Tag Archives: iPSCs

Another way to study Alzheimer’s

Until I read the paper PLOS Genet. 14, e1007791 (2018)., I thought that this was a sure way to win Nobel prize.  It’s still pretty interesting.  The abstract in Science was misleading, implying that there was an APOE4 variant which was actually protective against Alzheimer’s disease. That would have been fantastic, as it would provide a clue as to just what the APOE4 allele was doing to increase the risk of Alzheimer’s disease.

A huge amount of work has been done on APOE4.   Googling produced 433,000 results (0.46 seconds).  Theories abound but we still don’t know.

The authors studied Blacks and Puerto Ricans and found that if you inherited the APOE4 allele from an African source (rather than a European source), your chance of developing Alzheimer’s disease was significantly less.  A total of 1,766 African American and 220 Puerto Rican individuals with late-onset Alzheimer disease, and 3,730 African American and 169 Puerto Rican cognitively healthy individuals (> 65 years) participated in the study.

The numbers: ApoE ε4 alleles on an African background conferred a lower risk than those with a European ancestral background, regardless of population (Puerto Rican: OR = 1.26 on African background, OR = 4.49 on European; African American: OR = 2.34 on African background, OR = 3.05 on European background).

Note that the ORs are still up for Alzheimer’s if you have APOE4, but the differences are significant and certainly real given the size of the study.

The authors think it’s the area around the APOE  gene, rather than the total genetic background (African vs. European etc. etc.)

It still might be worth doing the following.  Take skin fibroblasts from all four types of people (Puerto Ricans with APOE4 on African background, Puerto Ricans with APOE4 on European background, Blacks with APOE4 on African background, APOE4 on a European background).

Make induced pluripotent stem cells (iPSCs) from them (the technology to do so is quite advanced). Differentiate these iPSCs into neurons  and others into glia (technology quite available).  Study protein and mRNA expression, epigenetic modifications in neurons and glia from all 4 groups.  This might tell you just what APOE4 was doing in high and lower risk people, and possibly might give a clue as to how it was increasing Alzheimer’s risk.

My hopes were really up, because the abstract in Science implied that APOE4 in Blacks and Puerto Ricans was actually absolutely rather than relatively protective, which would have given us some serious clues to Alzheimer pathogenesis, when APOE4 protective cells were contrasted with APOE4 increased risk cells.

Oh well.

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It ain’t the bricks, it’s the plan

Nothing better shows the utility (and the futility) of chemistry in biology than using it to explain the difference between man and chimpanzee. You’ve all heard that our proteins are only 2% different than the chimp, so we are 98% chimpanzee. The facts are correct, the interpretation wrong. We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

This is like saying Monticello and Independence Hall are just the same because they’re both made out of bricks. One could chemically identify Monticello bricks as coming from the Virginia piedmont, and Independence Hall bricks coming from the red clay of New Jersey, but the real difference between the buildings is the plan.

It’s not the proteins, but where and when and how much of them are made. The control for this (plan if you will) lies outside the genes for the proteins themselves, in the rest of the genome (remember only 2% of the genome codes for the amino acids making up our 20,000 or so protein genes). The control elements have as much right to be called genes, as the parts of the genome coding for amino acids. Granted, it’s easier to study genes coding for proteins, because we’ve identified them and know so much about them. It’s like the drunk looking for his keys under the lamppost because that’s where the light is.

We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

All the molecular biology you need to understand what follows is in the following post — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/

Briefly an enhancer is a stretch of DNA distinct from the DNA coding for a given protein, to which a variety of other proteins called transcription factors bind. The enhancer DNA and associated transcription factors, then loops over to the protein coding gene and ‘turns it on’ — e.g. causes a huge (megaDalton) enzyme called pol II to make an RNA copy of the gene (called mRNA) which is then translated into protein by another huge megaDalton machine called the ribosome. Complicated no? Well, it’s happening inside you right now.

The faces of chimps and people are quite different (but not so much so that they look frighteningly human). The cell paper studied cells which in embryos go to make up the bones and soft tissues of the face called Cranial Neural Crest Cells (CNCCs). How did they get them? Not from Planned Parenthood, rather they made iPSCs (induced Pluripotent Stem Cells — https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell) differentiate into CNCCs. Not only that but they studied both human and chimp CNCCs. So you must at least wonder how close to biologic reality this system actually is.

It’s rather technical, but they had several ways of seeing if a given enhancer was active or not. By active I mean engaged in turning on a given protein coding gene so more of that protein is made. For the cognoscenti, these methods included (1) p300 binding (2) chromatin accessibility,(3) H3K4Me1/K3K4me3 ratio, (4) H3K27Ac.

The genome is big — some 3,200,000,000 positions (nucleotides) linearly arranged along our chromosomes. Enhancers range in size from 50 to 1,500 nucleotides, and the study found a mere 14,500 enhancers in the CNCCs. More interestingly 13% of them were activated differentially in man and chimp CNCCs. This is probably why we look different than chimps. So although the proteins are the same, the timing of their synthesis is different.

At long last, molecular biology is beginning to study the plan rather than the bricks.

Chemistry has a great role in this and will continue to do so. For instance, enhancers can be sequenced to see how different enhancer DNA is between man and chimp. The answer is not much (again 2 or so nucleotides per hundred nucleotides of enhancer). The authors did find one new enhancer motif, not seen previously called the coordinator motif. But it was present in man in chimp. Chemistry can and should explain why changing so few nucleotides changes the proteins binding to a given enhancer sequence, and it will be important in designing proteins to take advantage of these changes.

So why is chemistry futile? Because as soon as you ask what an enhancer or a protein is for, you’ve left physicality entirely and entered the realm of ideas. Asking what something is for is an entirely different question than how something actually does what it is for.  The latter question  is answerable by chemistry and physics. The first question is unanswerable by them.  The Cartesian dualism of flesh and spirit is alive and well.

It’s interesting to see how quickly questions in biology lead to teleology.

Short and Sweet

Yamanaka strikes again. Citrulline is deiminated arginine, replacing a C=N-H (the imine) by a carbonyl C=O. An enzyme called PAD4 does the job. Why is it important? Because one of its targets is the H1 histone which links nucleosomes together. Recall that the total length of DNA in each and every one of our cells is 3 METERS. By wrapping the double helix around nucleosomes, the DNA is shortened by one order of magnitude.

So what? Well, at physiologic pH the imine probably binds another proton making it positively charged, making it bind to the negatively charged DNA phosphate backbone. Removing the imine makes this less likely to happen, so the linker doesn’t bind the double helix as tightly.

Duck soup for the chemist, but apparently no one had thought to look at this before.

This opens up the DNA (aka chromatin decondensation) for protein transcription. Why is Yamanaka involved? Because PAD4 is induced during cellular reprogramming to induced pluripotent stem cells (iPSCs), activating the expression of key stem cell genes. Inhibition of PAD4 lowers the percentage of pluripotent stem cells, reducing reprogramming efficiency. The paper is Nature vol. 507 pp. 104 – 108 ’14.

Will this may be nice for forming iPSCs, it should be noted that PAD4 is unregulated in a variety of tumors.