How little we know

Who would have thought that a random mutagenesis experiment throwing Ethyl Nitroso Urea (ENU) at unsuspecting mice looking for genes using a mutagenesis strategy to identify novel immune regulatory genes would point to a possible treatment for muscular dystrophy? When the experimenters looked at the mutated offspring, they found that the muscles appeared unusually red.

What happened?

You need to know a bit more about muscles. On a very simplistic level there are only two types of muscle fibers, red and white. Carnivores eating chicken know about dark meat and white meat. The dark meat is composed of red fibers, which have that appearance because of large numbers of mitochondria (which are full of iron) giving them the same red appearance as blood (which is also full of iron). In both cases the iron is bound by porphyrin rings. As one might expect, these muscles consume a lot of energy, being postural for the most part. The white meat made of white fibers has muscle which can contract very quickly and strongly, for flight and fight. They don’t have nearly the endurance of red muscle, because they can’t produce energy for the long term.

Humans have the two types of muscle fibers mixed up in each of our muscles.

The ENU had produced a mutation in something called fnip1 (Folliculin INteracting Protein 1). What’s folliculin? It prevents a gene transcription factor (TFE3) from getting into the nucleus. Folliculin prevents an embryonic stem cell from differentiating. It is mutated in the Birt Hogg Dube syndrome which is characterized by many benign hair follicle tumors. What in the world does this have to do with muscular dystrophy? It’s not something someone would start investigating looking for a cure is it? Knock out both copies of folliculin and the embryo dies in utero.

It gets deeper.

What does Fnip1 do to folliculin? It, and its cousin fnip2 form complexes with folliculin. The complex binds an enzyme called AMPK (which is turned on by energy depletion in the cell. AMPK phosphorylates both fnip1 and folliculin. Folliculin binds and inhibits AMPK.

So animals lacking fnip1 have a more activated AMPK. So what? Well AMPK activates a transcriptional coactivator called PGC1alpha (you don’t want to know what the acronym stands for). This ultimately results in production of more mitochondria (recall that AMPK is an energy sensor, and one of the main functions of mitochondria is to produce energy, lots of it).

This ultimately means more red muscle fibers. There is a mouse model of Duchenne dystrophy called the mdx mouse (which has a premature termination codon in the dystrophin protein, resulting in a protein only 27% as long as it should be. That still leaves a lot, as normal dystrophin contains 3,685 amino acids. Knocking out fnip1 in the mdx mice improved muscle function. Impressive !!

I’m quite interested in this sort of work, as I ran a muscular dystrophy clinic from ’72 to ’87 and watched a lot of kids die. The major advance during that time wasn’t anything medical. It came from engineering — lighter braces using newer materials allowed the kids to stay out of wheelchairs longer.

You can read all about it in Proc. Natl. Acad. Sci. vol. 112 pp. 424 – 429 ’15 ] Clearly we know a lot (AMPK, dystrophin, PGC1alpha, fnip1, fnip2, folliculin, TFE3), but what we didn’t know was how in the world they function together in the cell. We’re sure to learn a lot more, but this whole affair was uncovered when looking for something else (immune regulators) using the bluntest instrument possible (throw a mutagen at an animal and see what happens). No one applying for a muscular dystrophy grant would dare to offer the original work as a rationale, yet here we are.

So directed research isn’t always the way to go. Although we know a lot, we still know very little.

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