The brain just got a lot more complicated

People have been studying the locus coeruleus (LC) for at least 60 years.  For one thing it is easy to see on dissection of the brain (it’s blue — think cerulean blue).  For another it’s contains the largest collection of neurons using norepinephrine as a neurotransmitter (about which much more later) whose effects on arousal, mood, addiction and psychiatric disease have long been known.  So anything altering LC activity is likely to be important clinically.

Neuroscience has largely concentrated on just a few neurotransmitters — glutamic acid, the major excitatory neurotransmitter in brain which opens ion channels causing neurons to fire and gamma amino butyric acid (GABA) the major inhibitory neurotransmitter which shuts ion channels inhibiting neurons from firing.  Then there are the volume neurotransmitters (dopamine, serotonin, norepinephrine, histamine, acetyl choline) about which much more later.

Lastly, there are the peptide neurotransmitters, of which the brain’s own opiates (the enkephalins) are the best known and studied.  There are tons of them —  over 100 are known — https://en.wikipedia.org/wiki/Neuropeptide#:~:text=There%20are%20over%20100%20known,molecules%20in%20the%20nervous%20system.

Technology has marched on and it is now possible to isolate a single neuron and study the messenger RNA (mRNA) it is making.  Not to leave anyone behind, we assume that if the cell is making mRNA coding for a protein, the ribosome will grab the mRNA and make the protein.  This is a major advance, since you don’t have to test for each of the 20,000 different proteins the genome codes for (and we don’t have tests for all of them). What distinguishes the wildly different cell types in our body is the collection of proteins they make, and the way they organize the membranes of the cell.  Each cell type has a different collection of proteins.

Enter Proc. Natl. Acad. Sci. vol. 120 e2222095120 ’23 — which used single cell RNA sequencing (scRNA-seq) on neurons in the rodent locus coeruleus (which has only 3,000 neurons, unlike man which has 50,000.

Based on the RNA found, they were able to study LC neurons making norepinephrine (as judged by presence of mRNAs for the enzymes making it).   The staggering finding (which the authors don’t make much of) is that, as a group,  LC neurons using norepinphrine express mRNAs for 19 different neurotransmitters and 30 neuropeptide receptors.   It is hard for me to find out the maximum or mean number of neurotransmitters and receptors expressed in and on a single neuron (I have written the authors on this point), but we do know that expression of norepinephrine and the neuropeptide galanin is common in this group.

But forget that.  It is reasonable to assume that if a neuron expends the metabolic energy to transcribe a gene for a neurotransmitter receptor into mRNA,  makes the protein corresponding to the mRNA and inserts it into the neuronal membrane — that it will respond to the neurotransmitter.

To respond to a neurotransmitter/neuropeptide a neuron must have a receptor, just as to respond to Ni Hao you must have the language receptor (understand the language) for Chinese.

We’re not in the Kansas of glutamic acid, GABA, norepinephrine, dopamine, serotonin, histamine, acetyl choline any more.  The complexity of 3o different neurotransmitters/neuropeptide producing effects on the 3,000 cells of the locus coeruleus is staggering.   Almost certainly someone is doing a similar study on the cerebral cortex.

Life becomes even more complex if a given norepinephrine neuron expresses multiple receptors (as I think they do — we’ll see what the author says).

Now a bit about why norepinephrine neurons are so important.

The locus coeruleus sends fibers all over the brain, releasing norepinephrine everywhere, and not just at synapses.  This is called volume neurotransmission. Most places on the axons of a LC neuron showing synaptic vesicles (where norepinephrine is found), don’t have a dendrite or any sort synaptic specialization next to them.  So the LC innervates the whole brain, in the same way that our brain innervates our muscles.  Stimulate the LC of a rat and the brain is flooded with norepinephrine and the animal wakes up.  Think of it as the sprinkler system of an office building.

This means that the length of axons of neurons acting by volume neurotransmission (this includes dopamine, serotonin, acetyl choline and histamine) must be enormous.  Here’s one reference for dopamine — “Individual neurons of the pars compacta (which uses dopamine as a neurotransmitter) are calculated to give rise to 4.5 meters of axons once all the branches are summed”  — [ Neuron vol. 96 p. 651 ’17 ].”   That’s just one cell doing all that.

I can’t find an actual source, so it may be a neuroscience urban myth, that every neuron is very close (within a few neuronal cell body diameters) to an axon of a volume transmitting neuron.  If anyone knows a source please write a comment.

 

The old year goes out with a bang

A huge amount of cellular genomics will have to be redone if the following paper is replicated. Remember “Extraordinary claims require extraordinary evidence.” Carl Sagan.

What’s all the shouting about? Normally when you think about messenger RNA (mRNA) as it exists in the cytoplasm after the initial transcript is significantly massaged in the nucleus, you think about the part that codes for amino acids. This ‘coding region’ is the part that is translated into amino acids by the ribosome. But mRNA is invariably larger having nucleotides at each end (3′ and 5′) which have other uses. These are called the 3′ Untranslated Region (3′ UTR) and 5′ Untranslated Region (5′ UTR).

So if you do single cell RNA sequencing (which we can do now) it shouldn’t matter what nucleotide sequence you search for (5′ UTR, 3′ UTR or the coding region) as all mRNA contains one of each.

Not so says this paper [ Neuron vol. 88 pp. 1149 – 1156 ’15 ].

Given the mRNA for a given protein in a single cell, using a probe for the 3’UTR and a probe for the coding sequence should give you the same abundance for both. That’s not what they found at all for single neurons from the brain. In some cases there was much more RNA coding for the 3’UTR than for the coding segment of a given mRNA for a protein. In others there was much less. Even more impressively is that the 3’UTR/(3’UTR + coding) ratio for a given protein varies between different parts of the brain. Obviously this ratio should be .5 given what we knew about mRNA in the past. The ratio has to be between 0 and 1.

Well they looked at a lot of proteins. The did find around 1,400 genes with a ratio of .5 (as expected), but they found 700 showing a ratio of .2 (lots more 3’UTR than coding sequence), and 1,100 showing a ratio of .8. Overall plotting the ratio vs. number of genes with that ratio gives something looking like a bell curve (Gaussian distribution).

It’s long been known that mRNA levels don’t exactly correlate with the levels of proteins made from them. If there’s lots of 3’UTRs around the authors found that there was relatively little protein made from the gene.

A variety of brain atlases have published mRNA abundances for various regions of the brain. If they just used one probe (as they probably did) this is clearly not enough.

The 3’UTRs may be acting as ceRNAs (competitive endogenous RNAs). These have been known for years — I’ve included a post of 3 years ago on the subject (at the end).

So this work (if replicated) throws everything we thought we knew about mRNA into a cocked hat. It’s why I love science, there’s always something really new to think about. Happy New Year !!!

Chemiotics II
Lotsa stuff, basically scientific — molecular biology, organic chemistry, medicine (neurology), math — and music
Why drug discovery is so hard: reason #20 — competitive endogenous RNAs

The chemist will appreciate le Chatelier’s principle in action in what follows. We are far from knowing all the players controlling cellular behavior. So how in the world will we find drugs to change cellular behavior when we don’t know all the things affecting it. The latest previously unknown cellular player to enter the lists are competitive endogenous RNAs (ceRNAs). For details see Cell vol. 147 pp. 344 – 357, 382 – 395 ’11. The background the pure chemist needs for what follows can all be found in the category “Molecular Biology Survival Guide.

Recall that microRNAs are short (20 something) polynucleotides which bind to the 3′ untranslated region (3′ UTR) of mRNA, and either (1) inhibit its translation into protein (2) cause its degradation. In each case, less of the corresponding protein is made. The microRNA and the appropriate sequence in the 3′ UTR of the mRNA form an RNA-RNA double helix (G on one strand binding to C on the other, etc.). Visualizing such helices is duck soup for a chemist.

Molecular biology is full of such semantic cherry bombs as nonCoding DNA (which meant DNA which didn’t cord for protein), a subset of Junk DNA. Another is the pseudogene — these are genes that look like they should code for protein, except that they don’t because of lack of an initiation codon or a premature termination codon. Except for these differences, they have the nucleotide sequence to code for a known protein. It is estimated that the human genome contains as many pseudogenes (20,000) as it contains true protein coding genes [ Genome Res. vol. 12 pp. 272 – 280 ’02 ]. We now know that well over half the genome is transcribed into mRNA, including the pseudogenes.

PTEN (you don’t want to know what it stands for) is a 403 amino acid protein which is one of the most commonly mutated proteins in human cancer. Our genome also contains a pseudogene for it (called PTENP). Interestingly deletion of PTENP (not PTEN) is found in some cancers. However PTENP deletion is associated with decreased amounts of the PTEN protein itself, something you don’t want as PTEN is a tumor suppressor. How PTEN accomplishes this appears to be fairly well known, but is irrelevant here.

Why should loss of PTENP decrease PTEN itself? The reason is because the mRNA made from PTENP, even though it has a premature termination codon, and can’t be made into protein, is just as long, so it also contains the 3’UTR of PTEN. This means PTENP is sopping up microRNAs which would otherwise decrease the level of PTEN. Think of PTENP mRNA as a sponge.

Subtle isn’t it? But there’s far more. At least PTENP mRNA closely resembles the PTEN mRNA. However other mRNAs coding for completely different proteins, also have binding sites in their 3’UTR for the microRNA which binds to the 3UTR of PTEN, resulting in its destruction. So transcription of a completely different gene (the example of ZEB2 is given) can control the abundance of another protein. Essentially its mRNA is acting as a sponge, sopping up the killer microRNA.

It gets worse. Most microRNAs have binding sites on the mRNAs of many different proteins, and PTEN itself has a 3’UTR which binds to 10 different microRNAs.

So here is a completely unexpected mechanism of control of protein levels in the cell. The general term for this is competitive endogenous RNA (ceRNA). Two years ago the number of human microRNAs was thought to be around 1,000. Unlike protein coding genes, it’s far from obvious how to find them by looking at the sequence of our genome, so there may be quite a few more.

So most microRNAs bind the 3’UTR of more than one protein (the average number is unclear at this point), and most proteins have binding sites for microRNAs in their 3’UTR (again the average number is unclear). What a mess. What subtlety. What an opportunity for the regulation of cellular function. Who is going to be smart enough to figure out a drug which will change this in a way that we want. Absence of evidence of a regulatory mechanism is not evidence of its absence. A little humility is in order.