid levels in the brains of the patients, which should be good news, but at the same time these reductions have not led to any real improvements in their cognitive state. Not even a slowdown in the rate of developing Alzheimer’s symptoms.

If you had time-traveled back to the mid-1990s and told people that antibody therapies would actually have cleared brain amyloid in Alzheimer’s patients, people would have started celebrating – until you hit them with the rest of the news. Of course, as I’ve often said, if you’d time-traveled back 30 years and told me (while I was working in the field) that we’d still be arguing about the amyloid hypothesis itself in 2022, I would have been profoundly displeased, to put it judiciously.

These failures have led to a whole list of explanatory, not to say exculpatory hypotheses: perhaps the damage had already been done by the time people could be enrolled in a clinical trial, and patients needed to be treated earlier (much, much earlier). Or we had somehow picked the wrong kind of Alzheimer’s patients – the disease might well stratify in ways that we couldn’t yet detect, and we needed to wait for better ways to pick those who would benefit.

Maybe the different forms of beta-amyloid (different lengths and different aggregation/oligomerization states) were not being targeted correctly: we had raised antibodies to the wrong ones, and when we zeroed in on the right one we would see some real clinical action.

Oligomers As An Explanation, And the *56 Species

That’s where the currently contested work really comes into the picture. There had been a lot of work (and a lot of speculation) about the possibility of there being hard-to-track-down forms of amyloid that were the real causative agent of Alzheimer’s. You can find plenty of papers from the late 1990s and early 2000s on the idea of pathogenic soluble amyloid oligomers, oligimerization state as correlated with disease, all that sort of thing. It was certainly not an unexplored idea.

But this 2006 paper did indeed get a lot of attention, because it took the idea further than many other research groups had. It was from the lab of Karen Ashe at Minnesota, highlighting work from Sylvain Lesné in her lab on a form of amyloid called AB*56. Isolating this species from a transgenic mouse model and injecting it into young rats caused them to start exhibiting memory defects in turn.

Now that’s the kind of smoking gun you want to see, especially in a field as murky and tangled as this one. That paper has been cited well over 2000 times since then, and Lesné has since produced a large number of papers following up on this idea and its ramifications. He’s running his own research group now, naturally, and Ashe’s group has also continued to work on amyloid oligomers, as have (by now) many others.

But some of these later Lesné papers had been flagged on the PubPeer site for potential image doctoring, and the Science report linked to in the first paragraph of this blog details the efforts of neuroscientist Matthew Schrag at Vanderbilt to track these things down. He was originally hired by two other neuroscientists who also sell biopharma stocks short  – my kind of people, to be honest – to investigate published research related to Cassava Sciences and their drug Simufilam, and that work led him deeper into the amyloid literature.

Now Cassava is a story of their own, and I have frankly been steering clear of it, despite some requests. To me, it’s an excellent example of a biotech stock with a passionate (and often flat-out irrational) fan club. In such cases, if you say bad things about a beloved stock then plenty of helpful strangers will point out that you are an idiot, a shill, an evil agent of the moneyed interests, and much, much more.

There’s a detailed sidebar in the Science article on Cassava and on simufilam, which I recommend to anyone who wants to catch up on that aspect. That compound is supposed to restore the function of the protein Filamin A, which is supposed to be beneficial in Alzheimer’s, and my own opinion is that neither the published work on this compound nor the conduct of the company inspires my trust. There’s an ongoing investigation into the work at CUNY, and perhaps I’ll return to the subject once it concludes.

But Schrag’s dive into the Alzheimer’s literature put him onto allegations of image manipulation in the amyloid field as well, and that’s why we find ourselves in the current situation. Schrag (and others on PubPeer) have found what looks like a long trail of image manipulation in Lesné’s papers, particularly the ever-popular duplication of Western blots to produce bands where you need them.

The Science article illustrates some of these, and it looks bad: protein bands showing up in different places with exactly the same noise in their borders, apparent copy-and-past border lines, etc. Schrag has filed a long, detailed whistleblower report with the NIH and alerted numerous journal editors, and some of the papers are already being flagged with Expressions of Concern while the matter is being investigated.

Science had Schrag’s findings re-evaluated by several neuroscientists, by Elisabeth Bik, a microbiologist and extremely skilled spotter of image manipulation, and by another well-known image consultant, Jana Christopher.

Those last two even found more examples that Schrag himself had missed. Apparently everyone agrees that Lesné’s work is full of trouble.

Phrases like “shockingly blatant” and “highly egregious” are quoted, and it looks like key parts of the experimental evidence in these papers is nothing more than cut-and-paste jobs assembled to show the desired result.

This is taken from a long document. Read the rest here: science.org

Header image: News Medical

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