Category Archives: Medicine in general


The innate immune system is intrinsically fascinating, dealing with invaders long before antibodies or cytotoxic cells are on the scene.  It is even more fascinating to a chemist because it works in part by forming amyloid inside the cell.  And you thought amyloid was bad.

The system becomes even more fascinating because blocking one part of it (RIPK1) may be a way to treat a variety of neurologic diseases (ALS, MS,Alzheimer’s, Parkinsonism) whose treatment could be improved to put it mildly.

One way to deal with an invader which has made it inside the cell, is for the cell to purposely die.  More and more it appears that many forms of cell death are elaborately programmed (like taking down a stage set).

Necroptosis is one such, distinct from the better known and studied apoptosis.   It is programmed and occurs when a cytokine such as tumor necrosis factor binds to its receptor, or when an invader binds to members of the innate immune system (TLR3, TLR4).

The system is insanely complicated.  Here is a taste from a superb review — unfortunately probably behind a paywall — — PNAS vol. 116 pp. 9714 – 9722 ’19.

“RIPK1 is a multidomain protein comprising an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The intermediate domain of RIPK1 contains an RHIM [receptor interacting protein (rip) homotypic interaction motif] domain which is important for interacting with other RHIM-containing proteins such as RIPK3, TRIF, and ZBP1. The C-terminal DD mediates its recruitment by interacting with other DD-containing proteins, such as TNFR1 and FADD, and its homodimerization to promote the activation of the N-terminal kinase domain. In the case of TNF-α signaling, ligand-induced TNFR1 trimerization leads to the assembly of a large receptor-bound signaling complex, termed Complex I, which includes multiple adaptors (TRADD, TRAF2, and RIPK1), and E3 ubiquitin ligases (cIAP1/2, LUBAC complex).”

Got that?  Here’s a bit more

“RIPK1 is regulated by multiple posttranslational modifications, but one of the most critical regulatory mechanisms is via ubiquitination. The E3 ubiquitin ligases cIAP1/2 are recruited into Complex I with the help of TRAF2 to mediate RIPK1 K63 ubiquitination. K63 ubiquitination of RIPK1 by cIAP1/2 promotes the recruitment and activation of TAK1 kinase through the polyubiquitin binding adaptors TAB2/TAB3. K63 ubiquitination also facilitates the recruitment of the LUBAC complex, which in turn performs M1- type ubiquitination of RIPK1 and TNFR1. M1 ubiquitination of Complex I is important for the recruitment of the trimeric IκB kinase complex (IKK) through a polyubuiquitin-binding adaptor subunit IKKγ/NEMO . The activation of RIPK1 is inhibited by direct phosphorylation by TAK1, IKKα/β, MK2, and TBK1. cIAP1 was also found to mediate K48 ubiquitination of RIPK1, inhibiting its catalytic activity and promoting degradation.”

So why should you plow through all this?  Because inhibiting RIPK1 reduces oxygen/glucose deprivation induced cell death in neurons, and reduced infarct size in experimental middle cerebral artery occlusion.

RIPK1 is elevated in MS brain, and inhibition of it helps an animal model (EAE).  Mutations in optineurin, and TBK1 leading to familial ALS promote the onset of RIPK1 necroptosis

Inflammation is seen in a variety of neurologic diseases (Alzheimer’s, MS) and RIPK1 is elevated in them.

Inhibitors of RIPK1 are available and do get into the brain.  As of now two RIPK1 inhibitors have made it through phase I human safety trials.

So it’s time to try RIPK1 inhibitors in these diseases.  It is an entirely new approach to them.  Even if it works only in one disease it would be worth it.

Now a dose of cynicism.  Diseased cells have to die one way or another.  RIPK1 may help this along, but it tells us nothing about what caused RIPK1 to become activated.  It may be a biomarker of a diseased cell.  The animal models are suggestive (as they always are) but few of them have panned out when applied to man.



Forgotten but not gone — take III

It’s pretty clear that life originated in the RNA world.  Consumed by thinking of proteins, enzymes, DNA etc. we tend to forget that there is a lot of RNA out there doing things we didn’t suspect.  Here are two more examples, one of which may explain why even genes coding  for proteins are relatively free of codons transcribed into amino acids.  The champ of course is dystrophin, discussed in the last post —  The gene is a monster with  2,220,233 nucleotides coding for just 3,685 amino acids, meaning that less than 1/200th of the gene is actually coding for protein. The work below should make us think about just what else the 199/200th of dystrophin might be doing,

Unsuspected use of RNA #1.   [ Neuron vol. 102 pp. 507 – 509, 553 – 563 ’19 ]  The Tumor protein p53 inducible nuclear protein 2 (Tp53inp2) gene codes for a low complexity protein of 222 amino acids, all in one exon.  However the ‘3 untranslated region (3’UTR)  of the RNA for it is nearly 5 times longer (3,121 nucleotides) vs. 666 amino acid coding nucleotides.  The protein is made from the mRNA in some cells, but not in sympathetic neurons, even though the mRNA for Tp53inp2 is the most abundant RNA in the axons of these neurons.

Why do animals lick their wounds?  Because their saliva contains nerve growth factor (NGF) among other things.  NGF is crucial for the growth of sympathetic neuron axons, and their very survival in embryonic life.  It is a protein, which binds to a receptor for it (TrkA) on the axon membrane.  The receptor/NGF complex is then internalized and transported back to the nucleus turning on the genes necessary for axon growth and cell survival.

Even though the mRNA for Tp53inp2 is NOT translated into protein in the axon, it is crucial for the internalization of TrkA/NGF.

People have studied proteins whose function it is to bind RNA for years.  They are called RBPs (RNA Binding Proteins), and our genome has 750 of them.  200 RBPs are associated with genetic disease.  This work turns everthing on its head.  Here is an RNA whose function it is to bind a protein (e.g. TrkA).

How many more mRNAs have nonCoding (for protein) parts with other functions?

Unsuspected use of RNA #2. Circular RNAs had been missed for years (although known since 1976).  The classic sequencing methods isolate only RNAs with characteristic tails (such as polyAdenine).  Circular RNAs don’t have any.    They are formed by back splicing of 3′ end of exon N to the 5′ end of exon N.  Fortunately this is only 1% as efficient as the normal way.

So what?  Circular RNAs are crucial in the innate immune response to microbial invaders.  Double stranded DNA belongs inside the nucleus.  When it gets into the cytoplasm when some organism brings it there,it binds to Protein Kinase R (PKR) activating it so it phosphorylates eukaryotic initiation factor 2 (eiF2) bringing protein synthesis to a screeching halt.

This means that the cell needs a mechanism to keep PKR quiet.  This is where circular RNAs come in   [ Cell vol. 177 pp. 797 – 799, 865 – 880 ’19 ].  If the nucleotides in the circle can reach across the circle and base pair with each other forming a duplex of any length, it will bind to PKR inhibiting it.  Most circular RNAs are expressed at only a handful of copies/cell, the cell containing just 10,000 of them.

The work found that overexpression of a single circular RNA able to form duplexes (dsRNA) inhibits PKR.  Over expression of linear RNA of the same sequence does not, nor does overexpression of circular RNA which can’t form dsRNA.

So when an invader with dsDNA or dsRNA gets into the cell, RNAase L, a cytoplasmic endonuclease is activated, cleaving circular RNA, and uninhibiting PKR.

So it’s back to the drawing board for mRNA and those parts (introns, 3’UTRs) we didn’t think were doing anything.  Perhaps that’s why there are so many of them, and why they take up more room in mRNA and genes than the ones coding for amino acids.  Also it’s time to look at RNAs as protein binders and modifiers, rather than the other way around as we have been doing.

Here’s a link to an earlier member of the series —

Duchenne muscular dystrophy — a novel genetic treatment

Could the innumerable genetic defects underlying Duchenne muscular dystrophy all be treated the same way?  Possibly.  Paradoxically, the treatment involves actually making the gene  even worse.

Understanding how and why this might work involves a very deep dive into molecular biology.  You might start by looking at the series of five background articles I wrote — start at and follow the links.

I have a personal interest in Duchenne muscular dystrophy because I ran such a clinic from ’72 to ’87 watching young boys and adolescents die from it.  The major advance during that time, was NOT medical or anything I did, but lighter braces, so the boys could stay ambulatory longer.  Things have improved as survival has improved by a decade so they die in their late 20s.

So lets start.  Duchenne muscular dystrophy is caused by a mutation in the gene coding for dystrophin, a large (3,685 amino acids) protein which ties the contractile apparatus of the muscle cell (actin and myosin) to the cell membrane. Although it isn’t the largest protein we have — titin, another muscle protein with 34,350 amino acids is, the gene for dystrophin is the largest we have, weighing in at 2,220,233 nucleotides.  This is why Duchenne is one of the most common diseases due to a defect in a single gene, the gene is so large that lots of things can (and do) go wrong with it.

The gene comes in 79 pieces (exons) which account for under 1/200 of the nucleotides of the gene.  The rest must be spliced out and discarded.  Have a look at  to see what can go wrong — the commonest is deletion of parts of the gene (60 – 70% of cases), followed by duplication of other parts (10% of cases) with the rest being mutations that change one amino acid to another.

Duchenne isn’t like cystic fibrosis where some 600 different mutations in the causative CFTR gene were known by 2003 but with 90% of cases due to just one.  So any genetic treatment for that young boy sitting in front of you had better be personalized to his particular mutation.

Or should it?

Possibly not.  We’ll need to discuss 3 things first

l. Nonsense Mediated Decay (NMD)

2. Nonsense Induced Transcriptional Compensation (NITC).

3. The MDX mouse model of Duchenne muscular dystrophy

Nonsense mediated decay.  Nonsense is a poor term, because the 3 nonSense codons (out of 64 possible) tell the ribosome to stop translating mRNA into protein and drop off the mRNA.  That isn’t nonsense.  I prefer stop codon, or termination codon

An an incredibly clever piece of business tells the ribosome (which is after all an inanimate object) when a stop codon occurs too early in the mRNA when there are a bunch of codons afterwards needed to make up the whole protein.

Lets go back to dystrophin and its 79 exons, and the fact that 99.5% of the gene is made of introns which are spliced out.   Remember the mRNA starts at the 5′ end and ends at the 3′ end.  The ribosome reads and translates it from 5′ to 3′. When an intron is spliced out, a protein complex of several proteins is placed on the mRNA some 20 – 24 basepairs 5′ to the splice site (this happens in the nucleus way before the mRNA gets near a ribosome in the cytoplasm).  The complex is called the Exon Junction Complex (EJC). The ribosome then happily munches along the mRNA from 5′ to 3′ knocking off the EJCs as it moves, until it hits a termination codon and drops off.

Over 95% of  genes do not have introns after the termination codon.  What happens if it does? Well then it is called a premature termination codon (PTC) and there is usually an EJC 3′ (downstream) to it.  If a termination codon is present 50 -55 nucleotides 5′ (upstream) to an EJC then NMD occurs.

Whenever any termination codon is reached, release protein factors (eRF1, eRF3, SMG1) bind to the mRNA.  It there is an EJC around (which there shouldn’t be) the interaction between the two complexes triggers phosphorylation of one of EJC proteins, triggering NMD.

So that’s how NMD happens, when there is a PTC.  Clever no?

Nonsense Induced Transcriptional Compensation (NITC).  I realize that this is a lot to throw at you, but a treatment for Duchenne is worth the effort (not to mention other genetic diseases in which the mechanism to be described also applies).

NITC is something I never heard about until two papers appearing in the 13 April Nature (vol. 568 pp. 179 – 180 (editorial), 193 – 197, 259 – 263).  Ever since we could knock out by placing a PTC early (near the 5′ end) of the gene we’ve been surprised by some of the results –e.g. knocking out some genes thought to be crucial had little or no effect.  Other technologies which didn’t affect the gene, but which decreased the expression of the mRNA (such as RNA interference, aka Post-Transcriptional gene silencing — PTGS) did have big phenotypic effects.

This turns out to be due NITC, which turns out to be due to increased transcription of genes which are ancestrally related to the mutant. Gene.  Hard to believe.

Time to go back to NMD.  It doesn’t break mRNA down nucleotide by nucleotide, but fragments it.  These fragments get into the nucleus, and bind to complementary genomic sequences of the PTC gene, and also to genes ancestrally related to the mutant gene (so they’ll have similar nucleotide sequences). Then epigenetics takes over because the fragments recruit the COMPASS complex which catalyzes the formation of H3K4Me3 which is part of the histone code which helps turn on transcription of the gene.  The sequence similarity of ancestrally related genes, allows them and only them to be turned on by NITC.  Even cleverer than finding a PTC by the ribosome.

Something so incredible needs evidence.  Well heterozygotic zebrafish can bemade to have one normal gene and one with a PTC. What do you think happens?  The normal gene is upregulated (e.g. more is made).  Pretty good.

Finally the Mdx mouse.  I’ve been reading about it for years.  It has a PTC in exon 23 of the dystrophin gene, resulting in a protein only 27% as long as it should be.  All sorts of therapeutic maneuvers have been tried on it.  Now any drug development chemist will tell you that animal models are lousy, but they’re all we’ve got.

The remarkable thing about the mdx mouse, is that they don’t get weak.  They do have muscle pathology.  All the verbiage above probably explains why.

So to treat ALL forms of Duchenne put in a premature termination codon (PTC) in exon #23 of the human gene. It should work as there are  4 dystrophin related proteins scattered around the genome — their names are — utrophin, dystrophin related protein 2 (DRP2), alpha dystrobrevin, and beta dystrobrevin

There is an even better way to look for a place to put a PTC in the dystrophin gene.  Our genomes are filled with errors — for details see —

There are lots of very normal people around with supposedly lethal mutations (including PTCs) in their genomes.  Probably scattered about various labs are at least 1,000,000 exome sequences in presumably normal people.  I’m not sure how much clinical information about them is available (other than that they are normal).  Hopeful their sex is.  Look at the dystrophin gene of normal males (females can be perfectly healthy carrying a mutant dystrophin gene as it is found on the X chromosome and they have 2) and see if PTCs are to be found.  You can’t have a better animal model than that.

At over 1,000 words this is the longest post I’ve written, and hopefully the most useful.

Why drug development is hard #32 and #33

The bloodbath among drug chemists continues (see Derek’s recent posts — because drug development is very hard and success is rare. Two nearly back to back papers in PNAS show just how hard drug development is (and why).

Animal models of human disease have a poor track record in pointing to new drugs.  One reason is that humans have new genes that animals don’t. One example is the horribly named CHRFAM7A, a dominant negative inhibitor of the alpha7 nicotinic cholinergic receptor [ PNAS vol. 116 pp. 7932 – 7940 ’16 ].

Alpha7 is found on macrophages where it exerts an anti-inflammatory action. Alpha7 agonists work beautifully in rodent inflammatory disease models.  They crashed and burned in human trials.  Why?  Because CHRFAM7A  binds to Alpha7 blocking the ability of acetyl choline to bind to it.  It is a totally new gene for man. It arose when 5 exons of the UL kinase 4 gene on chromosome #3 translocated nd then fused with the Dupa gene, which itself originated with 5 exons partially duplicated from the 10 exon alpha7 gene on the forward strand of chromosome #15.  So CHRFAM7A in close proximity to alpha7 (about which much more in the next post) and structurally similar to it.

[ PNAS vol. 116 pp. 7957 – 7962 ’19 ] Practically next door is a paper about MI-2, a drug thought to be useful in a (fortunately) rare brain tumor of childhood — diffuse intrinsic pontine glioma (maybe 3 cases in 38 years of practice).  Menin is a tumor suppressor lacking in a less rare syndrome (Type I Multiple Endocrine Neoplasia). MI-2 inhibits menin, but this paper shows that this isn’t its mechanism of action. Rather it inhibits an enzyme on the biosynthetic route to cholesterol (lanosterol synthetase).  So even when you think you know what a drug should be doing (which is probably why MI-2 was developed), that may not be how it works.

Forgotten but not gone — take II

The RNA world from whence we sprang strikes again, this time giving us a glimpse into its own internal dynamic.  18 months ago I wrote the following post — which will give you the background to follow the latest (found at the end after the (***)

Life is said to have originated in the RNA world.  We all know about the big 3 important RNAs for the cell, mRNA, ribosomal RNA and transfer RNA.  But just like the water, sewer, power and subway systems under Manhattan, there is another world down there in the cell which doesn’t much get talked about.  These areRNAs, whose primary (and possibly only) function is to interact with other RNAs.

Start with microRNAs (of which we have at least 1,500 as of 12/12).  Their function is to bind to messenger RNA (mRNA) and inhibit translation of the mRNA into protein.  The effects aren’t huge, but they are a more subtle control of protein expression, than the degree of transcription of the gene.

Then there are ceRNAs (competitive endogenous RNAs) which have a large number of binding sites for microRNAs — humans have a variety of them all with horrible acronyms — HULC, PTCSC3 etc. etc. They act as sponges for microRNAs keeping them bound and quiet.

Then there are circular RNAs.  They’d been missed until recently, because typical RNA sequencing methods isolate only RNAs with characteristic tails, and a circular RNA doesn’t have any.  One such is called CiRS7/CDR1) which contain 70 binding sites for one particular microRNA (miR-7).  They are unlike to be trivial.  They are derived from 15% of actively transcribed genes.  They ‘can be’ 10 times as numerous as linear RNAs (like mRNA and everything else) — probably because they are hard to degrade < Science vol. 340 pp. 440 – 441 ’17 >. So some of them are certainly RNA sponges — but all of them?

The latest, and most interesting class are the nonCoding RNAs found in viruses. Some of them function to attack cellular microRNAs and help the virus survive. Herpesvirus saimiri a gamma-herpes virus establishes latency in the T lymphocytes of New World primates, by expressing 7 small nuclear uracil-rich nonCoding RNAs (called HSURs).  They associate with some microRNAs, and rather than blocking their function act as chaperones < Nature vol. 550 pp. 275 – 279 ’17 >.  They HSURs also bind to some mRNAs inhibiting their function — they do this by helping miR-16 bind to their targets — so they are chaperones.  So viral Sm-class RNAs may function as microRNA adaptors.

Do you think for one minute, that the cell isn’t doing something like this.

I have a tendency to think of RNAs as always binding to other RNAs by classic Watson Crick base pairing — this is wrong as a look at any transfer RNA structure will show.  Far more complicated structures may be involved, but we’ve barely started to look.

Then there are the pseudogenes, which may also have a function, which is to be transcribed and sop up microRNAs and other things — I’ve already written about this —  Breast cancer cells think one (PTEN1) is important enough to stop it from being transcribed, even though it can’t be translated into protein.


[ Proc. Natl. Acad. Sci. vol. 116 pp. 7455 – 7464 ’19 ] The work reports a fascinating example of that early world in which the function of one denizen (a circular RNA called cPWWP2A) binds to another denizen of that world (microRNA 579 aka miR-579) acting as a sponge sopping up so it can’t bind to the mRNAs for angiopoetitin1, occludin and SIRT1.

So what you say?  Well it may lead to a way to treat diabetic retinopathy. How did they find cPWWP2A?  They used the Shanghai BIotechnology Company Mouse Circular RNA microArray which measures circular RNAs.  They found that 400 or so that were upregulated in diabetic retinopathy and another 400 or so that were downregulated.  cPWWP2A was on of the 3 top upregulated circular RNAs in diabetic retinopathy.  cPWWP2A comes from (what else?) PWWP2A, a gene coding for a protein which specifically binds the histone protein H2A.Z.

Overexpression of cPWW2PA or inhibition of miR-579 improves retinal vascular dysfunction in experimental diabetes.

So here is all this stuff going on way down there in the RNA world, first interacting with other players in this world and eventually reaching up to the level we thought we knew about and controlling gene expression.  It’s sort of like DOS (Disc Operating System) still being important in Windows.

How much more stuff like this is to be discovered controlling gene expression in us is anyone’s guess

How complicated can neuropharmacology be?

A revolution is occurring in our thinking about the neurochemistry and treatment of depression.  Spectacular therapeutic results with ketamine imply that neurotransmission with glutamic acid is involved (see the older post below for the background)  In addition gamma amino butyric acid (GABA) may also be a player.  That’s why a recent review [ Neuron vol. 102 pp. 75 – 94 ’19 ] is worth a careful reading.

Like all new fields, early results are particularly confusing. In particular the statement was made that in addition to NMDA receptor blockers (such as ketamine) positive allosteric modifiers (PAMs) of the NMDAR also are therapeutic in depression (the latter in animal models only, a phase III trial in depression having failed).

So I wrote the lead author ”

Great review, but how do you reconcile the rapid antidepressant action of the NMDAR blocker ketamine and friends and an NMDAR PAM (positive allosteric modifier)”

I got the following back —

We have data indicating that ketamine blocks NMDA receptors on GABA neurons resulting in disinhibition and increased synaptic activity of principle neurons, whereas the PAM (rapastinel) acts directly on NMDA receptors on principle neurons to produce a similar downstream effect

It didn’t make sense that drugs having opposite effects on the same therapeutic target (the NMDAR) would have the same therapeutic effect.

So I wrote

If I understand you correctly, this implies that the subunit composition of the NMDARs at the two sites (GABA interneurons and principal neurons) is different.

I got the following back, which is positively Talmudic in its logical intricacy.

It could be the same receptor complex; because ketamine is an open channel blocker the GABA neurons, which are more active, would be more sensitive because activity is required to remove the Mg+2 block in the channel and thereby allow ketamine to enter and block the channel. The PAM does not require activity and could act at directly on principle neurons.

If this is correct, a lot of neuropharmacology on drug effects will require rethinking.  What does the readership think?

Stock tip — update

The FDA approved esketamine (Spravato) last week (see copy of original post at the end).  I had recommended buying Johnson and Johnson if the FDA approved it.  I think it’s a good long term buy, but there is no rush for the following reason — Esketamine is not a drug you can get a prescription for and take on you own. Because of the psychiatric side effects it must be administered in a SPRAVATO REMS.

Risk Evaluation and Mitigation Strategy (REMS): SPRAVATO™ is available only through a restricted program called the SPRAVATO™ REMS because of the risks of serious adverse outcomes from sedation, dissociation, and abuse and misuse.

Important requirements of the SPRAVATO™ REMS include the following:

  • Healthcare settings must be certified in the program and ensure that SPRAVATO™ is:
    • Only dispensed in healthcare settings and administered to patients who are enrolled in the program.
    • Administered by patients under the direct observation of a healthcare provider and that patients are monitored by a healthcare provider for at least 2 hours after administration of SPRAVATO™.
  • Pharmacies must be certified in the REMS and must only dispense SPRAVATO™ to healthcare settings that are certified in the program.

So you can’t go to some shady practitioner who’ll say you have treatment resistant depression and get some (e.g. the pill pushers for opiates, ‘medical’ marihuana  etc. etc.)

So there aren’t going to be hordes of users right away, although the stuff I’ve read implies that there will be eventually.

If you have a subscription to Cell have a look at vol. 101 pp. 774 – 778 ’19 by the guys at Yale who did some of the original work.  If not content yourself with this.

They are refreshingly honest.

Was the Discovery of Ketamine’s Antidepressant Serendipitous?Of course. However, its discovery emerged from the testing of a novel mechanistic hypothesis related to the pathophysiology of depression.”

Basically the authors rejected the regnant theory of depression, namely that the cause was to be found in monoamine neurotransmission (e.g. by dopamine, norepinephrine, serotonin).  There was some evidence that the cerebral cortex was involved in depression (not just the monamine nuclei of the brainstem), so they looked at the two major neurotransmitters in brain (glutamic acid, and GABA), and chose to see what would happen if they blocked one of the many receptors for glutamic acid, the NMDA receptor.  They chose ketamine to do this.
Here’s what they found,  A single dose of ketamine produced antidepressant effects that began within hours peaked in 24 – 72 hours and dissipated within 2 weeks (if ketamine wasn’t repeated).  This was in 50 – 75% people with treatment resistant depression.  Remarkable 1/3 of treated patients went into remission.    There simply has never been anything like this, which is why I thought the drug would be a blockbuster.
There is a lot of speculation about just which effect of esketamine is crucial (increase in glutamic acid release with AMPAR stimulation, brain derived neurotrophic factor (BDNF) release, TrkB receptor stimulation, mTORC1 activation, local protein synthesis, restoration of functional connectivity in functional MRI.   In animals one sees a rapid proliferation of dendritic spines.
As promised – here’s a copy of the first post

Stock tip

The past performance of stock recommendations is no guarantee that it will continue — which is fortunate as my first tip (ONTX) was a disaster.  I knew it was a 10 to one shot but with a 100 to 1 payoff.  People play the lottery with worse odds.  Anyway ONTX had a rationale — for the gory details see —

For those brave souls who followed this recommendation (including yours truly) here’s another.

On 4 March 2019 if the FDA approves esketamine for depression, buy Johnson and Johnson.  Why?  Some people think that no drug for depression works that well, as big Pharma in the past only was reporting positive studies.  The following is from Nature 21 February 2019.

Depression drug A form of the hallucinogenic party drug ketamine has cleared one of the final hurdles towards clinical use as an antidepressant. During a 12 February meeting at the US Food and Drug Administration (FDA) in Silver Spring, Maryland,an independent advisory panel voted 14 to 2 in favour of recommending a compound known as esketamine for use in treating depression.

What’s so hot about esketamine?  First its mechanism of action is completely different than the SSRIs, Monoamine oxidase inhibitors, or tricyclic antidepressants.

As you likely know, antidepressants usually take a few weeks to work at least in endogenous depression.  My clinical experience as a neurologist is slightly different, as I only used it for patients with disease I couldn’t help (end stage MS etc. etc.) where the only normal response to the situation was depression.  They often helped patients within a week.

I was staggered when I read the following paper back in the day.  But there was no followup essentially.

archives of general psychiatry volume 63 pp. 856 – 864 2006
The paper is not from St. Fraudulosa Hospital in Plok Tic, but from the Mood Disorders Research Unit at the National Institute of Mental Health.
Here are the basics from the paper

Patients  Eighteen subjects with DSM-IV major depression (treatment resistant).

Interventions  After a 2-week drug-free period, subjects were given an intravenous infusion of either ketamine hydrochloride (0.5 mg/kg) or placebo on 2 test days, a week apart. Subjects were rated at baseline and at 40, 80, 110, and 230 minutes and 1, 2, 3, and 7 days postinfusion.

Main Outcome Measure  Changes in scores on the primary efficacy measure, the 21-item Hamilton Depression Rating Scale.

Results  Subjects receiving ketamine showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week. The effect size for the drug difference was very large (d = 1.46 [95% confidence interval, 0.91-2.01]) after 24 hours and moderate to large (d = 0.68 [95% confidence interval, 0.13-1.23]) after 1 week. Of the 17 subjects treated with ketamine, 71% met response and 29% met remission criteria the day following ketamine infusion. Thirty-five percent of subjects maintained response for at least 1 week.

Read this again: showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week.

This is absolutely unheard of.  Yet the paper essentially disappeared.

What is esketamine?  It’s related to ketamine (a veterinary anesthetic and drug of abuse) in exactly the same way that a glove for your left hand is related to a right handed glove.  The two drugs are optical isomers of each other.

What’s so important about the mirror image?  It means that esketamine may well act rather differently than ketamine (the fact that ketamine worked is against this).  The classic example is thalidomide, one optical isomer of which causes horrible malformations (phocomelia) while the other is a sedative used in the treatment of multiple myeloma and leprosy.

If toxic side effects can be avoided, the market is enormous.  It is estimated that 25% of women and 10% of men will have a major depression at some point in their lives.

Initially, Esketamine ( SPRAVATOTM)  will likely be limited to treatment resistant depression.  But depressed people will find a way to get it and  their docs will find a way to give it.  Who wants to wait three weeks.  Just think of the extremely sketchy ‘medical indications’ for marihuana.


How to treat Alzheimer’s disease

Let’s say you’re an engineer whose wife has early Alzheimer’s disease.  Would you build the following noninvasive device to remove her plaques?  [ Cell vol. 177 pp. 256 – 271 ’19 ] showed that it worked in mice.

Addendum 18 April — A reader requested a better way to get to the paper — Here is the title — “Multisensory Gamma Stimulation Ameliorates Alzheimer’s Associated Pathology and Improves Cognition”.  It is from MIT — here is the person to correspond to  —Correspondence —

The device emits sound and light 40 times a second.  Exposing mice  to this 1 hour a day for a week decreased the number of senile plaques all over the brain (not just in the auditory and visual cortex) and improved their cognition as well.

With apologies to Steinbeck, mice are not men (particularly these mice which carry 5 different mutations which cause Alzheimer’s disease in man).  Animal cognition is not human cognition.  How well do you think Einstein would have done running a maze looking for food?

I had written about the authors’ earlier work and a copy of that post will be found after the ****.

What makes this work exciting is that plaque reduction was seen not only  in the visual cortex (which is pretty much unaffected in Alzheimer’s) but in the hippocampus (which is devastated) and the frontal lobes (also severely affected).  Interestingly, to be effective, both sound and light had to be given simultaneously

Here are the details about the stimuli  —

“Animals were presented with 10 s stimulation blocks interleaved with 10 s baseline periods. Stimulation blocks rotated between auditory-only or auditory and visual stimulation at 20 Hz, 40 Hz, 80 Hz, or with random stimulation (pulses were delivered with randomized inter-pulse intervals determined from a uniform distribution with an average interval of 25 ms). Stimuli blocks were interleaved to ensure the results observed were not due to changes over time in the neuronal response. 10 s long stimulus blocks were used to reduce the influence of onset effects, and to examine neural responses to prolonged rhythmic stimulation. All auditory pulses were 1 ms-long 10 kHz tones. All visual pulses were 50% duty cycle of the stimulation frequency (25 ms, 12.5 ms, or 6.25 ms in length). For combined stimulation, auditory and visual pulses were aligned to the onset of each pulse.”

The device should not require approval by the FDA unless a therapeutic claim is made, and it’s about as noninvasive as it could be.

What could go wrong?  Well a flickering light could trigger seizures in people subject to photic epilepsy (under 1/1,000).

Certainly Claude Shannon who died of Alzheimer’s disease, would have had one built, as would Fields medal winner Daniel Quillen had he not passed away 8 years ago.

Here is the post of 12/16 which has more detail



Will flickering light treat Alzheimer’s disease ?

Big pharma has spent zillions trying to rid the brain of senile plaques, to no avail. A recent paper shows that light flickering at 40 cycles/second (40 Hertz) can do it — this is not a misprint [ Nature vol. 540 pp. 207 – 208, 230 – 235 ’16 ]. As most know the main component of the senile plaque of Alzheimer’s disease is a fragment (called the aBeta peptide) of the amyloid precursor protein (APP).

The most interesting part of the paper showed that just an hour or so of light flickering at 40 Hertz temporarily reduced the amount of Abeta peptide in visual cortex of aged mice. Nothing invasive about that.

Should we try this in people? How harmful could it be? Unfortunately the visual cortex is relatively unaffected in Alzheimer’s disease — the disease starts deep inside the head in the medial temporal lobe, particularly the hippocampus — the link shows just how deep it is -

You might be able to do this through the squamous portion of the temporal bone which is just in front of and above the ear. It’s very thin, and ultrasound probes placed here can ‘see’ blood flowing in arteries in this region. Another way to do it might be a light source placed in the mouth.

The technical aspects of the paper are fascinating and will be described later.

First, what could go wrong?

The work shows that the flickering light activates the scavenger cells of the brain (microglia) and then eat the extracellular plaques. However that may not be a good thing as microglia could attack normal cells. In particular they are important in the remodeling of the dendritic tree (notably dendritic spines) that occurs during experience and learning.

Second, why wouldn’t it work? So much has been spent on trying to remove abeta, that serious doubt exists as to whether excessive extracellular Abeta causes Alzheimer’s and even if it does, would removing it be helpful.

Now for some fascinating detail on the paper (for the cognoscenti)

They used a mouse model of Alzheimer’s disease (the 5XFAD mouse). This poor creature has 3 different mutations associated with Alzheimer’s disease in the amyloid precursor protein (APP) — these are the Swedish (K670B), Florida (I716V) and London (V717I). If that wasn’t enough there are two Alzheimer associated mutations in one of the enzymes that processes the APP into Abeta (M146L, L286V) — using the single letter amino acid code – Then the whole mess is put under control of a promoter particularly active in mice (the Thy1 promoter). This results in high expression of the two mutant proteins.

So the poor mice get lots of senile plaques (particularly in the hippocampus) at an early age.

The first experiment was even more complicated, as a way was found to put channelrhodopsin into a set of hippocampal interneurons (this is optogenetics and hardly simple). Exposing the channel to light causes it to open the membrane to depolarize and the neuron to fire. Then fiberoptics were used to stimulate these neurons at 40 Hertz and the effects on the plaques were noted. Clearly a lot of work and the authors (and grad students) deserve our thanks.

Light at 8 Hertz did nothing to the plaques. I couldn’t find what other stimulation frequencies were used (assuming they were tried).

It would be wonderful if something so simple could help these people.

For other ideas about Alzheimer’s using physics rather than chemistry please see —

Apologies to Hamlet

Apologies to Shakespeare and Hamlet.  Serotonin does “more things in heaven and Earth, Horatio, than are dreamt of in your philosophy.”  How about chemically modifying histones?We all know about serotonin and depression (or at least we think we know).  Block serotonin reuptake by the releasing neuron and bingo you’ve  cured depression (sometimes).  Do not ask the lecturer which of the 15 known serotonin receptors in the brain the increased serotonin actually binds to and what effects the increased levels produce after binding (and which are important for the alleviation of depression).The two body organs producing the most serotonin are the brain and the gut.  Chemical modification of proteins by serotonin has been known for 10 years.  The enzyme responsible is transglutaminase2, it takes the NH2 group of serotonin and replaces the NH2 of glutamine with it — forming an isopeptide bond.

Interestingly, the serotonylation of histones is quite specific.  Only glutamine #5 on histone H3 is modified this way.  For the reaction to occur lysine #4 on histone H3 must be trimethylated (H3K4Me3) — now you can begin to see the combinatorial possibilities of the various histone modifications known.  Over 130 post-ranslational modifications of histones were known by 2013 [ Cell vol. 155 p. 42 ’13 ].

The H3K4Me3Q5Ser is enriched in euchromatin and correlates with permissive gene expression.  Changing glutamine #5 to something else so it can’t be serotonylated changes the transcription pattern, and deficits in cellular differentiation.  You can read more about it in Nature vol. 567 pp. 464 – 465, 535 – 539 ’19 ]

Yet another mechanism of gene regulation

A snippet of RNA from an intron in a gene can bind to an upstream regulatory element forming a triple helix and shut off transcription of the gene.  Rather amazing don’t you think?  Yet exactly was found in a far from obscure gene, the beta globin gene of hemoglobin on chromosome #11 [ Proc. Natl. Acad. Sci. vol. 116 pp. 6130 – 6139 ’19 ].

We’re talking large segments of DNA.  There are five genes for the beta subunit of hemoglobin located from 5′ to 3′ as epsilon, gammaG, gammaA, delta and beta.  The first 4 are expressed during fetal development.  Beta globin is the one found in our red blood cells.  The regulatory element controlling all 5 is found FIFTY kiloBases upstream from the beginning (5′ end) of beta globin.

The regulatory region is called the locus control region (LCR)and stretches over 20+ kiloBases.  It has 7 sites where transcription factors bind (called hypersensitive sites HS1 — HS7).  The hypersensitivity comes from the fact the chromosome is relative ‘open’ at these places and not compacted, so that an enzyme (DNAase I) can break the chromosome.

So after the beta globin gene is transcribed, the introns are spliced out, and the RNA from the second intron binds to HS2 forming a triple helix and displacing transcription factors bound there (USF2, GATA1, TAL1) which recruit RNA polymerase II (Pol II)  In the normal course of events the whole mess would then march around the genome and eventually hit the promoter of beta globin (at least 50 kiloBases away) and turn on transcription.

This seems to be yet another mechanism of gene regulation.  Just how widespread this is, isn’t known, but most protein coding genes have introns.  Stay tuned.

Molecular biology is fascinating

Another research idea yours for the taking

How many of our 20,000 or so protein coding genes are essential for human existence?  There is a way to find out with no human experimentation whatsoever.  Even better, probably all the data is out there.  Looking at it the right way, finding and collating it is where you come in.  Be warned, it would be a lot of work.

Previous work [ Science vol. 350 pp. 1028 – 1029, 1092 – 1096, 1096 – 1101 ’15 ] came up with the idea that only 2,000 or so of our protein coding genes were truly essential.  The authors cleverly looked at a ‘near haploid’ chronic myelogenous leukemia cell line (KBM7).  Then because only one copy of a gene was present, they systematically knocked out gene after gene using CRISPR and looked at viability.

Similar work in yeast stated that only 1,000 of its 6,000 protein coding genes were essential.

But this is single cell stuff.  What about living breathing people?

Where is this data?  How should it be interrogated?  See if you can figure it out before reading further.

Probably more has been done since Science vol. 337 pp. 64 – 69 ’12 sequenced just the portion of our genome coding for proteins (the exomes) in 1,351 Europeans and 1,088 Africans.  Each individual had 35 premature termination codons, meaning that the gene likely didn’t produce a functional protein.  The average person also had 13,595 single nucleotide polymorphisms (from the standard genome), and probably some of them a less than functional protein.

Do you see how you could use this sort of thing to find out which genes are essential to our existence?

People sequence exomes because it’s easy and because the exome accounts for only 2% of our genome.

My guess is that probably a million exomes have been sequenced thus far, if not more.

So all you have to do is look at all million exome sequences and all 20,000 protein coding genes, and see —

In one of the Sherlock Holmes stories the following dialog appears

Gregory (Scotland Yard): “Is there any other point to which you would wish to draw my attention?”
Holmes: “To the curious incident of the dog in the night-time.”
Gregory: “The dog did nothing in the night-time.”
Holmes: “That was the curious incident.”

The curious incident would be a gene which never (or rarely) had a premature termination codon in the 1,000,000 or so exomes.  That would imply that it was essential for the existence of a living breathing human being.

Cute !  Well I’m a retired neurologist with no academic affiliation — take the idea and run with it.

Addendum 31 Mar ’19 – I received the following comment from Bryan

You may be interested in reading this pre-print on the topic:
Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes

To which I replied
    • Bryan– thanks for the link. It was a good enough idea that the people at the Broad Institute had thought of it and carried it out. As people in grad school used to say when they got scooped on a paper — at least we were thinking well.

      It was hard to tell from reading the preprint whether there were genes with no pLoF (predicted loss of function) proving them essential. They do say that the 678 genes essential for human cell viability (characterized by CRISPR screening were ‘depleted’ for pLoF.