Tag Archives: Alzheimer’s disease

Cassava Sciences — the clinical reality underneath the stock gyrations.

The stock of Cassava Sciences (symbol SAVA) has undergone some wild gyrations this year.  On 14 September it traded at 41.70, today just two weeks later it is trading in the upper 60s.

The important thing to keep in mind, is that 1 year out on treatment with SAVA’s drug Simufilam 50 patients with mild Alzheimer disease were (as a group) slightly improved.  This is absolutely unprecedented.  The best that previous therapy could accomplish was a slightly slower rate of decline — see arshttps://science.sciencemag.org/content/sci/373/6555/624.full.pdf — for a recent review of other therapy attempts.  So Cassava’s results are unprecedented.   While Alzheimer (and other dementia) patients fluctuate from day to day (like the tides from minute to minute) at the end of a year they are all worse.

These results have not been attacked, unlike their data on the effect of Simufilam on biomarkers which has been criticized by a person of standing — Elizabeth Bik — https://scienceintegritydigest.com/2021/08/27/cassava-sciences-of-stocks-and-blots/#more-2692.

But that’s irrelevant and guilt by association at best.  As a clinical neurologist, no one was ever brought to see me because of their biomarkers.

They have released part of their 1 year results — https://www.cassavasciences.com/news-releases/news-release-details/cassava-sciences-announces-top-line-results-12-month-interim.  There is a lot more that I’d like to know, but a press release is not a detailed scientific paper.

What follows is a lot of commentary and speculation about the 1 year data which we haven’t seen yet.

The results concern the first 50 patients to complete one year on the drug.  The dropout rate is stated to be under 10%.  Presumably this includes death, in a cohort (presently at around 200) with a significant mortality.  It would be interesting to know how many patients on entry made it to one year.

As a clinical neurologist I was particularly impressed with part of their data at 9 months.  Here’s a link — keep it handy — https://www.cassavasciences.com/static-files/13794384-53b3-452c-ae6c-7a09828ad389.

They measured cognitive changes by something called ADAS-Cog — a full description can be found in the following post — https://luysii.wordpress.com/2021/08/25/cassava-sciences-9-month-data-is-probably-better-than-they-realize/

ADAS-Cog score counts errors, so a perfect score would be 0, and a terrible score would be 70.  The range of deficit on entry was 16 – 26 (but possibly on something else called the MMSE) — this is what the 1 year results used.  The 9 month results used ADAS-Cog.  Perhaps they are actually the same thing — I don’t know.

On the link — https://www.cassavasciences.com/static-files/13794384-53b3-452c-ae6c-7a09828ad389 — look at the diagram titled “Individual Patient Changes in ADAS-Cog (N = 50).

There were 5 patients out of 50 at 9 months with improvements of 11 – 14, which would mean that they were pretty close to normal if their entry score was 16 and 50% improved if their score was 26.  From here out I’m just calling them ‘the 5’.

The 9 month report doesn’t discuss this, and only a clinician would know, but this is the way neurologic patients respond to treatment.  Some do extremely well while others have no effect.  Why?  It’s probably because not really understanding causation, we classify patients clinically (it’s all docs have after all).

I ran a Muscular Dystrophy Clinic for 15 years back in the day.  The Muscular Dystrophy Association was founded by parents of weak kids.  They didn’t know that some weakness was due to the muscle itself (what we’re now calling muscular dystrophy), some was due to disease affected the nerves from the spinal cord to the muscle (what we call a neuropathy now) and others were due to disease of the cells in the spinal cord giving rise to the nerves to the muscle (motor neuron disease).  That all came later.

It is quite presumptuous to say that Alzheimer’s disease is just one thing.  Perhaps the 5 patients doing so very well had it from a different (as yet unknown) cause than the other 45.  Even so such a treatment would be worth having.

So here are a few questions for the folks at Cassava about their data

l. Some 16 different sites were involved in the open label study.  Were all of ‘the 5’  from the same site (doubtful — but if true, perhaps they tested ADAS-Cog differently, casting doubt on these results).

2. What were the ADAS-Cog scores initially on ‘the 5’.

3. What happened to ‘the 5’ in the past 3 months (did they maintain improvement, slide back, or improve further?)

4. We must have lots more people passing the 3, 6, 9 month markers.  Have their results paralleled that of the first 50 reaching the mileposts?   It would be very useful to know if there are now more than 5 with improvements over 10 in ADAS-Cog at 9 months.

The slightly slowing of improvement at 1 year relative to 9 months is typical of neurologic disease.  When L-DOPA was first available in the USA in 1970, some patients because so normal that you couldn’t tell they had Parkinson’s disease, and for a few years, neurologists (myself included) thought we were actually curing the disease.  Of course we weren’t and the underlying pathology of Parkinsonism (death of neurons using dopamine) continued unabated.  The L-DOPA just helped the surviving neurons function more efficiently.  Something similar may be going on with Simufilam and Alzheimer’s.

Now for some blue sky about Simufilam. Just as the gray hair on the head of an 80 year old looks the same under the microscope as one from a prematurely gray 30 year old, the brain changes of Alzheimer’s disease (the senile plaque)  are the same regardless of the age of onset.  Assuming that the senile plaque is in someway related to dementia (despite the lack of effect of therapies trying to remove it) and given that we all accumulate a few as we age, could Simufilam improve cognition in the elderly?   Would it then be intellectual viagra and the blockbuster drug of all blockbuster drugs.

 

Cassava Sciences 9 month data is probably better than they realize

My own analysis of the Cassava Sciences 9 month data shows that it is probably even better than they realize.

Here is a link to what they released — keep it handy https://www.cassavasciences.com/static-files/13794384-53b3-452c-ae6c-7a09828ad389.

I was unable to listen to Lindsay Burn’s presentation at the Alzheimer Association International Conference in July as I wasn’t signed up.  I have been unable to find either a video or a transcript, so perhaps Lindsay did realize what I’m about to say.

Apparently today 25 August there was another bear attack on the company and its data.  I’ve not read it or even seen what the stock did.  In what follows I am assuming that everything they’ve said about their data is true and that their data is what they say it is.

So the other day I had a look at what Cassava released at the time of Lindsay’s talk.

First some background on their study.  It is a report on the first 50 patients who had received Simulfilam for 9 months.  It is very important to understand how they were measuring cognition.  It is something called ADAS-Cog11

Here it is and how it is scored and my source — https://www.verywellhealth.com/alzheimers-disease-assessment-scale-98625

The original version of the ADAS-Cog consists of 11 items, including:1

1. Word Recall Task: You are given three chances to recall as many words as possible from a list of 10 words that you were shown. This tests short-term memory.

2. Naming Objects and Fingers: Several real objects are shown to you, such as a flower, pencil and a comb, and you are asked to name them. You then have to state the name of each of the fingers on the hand, such as pinky, thumb, etc. This is similar to the Boston Naming Test in that it tests for naming ability, although the BNT uses pictures instead of real objects, to prompt a reply.

3. Following Commands: You are asked to follow a series of simple but sometimes multi-step directions, such as, “Make a fist” and “Place the pencil on top of the card.”

4. Constructional Praxis: This task involves showing you four different shapes, progressively more difficult such as overlapping rectangles, and then you will be asked to draw each one. Visuospatial abilities become impaired as dementia progresses and this task can help measure these skills.

5. Ideational Praxis: In this section, the test administrator asks you to pretend you have written a letter to yourself, fold it, place it in the envelope, seal the envelope, address it and demonstrate where to place the stamp. (While this task is still appropriate now, this could become less relevant as people write and send fewer letters through the mail.)

6. Orientation: Your orientation is measured by asking you what your first and last name are, the day of the week, date, month, year, season, time of day, and location. This will determine whether you are oriented x 1, 2, 3 or 4.

7. Word Recognition Task: In this section, you are asked to read and try to remember a list of twelve words. You are then presented with those words along with several other words and asked if each word is one that you saw earlier or not. This task is similar to the first task, with the exception that it measures your ability to recognize information, instead of recall it.

8. Remembering Test Directions: Your ability to remember directions without reminders or with a limited amount of reminders is assessed.

9. Spoken Language: The ability to use language to make yourself understood is evaluated throughout the duration of the test.

10. Comprehension: Your ability to understand the meaning of words and language over the course of the test is assessed by the test administrator.

11. Word-Finding Difficulty: Throughout the test, the test administrator assesses your word-finding ability throughout spontaneous conversation.

What the ADAS-Cog Assesses

The ADAS-Cog helps evaluate cognition and differentiates between normal cognitive functioning and impaired cognitive functioning. It is especially useful for determining the extent of cognitive decline and can help evaluate which stage of Alzheimer’s disease a person is in, based on his answers and score. The ADAS-Cog is often used in clinical trials because it can determine incremental improvements or declines in cognitive functioning.2

Scoring

The test administrator adds up points for the errors in each task of the ADAS-Cog for a total score ranging from 0 to 70. The greater the dysfunction, the greater the score. A score of 70 represents the most severe impairment and 0 represents the least impairment.

The average score of the 50 individuals entering was 17 with a standard deviation of 8, meaning that about 2/3 of the group entering had scores of 9 to 25 and that 96% had scores of 1 to 32 (but I doubt that anyone would have entered the study with a score of 1 — so I’m assuming that the lowest score on entry was 9 and the highest was 25).  Cassava Sciences has this data but I don’t know what it is.

Now follow the link to Individual Patient Changes in ADAS-Cog (N = 50) and you will see 50 dots, some red, some yellow, some green.

Look at the 5 individuals who fall between -10 and – 15 and think about what this means.  -10 means that an individual made 10 fewer errors at 9 months than on entry into the study.  Again, I have no idea what the scores of the 5 were on entry.

So assume the worst and that the 5 all had scores of 25 on entry.  The group still showed a 50% improvement from baseline as they look like they either made 12, 13, or 14 fewer errors.  If you assume that the 5 had the average impairment of 17 on entry, they were nearly normal after 9 months of treatment.  That doesn’t happen in Alzheimer’s and is a tremendous result.   Lindsay may have pointed this out in her talk, but I don’t know although I’ve tried to find out.

Is there another neurologic disease with responses like this.  Yes there is, and I’ve seen it.

I was one of the first neurologists in the USA to use L-DOPA for Parkinsonism.  All patients improved, and I actually saw one or two wheelchair bound Parkinsonians walk again (without going to Lourdes).  They were far from normal, but ever so much better.

However,  treated mildly impaired Parkinsonians became indistinguishable from normal, to the extent that I wondered if I’d misdiagnosed them.

12 to 14 fewer errors is a big deal, an average decrease of 3 errors, not so much, but still unprecedented in Alzheimer’s disease.   Whether this is clinically meaningful is hard to tell.  However, 12 month data on the 50 will be available in the fourth quarter of ’21, and if the group as a whole continues to improve over baseline it will be a very big deal as it will tell us a lot about Alzheimer’s.

Cassava Sciences has all sorts of data we’ve not seen (not that they are hiding it).  Each of the 50 has 4 data points (entry, 3, 6 and 9 months) and it would be interesting to see the actual scores rather than the changes between them in all 50.  Were the 5 patients with the 12 – 14 fewer errors more impaired (high ADAS-Cog11 score in entry) or less.

Was the marked improvement in the 5 slow and steady or sudden?   Ditto for the ones who deteriorated or who got much worse or who slightly improved.

Even if such dramatic improvement is confined to 10% of those receiving therapy it is worth a shot to give it to all.  Immune checkpoint blockade has dramatically helped some patients with cancer  (far from all), yet it is tried in many.

Disclaimer:  My wife and I have known Lindsay since she was a teenager and we were friendly with her parents.  However, everything in this post is on the basis of public information available to anyone (and of course my decades of experience as a clinical neurologist)

 

What Cassava Sciences should do now

Apparently someone important didn’t like the way Cassava Sciences analyzed their data and their stock tanked again today..  Unfortunately all of this seems to be behind a paywall, and the someone important isn’t named.  I’d love a link if any reader knows of one — just put it in as a  comment below.

I’m not important, but I thought Cassava’s results were quite impressive.  They had enough cases and enough time for the results to be statistically significant

For one thing,  Cassava dealt with severely impaired people (see below) who would be expected to show greater neuronal dropout, senile plaques and neurofibrillary tangles, than recently diagnosed patients.   Neuronal loss is not reversible in man, despite hoards of papers showing the opposite in animals.

Since everything turns on ADAS-CoG, here is a link to a complete description along with some discussion — https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929311/

On a slide from Cassava’s presentation yesterday the ADAS-CoG average of the 50 patients on entry 9 months ago was 16.6.  With a perfect score of 70, it’s clear that these people were significantly impaired (please look at the test items to see how simple the tasks in ADAS-CoG actually are).    So an improvement of 3 points at 9 months  is significant, particularly since a drop of 5 points is expected each year — yes I’ve seen plenty of Alzheimer patients with ADAS-CoG scores of zero or close to it.

So an increase of 3 points in this group is about a16% improvement.

Here’s what Cassava should do now.  Their data should be re-examined as follows.  Split the ADAS-CoG scores into 3 groups: highest middle and lowest. Quartiles are usually used, but I don’t think 50 patients is enough to do this.  Then examine the median improvement in each of the three.  I’d use median rather than average as with small numbers in each group, a single outlier can seriously distort things — think of the survival of Stephen Hawking in a group of 12 ALS patients.

If the patients with the highest ADAS-CoG scores have the highest median improvement, there is no reason mildly impaired individuals should have a less than 16% improvement in their scores.  This means that a person with ADAS-CoG of 60 should achieve a perfect score of 70,  e.g. return to normal.

This would be incredibly useful for early Alzheimer’s disease.

There is a precedent for this.  Again it’s Parkinson’s disease.

As I mentioned in an earlier post, I was one of the first neurologists in the USA to use L-DOPA for Parkinsonism.  All patients improved, and I actually saw one or two wheelchair bound Parkinsonians walk again (without going to Lourdes).  They were far from normal, but ever so much better.

However,  treated mildly impaired Parkinsonians became indistinguishable from normal, to the extent that I wondered if I’d misdiagnosed them. These results were typical.   For a time, in the early 70s neurologists thought that we’d actually cured the disease.  It was a very heady time.  We were masters of the neurologic universe — schizophrenia was too much dopamine, Parkinsonism not enough. Bring on the next neurotransmitter, bring on the next disease.

We hadn’t cured anything of course, and the underlying loss of dopamine neurons in the substantia nigra continued.  Reality intruded for me with one such extremely normal appearing individual I’d diagnosed with Parkinsonism a few years earlier. He needed surgery, meaning that he couldn’t take anything by mouth for a while.  L-DOPA could only be given orally, and he looked quite Parkinsonian in a day or two.

If reanalysis of the existing data shows what I hope, Cassava Sciences should start another study in Alzheimer patients with ADAS-CoG scores of over 50.  If I’m right the results should be spectacular (and lead to immediate approval of the drug).

A little blue sky.  Sumafilam will then come to be known as intellectual Viagra, as all sorts of oldsters (such as yrs trly) will try to get it Alzheimer’s or no Alzheimer’s.

If you decided to buy Cassava Sciences yesterday everything went perfectly (except the price)

Yesterday I laid out the pros and cons of buying Cassava Sciences that day.  The post is reproduced below the ***

Everything I hoped for came true.  The 50 patients on Sumafilam were followed for 9 months and their ADAS-CoG score improved by 3 points.  This is unprecedented for any Alzheimer’s drug.  Historical controls show that Alzheimer patients lost 5 points a year on ADAS-CoG.  So this is a potential net gain with therapy vs. no therapyof  6 – 7 ADAS-CoG points.  Recall that a perfect ADAS-CoG score is 70.  I’ve been unable to find what the average score of 50 patients was on entry.  The paper isn’t published, but is public record results having been presented at conferences (such as today).  Recall that historical controls must be used as the study was open label (e.g. no concurrent controls).

Addendum 30 July:  Since everything turns on ADAS-CoG, here is a link to a complete description along with some discussion — https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929311/

On a slide from Cassava’s presentation yesterday the ADAS-CoG average of the 50 patients on entry 9 months ago was 16.6.  With a perfect score of 70, it’s clear that these people were significantly impaired (please look at the test items to see how simple the tasks in ADAS-CoG actually are).    So an improvement of 3 points at 9 months is significant, particularly since a drop of 5 points is expected each year — yes I’ve seen plenty of Alzheimer patients with ADAS-CoG scores of zero or close to it. 

However using historical controls is a no no particularly in neurology and cardiology.

Why?

From an old post “MDs gradually woke up to the fallacy of using historical rather than concurrent controls particularly in studies of therapies to prevent heart attack and stroke, as the rates of both dropped significantly in the past 50 years, and survival from individual heart attacks and strokes also improved.

 

However, I think using ADAS-CoG is OK in Alzheimer’s as we’re  talking about a disorder with no useful therapy.

 

I’m pleased that I saw the possibility of continued improvement in cognition in yesterday’s post.

 

So all my hopes for the drug came true, yet the stock tanked, closing at 103 down 32 points (down 24%) !

 

Why?  Well, in the past few months, all companies with drugs for Alzheimer’s disease have been fluctuating in price together, and one of them (to remain nameless to protect the innocent) had the temerity to release a 25 day study today on their drug based on 14 patients.  The stock was down 60%.

 

So Cassava got tarred with this brush.

 

Another likely reason is that the rise in Cassava was fueled by very small investors.  If you watched the transactions on a day SAVA was soaring, the purchases were rarely over 200.  So many of them were likely buying because others were.  So they sold when others were.  Lemmings anyone?

 

Nonetheless, SAVA’s data is much better than Biogen’s awful (and expensive) Aduhelm, so that Sumafilam is almost certain to be approved (1) if the data continue to be good (2) if a controlled trial controlled underway produces the same result.

 

So I think, in the long run, that the stock has a bright future, but as John Keynes said “In the long run we are all dead”

 

*** Yesterday’s post

 

Should you buy Cassava Sciences today?

Tomorrow Cassava Sciences will announce the interim results of an open label trial of its Alzheimer drug Sumafilam in 50 patients receiving the drug for 9 months. Should you buy the stock today?

The stock (symbol SAVA) has had a huge run this year starting at 7 and closing yesterday 27 July ’21 at 127.50.

I’ve been interested in the stock for several reasons

l. As a neurologist, I’ve watched patients, family members and friends deteriorate and die, being totally unable to help them.

2. I’ve known one of the principals in the company since she was a teenager in Montana — Lindsay Burns https://luysii.wordpress.com/2021/02/02/montana-girl-does-good-real-good/

3. Sumafilam is thought to work by a completely different mechanism of action than previous approaches (all of which have failed to produce a useful drug)– https://luysii.wordpress.com/2021/03/25/the-science-behind-cassava-sciences-sava/

In fact some of these therapies have actually made Alzheimer’s worse [ Nature Reviews Drug Discovery vol. 18 p. 327 ’19 ]

Tomorrow’s results should move the stock significantly.  If there is no improvement in cognition the stock will plummet.  If there is improvement the stock should soar, at least double again.  Why? Because we have no useful therapy.  Forget Biogen’s drug Aduhelm — the FDA advisory committee resigned in protest after the drug was approved, as the evidence for help was minimal at best.

Of course I’m rooting for the drug as a clinician and as a friend of Lindsay.

There is some evidence that the results tomorrow will show that the drug helps

A prior analysis after six months showed patients taking Cassava’s medication had a 10% improvement on cognition and 29% improvement on an inventory of dementia-related behavior, like delusions and anxiety.

 

The author of the article didn’t realize just how unprecedented these results are.  The numbers of patients (50) and the time (6 months) are long enough to make statistical fluke unlikely.

 

It is even possible that the patients will continue to improve — from the 6 month results, in which case the stock will go bananas.

 

Here’s why.
This isn’t in the books, but there is a precedent for continued improvement on Sumafilam based on my clinical experience with Parkinson’s disease.

 

I was one of the first docs able to prescribe L-DOPA for Parkinsonism in 9/70.  L-DOPA was released in the USA that month, after unconsciounable delay by the FDA.  I’d just left the Air Force and was starting to finish up my neurology residency at the University of Colorado.  The chief (James Austin) called me in and tasked me with setting up the brand new L-DOPA clinic.

 

 
We didn’t know what the drug would do, so we proceeded very cautiously.  Giving a little, watching, waiting, giving a little more, watching, waiting.  Wash rinse repeat.  The results were dramatic, as (like current therapy for Alzheimer’s disease), previous therapy was lousy. 

 

What became apparent to me, was that patients continued to improve ON THE SAME DOSE.   One of the mistakes GPs would make in subsequent years was increasing the dose quickly, since improvement was continuing (on the theory that if a little is good more would be better).  This pushed patients into toxicity (reversible fortunately). 

 

Something similar happens with all the antidepressants we have (except the ketamine derivatives).  You almost never see improvement in the first week or two. 

 

Do I know what tomorrow’s results will be?  Do I have inside information?  No.  Both my wife’s parents had decades long careers at the Securities and Exchange Commission (SEC), and I well know how they regard trading on inside information.

 

So these thoughts are just educated guesses.  If you are trying to decide whether or not to buy the stock, I hope they will be helpful to you.  Full disclosure: I do have a small position in the stock and am anxiously awaiting tomorrow’s results.

Nightmare on Wall Street

I’ve written several posts about Cassava Biosciences (symbol SAVA) and their potential drug for Alzheimer’s (see the end). The recent approval of Biogen’s ineffective (but highly lucrative) therapy Aducanumab for the disease brings forth the following nightmare. At a cost of > $50,000/year and millions of desperate famililes, Biogen will soon be rolling in money. The Cassava drug is orally available and should cost a fraction of that. Even better — it may actually work, although I think serious side effects are likely. Given the sketchy data getting Aducanumab through the FDA, Cassava’s drug represents a real threat to Biogen.

It will be perfectly legal for Biogen to outright buy Cassava and stop development. They will have the money. They won’t be able to do it on the sly, as any position of one company (or individual) in another greater than 5% of the value of the company must be reported to the SEC where it becomes public knowledge.

This from a cousin who is a stock market guru. His wife wasn’t available when I called being next door taking care of a woman with early Alzheimer’s, whose husband had to leave as his father suddenly passed away. She can’t be left alone. Such is the market for Aducanumab.

So will my friend Lindsay and her husband have the moral strength to resist Biogen?

Back in the day when I was in the service in Denver, a very wealthy stockbroker (who had brought the waterPik public) bought up many of beautiful old mansions on the west side of Cheeseman park. He then sold them to people he trusted (such as ourselves), so they wouldn’t be broken up into apartments (which was quite lucrative). I asked why the other people living on Humboldt street didn’t do the same. He said they had so much money they didn’t need character. The folks at Cassava don’t have a hell of a lot of money but hopefully they do have character.

Other posts on Cassava should you be interested are

The science behind Cassava Sciences (SAVA)

Do glia think? Take II

Do glia think Dr. Gonatas?  This was part of an exchange between G. Milton Shy, head of neurology at Penn, and Nick Gonatas a brilliant neuropathologist who worked with Shy as the two of them described new disease after new disease in the 60s ( myotubular (centronuclear) myopathy, nemaline myopathy, mitochondrial myopathy and oculopharyngeal muscular dystrophy).

Gonatas was claiming that a small glial tumor caused a marked behavioral disturbance, and Shy was demurring.  Just after I graduated, the Texas Tower shooting brought the question back up in force — https://en.wikipedia.org/wiki/University_of_Texas_tower_shooting.

Well that was 55 years ago, and we’ve learned a lot more about glia since.  

If glia don’t actually think, they may actually help neurons think better.  Since the brain is consuming 20% of your cardiac output as you sit there, it had better use the energy in the form of glucose  brought to it efficiently, and so it does, oxidizing it using oxygen (aerobic metabolism).  Glia on the other hand for reasons as yet unknown oxidize glucose anaerobically producing lactic acid (aerobic glycolysis). They transport the lactic acid to neurons and blocking transport impairs memory consolidation in experimental animals.  In fact aerobic glycolysis occurs in conditions of high synaptic plasticity and remodeling.  

The brain is 60% fat, some of which is cholesterol, which has to be made in the brain, as it doesn’t cross the blood brain barrier. Although neurons can synthesize cholesterol from scratch, most synthesis of cholesterol in the brain occurs in astrocytes.  It is than carried to neurons by apolipoprotein E.  As you are doubtless aware, apolipoprotein E (APOE) comes in three flavors 2, 3 and 4, and having two copies of APOE4 increases your risk of Alzheimer’s disease. 

But APOE does much more than schlep cholesterol to neurons according to a recent paper [ Neuron vol. 109 pp. 907 – 909, 957 – 970 ’21 ] Inside the particles are microRNAs.  You’ll recall that microRNAs decrease  the expression of proteins they target by binding to the messenger RNA (mRNA) for the targeted protein triggering its destruction. 

The microRNAs inside APOE suppress enzymes involved in de novo neuronal cholesterol biosynthesis (why work making cholesterol when the astrocyte is giving to you for free?).

This is unprecedented.  Passing metabolites (lactic acid, cholesterol) to neurons is one thing, but changing neuronal protein expression is quite another. 

Passing microRNAs in exosomes has been well worked out between cells (particularly cancer cells) outside the brain, but that’s for another time. 

The uses and abuses of Molarity

Quick what does a one Molar solution of a protein look like?

Answer: It doesn’t. The average protein mass is 100 kiloDaltons — http://book.bionumbers.org/how-big-is-the-average-protein/. That’s 100,000 grams per mole (100 kilograms).

A mole of any chemical is Avogadro’s number of it — or 6.02 x 10^23.  The molar mass counts 1 gram for each hydrogen it contains, 12 for each carbon etc. etc. 

A 1 molar concentration of any chemical is its molecular mass dissolved in 1 liter of water, which is 1,000 cubic centimeters (cc.).  The density of water is pretty much the same between 32 and 212 Fahrenheit (or 1 – 100 centigrade).  

What is the molar concentration of water, e.g. how many moles of water are in a liter of water.  The molecular mass of water is 18 so there are 1000/18 = 55.6 moles of water per liter of water.  

Well you can’t get 220 pounds of our 100 kiloDalton protein into 2.2 pounds (1 kiloGram) of water.  You could decorate each of the 6.02 x 10^23 protein molecules with 55 waters. 

Why belabor the obvious?  Because numbers are infinitely divisible and it is possible to talk about concentrations given in moles which make no chemical sense. Why?  Because matter is not infinitely divisible.  Divisibility for chemists stops at the atom level. 

Now let’s do some biology.  Cell size is measured in microns or 10^-6 meters.   A liter is a cube 10 centimeters on a side, so it is 10^-3 cubic meters.  A cubic micron is 10^-18 cubic meters, so there are 10^15 cubic microns in a liter. 

Now lets put 1 molecule in our cubic micron and each and every cubic micron in a liter of water.  What is its concentration in moles?  Our liter contains 10^15 molecules of our chemical, so its Molar concentration is 10^15/6.02 *10^23or .16 x 10^-8  or 1.6 x 10^-9 or 1.6 nanoMolar.    So 1 cubic micron is the volume  at which concentration less than 1.6 nanoMolar make no sense. 

It should be noted that 1 cubic micron contains plenty of water molecules to dissolve our molecule.  The actual number:

55 x 6.02 x 10^23/10^15 = 331 x 10^8  = 3 x 10^10 of them.

Notice that the mass of the molecule makes no difference.  Molar means moles/liter and liter is just a volume.  The number of molecules is what is crucial. 

As the volume goes up 1 molecule/volume makes sense at lower and lower concentrations. 

At this point the physicist says ‘consider a spherical cow’.  The biologist doesn’t have to.  We have lymphocytes which are nearly spherical with diameters ranging from 6 to 14 microns. 

Call it 10 microns.  Then the volume of our lymphocyte is  4/3 * pi * 5^3 = 524 cubic microns (call it 1,000 cubic microns to make things easier).  Recall that a liter contains 10^15 cubic microns.  So a liter can contain at most 10^12 lymphocytes, or 10^12 of our molecules so their concentration is 10^12/6.02 * 10^23 or 1.6 x 10^-12 molar. or 1.6 picoMolar.   Molar concentrations lower than 1.6 picoMolar make no chemical or biological sense in volumes of 1000 cubic microns. 

Are there chemicals in the lymphocyte with concentrations that low?  Sure there are.  Each chromosome is a molecule, so in male lymphocytes there is exactly one X chromosome and one Y. 

Next up.  Is a dissociation constant (Kd) in the femtoMolar (10^-15 Molar) range biologically meaningful?   I’m not sure and am still thinking about it, but the answer has some relevance to Alzheimer’s disease. 

Montana girl does good, real good !

Montana is flyover country. Nobody smart lives there. We all know that.

But when I got there in 1972 an issue of Science contained an article by State Legislator about a modification of general relativity — https://en.wikipedia.org/wiki/Kenneth_Nordtvedt.  MIT grad, Harvard Junior Fellow etc. etc. 

Then there was the son of a doc I practiced with in Billings.   Honors physics at Billings Senior high school placed him in 2nd year physics at Harvard, from which he graduated in 4 years obtaining a masters in physics as well. 

Then there was a local boy, the Thiokol engineer who predicted the Challenger disaster and was over-ruled. 

The great thing about Montana was that no one ever bragged about this sort of thing.  There were so few people, that no one felt compelled to tell you about themselves, you’d find out about them soon enough.  The classic example was an excellent surgeon and friend I practiced with for 15 years.  Only on reading his obituary last year did I find out that he had a Fulbright after college.

Which brings me to Lindsay, a girl I first met when she was a high school student.  The family were ranchers with a beautiful spread on the east face of the Crazy mountains north of Big Timber.  I’m not sure how we first met — I don’t think I saw any of them as a patient.  But we all became friends and the galactic premiere of a cello sonata I wrote with a 19 year old secretary in a lumberyard was in their living room. 

The two least important things about Lindsay are that she was a centerfold and an olympic silver medalist in woman’s two person crew.  Don’t get excited about the centerfold bit, she was fully clothed, but for some reason the Harvard Alumni magazine had a 2 page picture on a field of daisys of her back in the 80’s when she was there. 

Lindsay went on to get a PhD from Cambridge and her work and that of her husband may have come up with something useful for Alzheimer’s disease.  I’ll talk about the science behind it in a future post.  But when the news broke today, the stock of her company hit 70  (it was around 7 at the beginning of the year).  For details please see — https://finance.yahoo.com/m/49fa6153-4235-3866-bff2-5a35470e54da/why-cassava-sciences-stock.html.

Couldn’t happen to a nicer girl.  Of course it didn’t just happen.  Decades of hard work went into it.  So as you fly across the country, look down.  Some people down there might be even smarter than you are. 

Maybe the senile plaque really is a tombstone

“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.” I’ve thought this way for years, and the quote is from 2012: https://luysii.wordpress.com/2012/07/26/research-on-alzheimers-disease-the-bad-news-the-good-news/.

We now have some evidence that the senile plaque may be just a tombstone marking a dead neuron. Certainly attempts to remove the plaques haven’t helped patients despite billions spent in the attempt.

A recent paper [ Proc. Natl. Acad. Sci. vol. 117 pp. 28625–28631 ’20 –https://www.pnas.org/content/pnas/117/46/28625.full.pdf ] not only provides a new way to look at Alzheimer’s disease, but immediately suggests (to me at least) a way to test the idea. If the test proves correct, it will turn the focus of Alzheimer disease research on its ear.

Not to leave anyone behind, the senile plaque is largely made of a small fragment (the aBeta peptide 40 or 42 amino acids) from a much larger protein (the amyloid precursor protein [ APP ] — with well over 800 amino acids). Mutations in APP with the net effect of producing more aBeta are associated with familial Alzheimer’s disease, as are mutations in the enzymes chopping up APP to form Abeta (presenilin1, etc.).

The paper summarizes some evidence that the real culprit is neuronal uptake of the Abeta peptide either as a monomer, or a collection of monomers (an oligomer) or even the large aggregate of monomers seen under the microscope as the senile plaque.

The paper gives clear evidence that a 30 amino acid fragment of Abeta by itself without oligomerization can be taken up by neurons. Not only that but they found the protein on neuronal cell surface that Abeta binds to as well.

Ready to be shocked?

The protein taking Abeta into the neuron is the prion protein (PrPC) which can cause mad cow disease, wasting disease of elk and all sorts of horrible neurologic diseases among them Jakob Creutzfeldt disease.

Now to explain a bit of the jargon which follows. The amino acids making up our proteins come in two forms which are mirror images of each other. All our amino acids are of the L form, but the authors were able to synthesize the 42 amino acid Abeta peptide (Abeta42 below) using all L or all D amino acids.

It’s time to let the authors speak for themselves.

“In previous experiments we compared toxicity of L- and D-Aβ42. We found that, under conditions where L-Aβ42 reduced cell viability over 50%, D-Aβ42 was either nontoxic (PC12) or under 20% toxic . We later showed that L-Aβ is taken up approximately fivefold more efficiently than D-Aβ (28), suggesting that neuronal Aβ uptake and toxicity are linked.”

Well, if so, then the plaque is the tombstone of a neuron which took up too much Abeta.

Well how could you prove this? Any thoughts?

Cell models are nice, but animal models are probably better (although they’ve never resulted in useful therapy for stroke despite decades of trying).

Enter the 5xFAD mouse — it was engineered to have 3 mutations in APP known to cause Familial Alzheimer’s Disease + 2 more in Presenilin1 (which also cause FAD). The poor mouse starts getting Abeta deposition in its brain under two months of age (mice live about two years).

Now people aren’t really sure just what the prion protein (PrPC) actually does, and mice have been made without it (knockout mice). They are viable and fertile, and initially appear normal, but abnormalities appear as the mouse ages if you look hard enough. But still . . .

So what?

Now either knock out the PrPC gene in the 5xFAD mouse or mate the two different mouse strains.

The genes (APP, presenilin1 and PrPC) are on different chromosomes (#21, #14 and #20 respectively). So the first generation (F1) will have a normal counterpart of each of the 3 genes, along with a pathologic gene (e.g. they will be heterozygous for the 3 genes).

When members of F1 are bred with each other we’d expect some of them to have all mutant genes. If it were only 2 genes on two chromosomes, we’d expect 25% of he offspring (F2 generation) to have all abnormal genes. I’ll leave it for the mathematically inclined to figure out what the actual percentage of homozygous abnormal for all 3 would be).

What’s the point? Well, it’s easy to measure just what genes a mouse is carrying, so it’s time to look at mice with a full complement of 5xFAD genes and no PrPC.

If these mice don’t have any plaques in their brains, it’s game, set and match. Alzheimer research will shift from ways to remove the senile plaque, to ways to prevent it by inhibiting cellular uptake of the abeta peptide.

What could go wrong? Well, their could be other mechanisms and other proteins involved in getting Abeta into cells, but these could be attacked as well.

If the experiment shows what it might, this would be the best Thanksgiving present I could imagine.

So go to it. I’m an 80+ year old retired neurologist with no academic affiliation. I’d love to see someone try it.

Amyloid

Amyloid goes way back, and scientific writing about has had various zigs and zags starting with Virchow (1821 – 1902) who named it because he thought it was made out of sugar.  For a long time it was defined by the way it looks under the microscope being birefringent when stained with Congo red (which came out 100 years ago,  long before we knew much about protein structure (Pauling didn’t propose the alpha helix until 1951).

Birefringence itself is interesting.  Light moves at different speeds as it moves through materials — which is why your legs look funny when you stand in shallow water.  This is called the refractive index.   Birefringent materials have two different refractive indexes depending on the orientation (polarization) of the light looking at it.  So when amyloid present in fixed tissue on a slide, you see beautiful colors — for pictures and much more please see — https://onlinelibrary.wiley.com/doi/full/10.1111/iep.12330

So there has been a lot of confusion about what amyloid is and isn’t and even the exemplary Derek Lowe got it wrong in a recent post of his

“It needs to be noted that tau is not amyloid, and the TauRx’s drug has failed in the clinic in an Alzheimer’s trial.”

But Tau fibrils are amyloid, and prions are amyloid and the Lewy body is made of amyloid too, if you subscribe to the current definition of amyloid as something that shows a cross-beta pattern on Xray diffraction — https://www.researchgate.net/figure/Schematic-representation-of-the-cross-b-X-ray-diffraction-pattern-typically-produced-by_fig3_293484229.

Take about 500 dishes and stack them on top of each other and that’s the rough dimension of an amyloid fibril.  Each dish is made of a beta sheet.  Xray diffraction was used to characterize amyloid because no one could dissolve it, and study it by Xray crystallography.

Now that we have cryoEM, we’re learning much more.  I have , gone on and on about how miraculous it is that proteins have one or a few shapes — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/

So prion strains and the fact that alpha-synuclein amyloid aggregates produce different clinical disease despite having the same amino acid sequence was no surprise to me.

But it gets better.  The prion strains etc. etc may not be due to different structure but different decorations of the same structure by protein modifications.

The same is true for the different diseases that tau amyloid fibrils produce — never mind that they’ve been called neurofibrillary tangles and not amyloid, they have the same cross-beta structure.

A great paper [ Cell vol. 180 pp. 633 – 644 ’20 ] shows how different the tau protofilament from one disease (corticobasal degeneration) is from another (Alzheimer’s disease).  Figure three shows the side chain as it meanders around forming one ‘dish’ in the model above.  The meander is quite different in corticobasal degeneration (CBD) and Alzheimers.

It’s all the stuff tacked on. Tau is modified on its lysines (some 15% of all amino acids in the beta sheet forming part) by ubiquitination, acetylation and trimethylation, and by phosphorylation on serine.

Figure 3 is worth more of a look because it shows how different the post-translational modifications are of the same amino acid stretch of the tau protein in the Alzheimer’s and CBD.  Why has this not been seen before — because the amyloid was treated with pronase and other enzymes to get better pictures on cryoEM.  Isn’t that amazing.  Someone is probably looking to see if this explains prion strains.

The question arises — is the chain structure in space different because of the modifications, or are the modifications there because the chain structure in space is different.  This could go either way we have 500+ enzymes (protein kinases) putting phosphate on serine and/or threonine, each looking at a particular protein conformation around the two so they don’t phosphorylate everything — ditto for the enzymes that put ubiquitin on proteins.

Fascinating times.  Imagine something as simple as pronase hiding all this beautiful structure.