Category Archives: Medicine in general

How general anesthesia works

People have been theorizing how general anesthesia works since there has been general anesthesia.  The first useful one was diethyl ether (by definition what lipids dissolve in).  Since the brain has the one of the highest fat contents of any organ, the mechanism was obvious to all.  Anesthetics dissolve membranes.  Even the newer anesthetics look quite lipophilic — isoflurane CF3CHCL O CF2H screams (to the chemist) find me a lipid to swim in.  One can show effects of lipids on artificial membranes but the concentrations to do so are so high they would be lethal.

Attention shifted to the GABA[A] receptor, because anesthetics are effective in potentiating responses to GABA  — all the benzodiazepines (valium, librium) which bind to it are sedating.  Further evidence that a protein is involved, is that the optical isomers of enflurane vary in anesthetic potency (but not by very much — only 60%).  Lipids (except cholesterol) just aren’t optically active.  Interestingly, alfaxolone is a steroid and a general anesthetic as well.

Well GABA[A] is an ion channel, meaning that its amino acids form alpha helices which span the membrane (and create a channel for ion flow).  It would be devilishly hard to distinguish binding to the transmembrane part from binding to the membrane near it. [ Science vol. 322 pp. 876 – 880 2008 ] Studied 4 IV anesthetics (propofol, ketamine, etomidate, barbiturate) and 4 gasses (nitrous  oxide, isoflurance, devoflurane, desflurane) and their effects on 11 ion channels — unsurprisingly all sorts of effects were found — but which ones are the relevant.

All this sort of stuff could be irrelevant, if a new paper is actually correct [ Neuron vol. 102 pp. 1053 – 1065 ’19 ].  The following general anesthetics (isoflurane, propofol, ketamine and desmedtomidine) all activate cells in the hypothalamus (before this anesthetics were thought to work by ultimately inhibiting neurons).  They authors call these cells AANs (Anesthesia Activated Neurons).

They are found in the hypothalamus and contain ADH.  Time for some anatomy.  The pituitary gland is really two glands — the adenohypophysis which secretes things like ACTH, TSH, FSH, LH etc. etc, and the neurohypophysis which secretes oxytocin and vasopressin (ADH) directly into the blood (and also into the spinal fluid where it can reach other parts of the brain.  ADH release is actually from the axons of the hypothalamic neurons.  The AANs activated by the anesthetics release ADH.

Of course the workers didn’t stop there — they stimulated the neurons optogenetically and put the animals to sleep. Inhibition of these neurons shortened the duration of general anesthesia.

Fascinating (if true).  The next question is how such chemically disparate molecules can activate the AANs.  Is there a common receptor for them, and if so what is it?

Happy fiscal new year !

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A sad (but brilliant) paper about autophagy

Over the past several decades I’ve accumulated a lot of notes on autophagy (> 125,000 characters).  It’s obviously important, but in a given cell or disease (cancer, neurodegeneration) whether it helps a cell die gracefully or is an executioner is far from clear.  Ditto for whether enhancing or inhibiting it in a given situation would be helpful (or hurtful).

A major reason for the lack of clarity despite all the work that’s been done can be found in the following excellent paper [ Cell vol. 177 pp.1682 – 1699 ’19 ].  Some 41 proteins are involved in autophagy in yeast and more in man.  Many are described as ATGnn (AuTophagy Gene nn).

Autophagy is a complicated business: forming a membrane, then engulfing various things, then forming a vacuole,  then fusing with the lysosome so that the engulfees are destroyed.

The problem with previous work is that if a protein was found to be important in autophagy, it was assumed to have that function and that function only.   The paper shows that core autophagy proteins are involved in (at least) 5 other processes (endocytosis, melanocyte formation, cytokinesis, LC3 assisted phagocytosis and translocation of vesicles from the Golgi to the endoplasmic reticulum).

Experiments deleting or  increasing a given ATGnn were assumed to produce their biological effects by affecting autophagy.

The names are unimportant.  Here is a diagram of 6 autophagy proteins forming a complex producing autophagy

1 2 3

4 5 6

So 2 binds to 1, 3 and 5

But in endocytosis

1 2 3

5

form an important complex

In cytokinesis the complex formed by

2 3

5

is important.

Well you get the idea.  Knocking out 2 has cellular effects on far more than autophagy.  So a lot of work has to be re-thought and probably repeated.

Notice that all 6 functions involve movement of membranes.  So just regard the 6 proteins as gears of different diameters which can form the guts of different machines as they combine with each other (and proteins specific to the other 5 processes mentioned) to move things around in the cell.

Set points, a mechanism for one at last.

Human biology is full of set points.  Despite our best efforts few can lose weight and keep it off.  Yet few count calories and try to eat so their weight is constant.  Average body temperature is pretty constant (despite daily fluctuations).  Neuroscientists are quite aware of synaptic homeostasis.

And yet until now, despite their obvious existence, all we could do is describe setpoints, not explain the mechanisms behind them.  Most ‘explanations’ of them were really descriptions.

Here is an example:

Endocrinology was pretty simple in med school back in the 60s. All the target endocrine glands (ovary, adrenal, thyroid, etc.) were controlled by the pituitary; a gland about the size of a marble sitting an inch or so directly behind the bridge of your nose. The pituitary released a variety of hormones into the blood (one or more for each target gland) telling the target glands to secrete, and secrete they did. That’s why the pituitary was called the master gland back then.  The master gland ruled.

Things became a bit more complicated when it was found that a small (4 grams or so out of 1500) part of the brain called the hypothalamus sitting just above the pituitary was really in control, telling the pituitary what and when to secrete. Subsequently it was found that the hormones secreted by the target glands (thyroid, ovary, etc.) were getting into the hypothalamus and altering its effects on the pituitary. Estrogen is one example. Any notion of simple control vanished into an ambiguous miasma of setpoints, influences and equilibria. Goodbye linearity and simple notions of causation.

As soon as feedback (or simultaneous influence) enters the picture it becomes like the three body problem in physics, where 3 objects influence each other’s motion at the same time by the gravitational force. As John Gribbin (former science writer at Nature and now prolific author) said in his book ‘Deep Simplicity’, “It’s important to appreciate, though, that the lack of solutions to the three-body problem is not caused by our human deficiencies as mathematicians; it is built into the laws of mathematics.” The physics problem is actually much easier than endocrinology, because we know the exact strength and form of the gravitational force.

A recent paper [ Neuron vol. 102 pp. 908 – 910, 1009 – 1024 ’19 ] is the first to describe a mechanism behind any setpoint and one of particular importance to the brain (and possibly to epilepsy as well).

The work was done at significant remove from the brain — hippocampal neurons grown in culture.  They synapse with each other, action potentials are fired and postsynaptic responses occur.  The firing rate is pretty constant.  Block a neurotransmitter receptor, and the firing rate increases to keep postsynaptic responses the same.  Increase the amount of neurotransmitter released by an action potential (neuronal firing) and the firing rate descreases.  This is what synaptic homeostasis is all about.  It’s back to baseline transmission across the synapse regardless of what we do, but we had no idea how this happened.

Well we still don’t but at least we know what controls the rate at which hippocampal neurons fire in culture (e.g. the setpoint).  It has to do with an enzyme (DHODH) and mitochondrial calcium levels.

DHODH stands for Di Hydro Orotate DeHydrogenase, an enzyme in mitochondria involved in electron transfer (and ultimately energy production).   Inhibit the enzyme (or decrease the amount of DHODH around) and the neurons fire less.  What is interesting about this, that all that is changed is the neuronal firing rate (e.g. the setpoint is changed).  However, there is no change in the intrinsic excitability of the neurons (to external electrical stimulation), the postsynaptic response to transmitter, the number of mitochondria, presynaptic ATP levels etc.

Even better, synaptic homeostasis is preserved.  Manipulations increasing or decreasing the firing rate are never permanent, so that changes back to the baseline rate occur.

Aside from its intrinsic intellectual interest, this work is potentially quite useful.  The firing rate of neurons in people with epilepsy is increased.  It is conceivable that drugs inhibiting DHODH would treat epilepsy.  Such drugs (teriflunomide) are available for the treatment of multiple sclerosis.

The paper has some speculation of how DHODH inhibition would lead to decreased neuronal firing (changes in mitochondrial calcium levels etc. etc) which I won’t go into here as it’s just speculation (but at least plausible spectulation).

Will flickering light treat Alzheimer’s disease ? — Take II

30 months ago, a fascinating paper appeared in which flickering light improved a mouse model of Alzheimer’s disease.  The authors (MIT mostly) have continued to extend their work.   Here is a copy of the post back then.  Their new work is summarized after the ****

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 -https://en.wikipedia.org/wiki/Hippocampus#/media/File:MRI_Location_Hippocampus_up..png

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 –http://www.biochem.ucl.ac.uk/bsm/dbbrowser/c32/aacode.html.hy1. 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 — https://luysii.wordpress.com/2014/11/30/could-alzheimers-disease-be-a-problem-in-physics-rather-than-chemistry/

****

The new work appears in two papers.

First [ Cell vol. 1777 pp. 256 – 271 ’19 ] 7 days of auditory tone stimuli at 40 cycles/second (40 Hertz) for just one hour a day reduced amyloid in the auditory cortex of the same pathetic mice described above (the 5XFAD mice).  They call this GENUS (Gamma ENtrainment Using sensory Stimuli).  Neurologists love to name frequencies in the EEG, and the 40 Hertz is in the gamma range.

The second paper [ Neuron vol. 102 pp. 929 – 943 ’19 ] is even better.  Alzheimer’s disease is characterized by two types of pathology — neurofibrillary tangles inside the remaining neurons and the senile plaque outside them.  The tangles are made of the tau protein, the plaques mostly of fragments of the amyloid precursor protein (APP).  The 5XFAD mouse had 3 separate mutations in the APP and two more in the enzyme that chops it up.

The present work looked at the other half of Alzheimer’s the neurofibrillary tangle.  They had mice with the P301S mutation in the tau protein found in a hereditary form of dementia (not Alzheimer’s) and also with excessive levels of CK-p25 which also results in tangles.

Again chronic visual GENUS worked in this (completely different) model of neurodegeneration.

This is very exciting stuff, but I’d love to see a different group of researchers reproduce it.  Also billions have been spent and lost on promising treatments of Alzheimer’s (all based on animal work).

Probably someone is trying it out on themselves or their spouse.  A EE friend notes that engineers have been trying homebrew transcranial magnetic and current stimulation using themselves or someone close as guineapigs for years.

What is legionella trying to tell us?

10 years out of Med School, a classmate in the Public Health service had to deal with the first recognized outbreak of Legionnaire’s disease, at the Bellevue Stratford hotel in Philly, about one air mile from Penn Med where we went.   The organism wasn’t known at the time and caused 182 cases with 29 deaths.  We’ve learned a lot more about Legionella Pneumophila since 1976 and the organism continues to instruct us.

The most recent lesson concerns one of the 300 or so proteins Legionella injects into a cell it attacks.  This is remarkable in itself.  The organism uses them to hijack various cellular mechanisms to build a home for itself in the cell (the LCV — Legionella Containing Vacuole).  Contrast this with diphtheria which basically uses one protein (diphtheria toxin) to kill the cell.

One of the 300 proteins is called SidJ and looks like a protein kinase (of which our genome has over 500).  However [ Science vol. 364 pp. 787 – 792 ’19 ] shows that SidJ carries out a different different reaction.SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases inhibiting them. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the gamma carboxyl group in the catalytic center of SidE a ubiquitin ligase.   This, instead of just esterifying the hydroxyl group of serine or threonine or tyrosine with the terminal phosphate of ATP as a kinase is supposed to do.

Why is this important? The only protein known to have polyglutamic acid added to it is tubulin, the protein from which microtubules (neurotubules to the neurologist).  The work is important because some of the 500+ protein kinases in our genome might be doing something else.  Has the chemistry each and every member of the group been studied?  Probably not..

The authors close with “In summary, our results underscore the diversity and catalytic versatility of the protein kinase superfamily. We propose that ATP-dependent ligation reactions may be a common feature among the vast diversity of eukaryotic protein kinase–like enzymes found in nature (25). There are more than 500 protein kinases in humans and our results suggest that they should be ex- amined for alternative activities.”

I couldn’t agree more.

RIPK1

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 — https://www.pnas.org/content/116/20/9714 — 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 — https://luysii.wordpress.com/2019/05/05/duchenne-muscular-dystrophy-a-novel-genetic-treatment/.  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 — https://luysii.wordpress.com/2019/04/15/forgotten-but-not-gone-take-ii/xa

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 https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ 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 http://www.dmd.nl.  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 — https://luysii.wordpress.com/2018/05/01/how-badly-are-thy-genomes-oh-humanity-take-ii/.

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 — https://blogs.sciencemag.org/pipeline/archives/2019/04/22/big-pharma-cuts-current-and-coming) 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. https://en.wikipedia.org/wiki/Transfer_RNA.  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 — https://luysii.wordpress.com/2010/07/14/junk-dna-that-isnt-and-why-chemistry-isnt-enough/.  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