My first blog post, so bear with me:-) Below is a link to access my first paper proposing that various chemically distinct compounds may actually share a common "indirect yet primary mechanism of action" of AMPK activation that may beneficially alter gene splicing in a number of genetic disorders. Interestingly, this paper (published in 2014) was the first to propose that metformin, a prototypical AMPK activator, may beneficially alter gene splicing by activating AMPK. As it turns out, a recent paper published in November of 2015 (check back for later posts) showed just that!
But first up, some background on how exactly I came up with the idea that AMPK activators that come in all shapes and stripes, structurally, can accomplish such a feat (for references to studies and other information, please visit my LinkedIn page via the link below and check out the posts).
As a patent attorney focused on life science and biotechnology inventions, I've always been intrigued by the interrelatedness of various systems of the body and how certain drugs, particularly metformin and rapamycin, appear to have an uncanny ability to positively affect and regulate numerous pathologies within the body but seemingly have different mechanisms of action (not really, as we shall see!). I was also super intrigued that both of these compounds could improve lifespan (how long you live) and healthspan (how healthy you are, i.e. life without disease) in several animal model organisms, with rapamycin increasing maximal lifespan in elderly normal mice even if given late in life. The ability of these compounds to ward off aging and disease led me to check out the accelerated aging disease Hutchinson-Gilford progeria syndrome (HGPS for short). What's interesting about this disease is that the symptoms of the disease result from the overwhelming use of a cryptic splice site in a gene known as LMNA, as opposed to the use of the normal splice site in the LMNA gene. The use of the cryptic splice site by the cell’s splicing machinery (i.e. the "cutting crew") causes the production of a truncated protein called progerin that causes the nucleus of the cell to become deformed, affecting many processes that occur in the nucleus, leading to symptoms of accelerating aging. HOWEVER, children with HGPS do use the normal splice site in the LMNA gene sparingly, producing a small amount of the resulting normal protein, Lamin A.
But here's the real kicker. Normal unaffected humans (you and I) have the same cryptic splice site in our LMNA genes as do HGPS children, but we use that splice site sporadically and the normal splice site predominantly. As we age however, the use of this cryptic splice increases and we slowly start to accumulate the toxic protein progerin over decades and thus experience the ravages of aging over an extended period of time.
With this information at hand, a thought immediately came to mind. If we have the same cryptic splice site as HGPS children but use it sporadically to make the same toxic progerin protein over decades, and if HGPS kids do use their normal splice site sporadically, could it be possible to "shift" the gene cutting crew from the cryptic splice site over to the normal splice site in both HGPS kids and in normal individuals, thus ameliorating accelerating aging defects and possibly slow down the aging process???
After doing some poking around in the research literature, the splicing factor SRSF1 constantly came up as a protein that is associated with alternative splicing or the use of splice sites other than the normal splice sites. SRSF1 basically acts as a foreman for the gene "cutting crew", directing the cutting crew to a specific splice site on the gene. The activity of SRSF1 is also concentration-dependent, meaning the more you have it, the greater its activity becomes. Although SRSF1 is a necessary for splicing reactions to occur, too much of it (or enhanced splicing activity) can lead to recognition and binding of cryptic splice sites by the gene cutting crew. Interestingly, a study showed that if you knockdown SRSF1, the amount of the toxic progerin protein decreases significantly. Bingo!! But how do you decrease the activity of SRSF1 safely? After all, it is necessary for most splicing reactions to occur.
My first thought was, ok, are there any endogenous inhibitors of SRSF1? And indeed there is. In comes the splicing-associated factor p32. p32 has been shown in numerous studies over the past few decades to inhibit the splicing activity of SRSF1. However, what's really cool about p32 is that it appears to act as a chaperone as well, translocating to various organelles in the cell, including the nucleus and mitochondria, helping proteins to do their job. Now, here's the connection between p32, aging, and AMPK. p32 spends most of its time in the mitochondria, a structure known as the powerhouse of the cell that makes most of the energy (in the form of ATP) necessary to perform almost all anabolic process in the cell. In fact, p32 is absolutely essential for the correct functioning and ATP production of mitochondria, as knockdown of p32 considerably inhibits the mitochondria’s ability to produce ATP. It's also pretty settled in the literature that optimal mitochondrial functioning is critical in promoting lifespan and healthspan in many different organisms and the activation of AMPK, which has been shown to be pivotal in increasing lifespan and healthspan, leads to a significant increase in mitochondrial biogenesis (i.e. the birth of new mitochondria), mitochondrial autophagy (the removal of old, worn-out mitochondria as well as other damaged organelles and proteins), and enhancement of mitochondrial oxidative phosphorylation.
So, if my reasoning was solid, compounds that could safely activate AMPK (despite what they looked like structurally) could possibly alter the activity of p32, leading to the inhibition of SRSF1 activity in the nucleus, mitochondrial biogenesis, amelioration of accelerated aging defects in HGPS kids, and slow the aging process in you and I. The anti-diabetic drug Metformin was an obvious choice. Metformin is basically a prototypical AMPK activator that improves lifespan and healthspan in many different organisms. BUT, can metformin actually alter gene splicing?? And can it do so through activating AMPK?? As of November of 2015, the answer is........you bet!!! Researchers in that study found that metformin corrected faulty gene splicing that was dependent on the activation of AMPK in a disease called myotonic dystrophy type 1 (DM1) using cells taken from patients and cells from embryos that had been diagnosed with the disease. Another HUGE takeaway from that study was that metformin also beneficially altered splicing of the insulin receptor gene in diabetic patients who didn't have DM1 but who were taking metformin. And the magic word: Bingo!!! Metformin and AMPK activation can beneficially alter gene splicing in genetic disorders as well as in normal humans.
But that's definitely not the end of the story. Remember, p32 is sort of a jack-of-all-trades, inhibiting the splicing activity of SRSF1 in the nucleus as well as boosting mitochondrial biogenesis and the generation of ATP. Interestingly, p32 is also critical for mitochondrial autophagy (i.e. mitophagy), the removal of worn-out mitochondria. A recent study showed that p32 was essential for mitophagy by acting as a chaperone for ULK1, a protein that is critical for the induction of autophagy. In fact, if you knockdown p32, mitophagy or autophagy (the general term for the removal of damaged cellular material) that is induced by the anti-aging drug rapamycin or starvation is significantly inhibited. On top of that, AMPK is known to activate ULK1 and induce autophagy/mitophagy. NOW, a pretty cool portrait starts to materialize: AMPK activation influences the activity of p32 to inhibit SRSF1, alters gene splicing, promotes mitochondrial biogenesis, and induces autophagy/mitophagy, all of which promote increased lifespan and healthspan in normal humans and potentially in children diagnosed with HGPS!!
But wait, of course there's more:-) The splicing factor SRSF1 has also been shown to promote faulty splicing of genes in the vascular system (e.g. VEGF, tissue factor, endoglin) that promotes endothelial senescence in normal humans. Kids with HGPS also almost invariably die from complications associated with a stroke or heart attack, implicating SRSF1 as player in cardiovascular senescence. Also, UV radiation, which promotes accelerated aging in many cells, both increases the activity of SRSF1 and increases the levels of the toxic protein progerin, again pointing the finger at SRSF1 as a key mediator in the promotion of faulty gene splicing and accelerated aging.
Lastly (and this where the fun really begins), as mentioned earlier, both rapamycin and starvation lost its ability to induce autophagy if p32 was knocked down. Interestingly, rapamycin has also been shown in studies to correct cellular aging defects in cells taken from HGPS patients by inducing autophagy! Hmmmm.....let us see. AMPK activates ULK1, thus inducing autophagy. p32 is needed as a chaperone for ULK1. Knockdown of p32 inhibits the induction of ULK1-mediated autophagy by rapamycin. Can you guess what my next prediction would be?!@#?
Yep, you guessed it. RAPAMYCIN MUST BE AN INDIRECT AMPK ACTIVATOR!! But there's one problem. No studies have shown rapamycin to activate AMPK in vivo (i.e. in a living animal) as I predicted in my paper, until............February of 2016. In a barn-burner of a study, researchers at the University of Washington showed that rapamycin indeed potently activated AMPK during the entire 10 week study in elderly normal mice. Rapamycin also transiently upregulated autophagy and mitochondrial biogenesis during the first two weeks. Again, a gorgeous molecular portrait is being put on display. AMPK activation by rapamycin is driving autophagy and mitochondrial biogenesis, both likely dependent on p32.
Interestingly, another compound that activates AMPK, sulforaphane (derived from broccoli sprouts), also corrects accelerated aging defects in HGPS cells by inducing autophagy. Also, retinoic acid, a vitamin A derivative that also activates AMPK, corrected accelerated aging defects in HGPS. Perhaps my favorite though is a recent study showing that methylene blue, a safe and inexpensive compound that 's been in use for decades, corrects accelerated aging defects in HGPS partly by enhancing mitochondrial biogenesis. Methylene blue also enhanced mitochondrial biogenesis in normal cells in that same study. Methylene blue has been shown in separate studies as well to activate AMPK in vivo and reduce senescence (i.e. aging-associated loss of cell division) in various cell types.
And there you have it!! Could it be possible to treat HGPS and normal aging with a variety of compounds that converge to indirectly activate AMPK?? Of course my answer is a resounding yes, but I'll let you be the judge:-)
Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation.
https://www.linkedin.com/pulse/potential-correction-gene-splicing-progeria-via-naturally-finley?trk=mp-reader-card
ABSTRACT
Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by an accelerated aging phenotype and an average life span of 13 years. Patients typically exhibit extensive pathophysiological vascular alterations, eventually resulting in death from stroke or myocardial infarction. A silent point mutation at position 1824 (C1824T) of the LMNA gene, generating a truncated form of lamin A (progerin), has been shown to be the cause of most cases of HGPS. Interestingly, this mutation induces the use of an internal 5’ cryptic splice site within exon 11 of the LMNA pre-mRNA, leading to the generation of progerin via aberrant alternative splicing. The serine-arginine rich splicing factor 1 (SRSF1 or ASF/SF2) has been shown to function as an oncoprotein and is upregulated in many cancers and other age-related disorders. Indeed, SRSF1 inhibition results in a splicing ratio in the LMNA pre-mRNA favoring lamin A production over that of progerin. It is our hypothesis that activation of AMP- activated protein kinase (AMPK), a master regulator of cellular metabolism, may lead to a reduction in SRSF1 and thus a decrease in the use of the LMNA 5’ cryptic splice site in exon 11 through upregulation of p32, a splicing factor-associated protein and putative mitochondrial chaperone that has been shown to inhibit SRSF1 and enhance mitochondrial DNA (mtDNA) replication and oxidative phosphorylation. AMPK activation by currently available compounds such as metformin, resveratrol, and berberine may thus have wide-ranging implications for disorders associated with increased production and accumulation of progerin.
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