Cancer drug used to successfully treat children with Progeria also reactivates latent HIV-1: Connection between AMPK, aging, and HIV-1 reactivation

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

And the evidence keeps rolling in:-) At this pace, I may be able to retire sooner (or later, hopefully sooner). Here's another recent LinkedIn post I authored explaining that a drug that was originally developed to treat cancer was successfully used to improve symptoms in children with Progeria. Interestingly, that same drug was also recently shown by researchers at the University of North Carolina and Case Western Reserve University (in collaboration with the drug company Merck) to reactivate latent HIV-1 in T cells taken directly from patients infected with HIV-1. One big coincidence?? I think not. This is just another example of researchers not realizing that many chemically distinct compounds converge on and indirectly activate a common pathway (AMPK) that connects and characterizes nearly every cell, system, and ultimately disease pathology within the body. Is it possible that normal aging, accelerated aging, infections that involve dormant viruses (e.g. HIV-1, HPV, Herpes, etc), and cancer are all connected and can be treated or possibly eradicated by activating a particular pathway that increases the lifespan and healthspan of nearly every organism that it's been tested in? If so, I think my mom will be pretty proud of me (if she actually believes me:-)

Cancer drug used to successfully treat children with Progeria also reactivates latent HIV-1: Connection between AMPK, aging, and HIV-1 reactivation

In an abstract presented in December of 2013 at the Sixth International Workshop on HIV Persistence during Therapy, researchers from the University of North Carolina and Case Western Reserve University (in collaboration with the pharmaceutical company Merck) reported that the use of an ultra-high throughput screen identified a class of compounds known as farnesyl-transferase inhibitors (FTIs) that reactivated latent HIV-1 expression and acted synergistically with other agents to reverse latency in T cell models and in resting CD4+ T cells isolated from patients infected with HIV-1 [1].

Interestingly, of the sub-classes of FTI’s identified in the screen that synergistically reactivated latent HIV-1, the FTI lonafarnib was specifically listed as a representative structure identified in the screen [1].  The identification of lonafarnib as a HIV-1 latency reversing agent is startling considering that lonafarnib, originally tested in preclinical studies as an anti-cancer agent, has recently been shown and reported to be beneficial in a clinical trial for the treatment of children diagnosed with Hutchinson-Gilford progeria syndrome (HGPS) [2,3].

The mechanism of action of FTI’s involves the inhibition of an enzyme known as farnesyl-transferase (FT). FT is an enzyme that adds a farnesyl group to proteins that bear a particular amino acid sequence [4]. The farnesyl group typically targets the protein to the cell membrane, often facilitating cellular signaling involving the farnesylated protein [4].  Within the context of HGPS, the protein lamin A becomes permanently farnesylated (also known as progerin) due to a gene mutation, leading to an excessive accumulation of progerin in the nuclear membrane and thus generating nuclear blebbing and dysfunctional nuclear processes [5].  Similarly, the oncoprotein Ras, a protein critical for cell cycle progression that is abnormally active in many cancers, is farnesylated by FT, thereby facilitating attachment of Ras to the cell membrane to promote signal transduction [4].

Although the primary mechanism of action of lonafarnib is the inhibition of FT, it is likely, similar to rapamycin and other compounds that are effective in treating progeria but also have immuno-modulatory effects, that inhibition of FT by lonafarnib is generating an indirect activation of the master metabolic regulator AMPK via the induction of cellular stress (i.e. anabolic pathway inhibition).  Indeed, FT is also involved in the mevalonate pathway, an anabolic pathway that is characterized by the biosynthesis of cholesterol [6].  Interestingly, statins, which function by inhibiting the rate limiting enzyme in the mevalonate pathway, HMG-CoA reductase, activate AMPK and have been shown to be efficacious in preclinical animal models of progeria [7,8].

In the abstract presented at the Sixth International Workshop on HIV Persistence during Therapy, the researchers noted that FTI’s act synergistically with the latency reversing agent JQ1 and protein kinase C (PKC) activators [1].  Interestingly, as AMPK activation is essential for T cell activation and thus efficient latent HIV-1 reactivation, JQ1 as well as the PKC activator bryostatin have both been shown to induce the phosphorylation and activation of AMPK in recent studies, implying that the FTI lonafarnib likely also induces AMPK activation [9,10].

Indeed, the abstract presented intriguing data that knock down of the beta subunit of the FT enzyme sensitized and constitutively reactivated latent HIV-1 in a Jurkat T cell model system [1].  Such evidence is provocative in the sense that the inhibition or knock down of mediators that function to promote anabolic processes, including HMG-CoA reductase (cholesterol biosynthesis), ATIC/AICART (purine metabolism), and mTOR (protein synthesis), lead to cellular stress-induced activation of AMPK [7,11,12].  Similarly, the inhibition of the beta subunit of FT, which is also involved in the anabolic mevalonate pathway, likely induces a cellular stress-induced activation of AMPK, potentially explaining a portion of the beneficial effects of lonafarnib in both HGPS and latent HIV-1 reactivation.

Lastly, the abstract also presented evidence that FTIs acted synergistically with the HDAC inhibitor vorinostat to reactivate latent HIV-1 in resting CD4+ memory T cells isolated from HIV-1 patients [1].  Interestingly, vorinostat has also been shown to induce AMPK activation in certain cancer cells [13].

Collectively, data from this abstract and the recent results published from the HGPS clinical trial provides additional evidence (reinforcing the implications of the figure below) that inhibition of FT by FTI’s such as lonafarnib leads to the induction of AMPK activation via the promotion of a cellular stress response, not unlike that of mTOR inhibition-induced AMPK activation by rapamycin [12].  The novel observation that chemically distinct compounds converge on single pathway to effectuate beneficial results in diseases as seemingly disparate as HGPS and HIV-1 reactivation is unprecedented.

 
https://www.linkedin.com/pulse/cancer-drug-used-successfully-treat-children-progeria-finley


References:

1. Farnesyl-transferase inhibitors: identification and validation of a class which reactivates HIV latent expression and is synergistic with other mechanisms in vitro. In: 6th intl workshop on HIV persistance during therapy report summary – report by David Margolis MD, UNC chapel hill and the collaboratory of AIDS researchers for eradication (CARE) – (12/15/13).  http://www.natap.org/2013/HIV/121913_01.htm, last accessed 04/24/2016.
2. Morgillo F, Lee HY. Lonafarnib in cancer therapy. Expert Opin Investig Drugs. 2006 Jun;15(6):709-19.
3. Gordon LB, Kleinman ME, Miller DT, et al. Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):16666-71.
4. Reuter CW, Morgan MA, Bergmann L. Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies?  Blood. 2000 Sep 1;96(5):1655-69.
5. Prokocimer M, Barkan R, Gruenbaum Y. Hutchinson-Gilford progeria syndrome through the lens of transcription. Aging Cell. 2013 Aug;12(4):533-43.
6. Marcuzzi A, De Leo L, Decorti G, Crovella S, Tommasini A, Pontillo A. The farnesyltransferase inhibitors tipifarnib and lonafarnib inhibit cytokines secretion in a cellular model of mevalonate kinase deficiency. Pediatr Res. 2011 Jul;70(1):78-82.
7. Sun W, Lee TS, Zhu M, et al. Statins activate AMP-activated protein kinase in vitro and in vivo. Circulation. 2006 Dec 12;114(24):2655-62.
8. Varela I, Pereira S, Ugalde AP, et al. Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging. Nat Med. 2008 Jul;14(7):767-72.
9. Wang H, Sharma L, Lu J, Finch P, Fletcher S, Prochownik EV. Structurally diverse c-Myc inhibitors share a common mechanism of action involving ATP depletion. Oncotarget. 2015 Jun 30;6(18):15857-70.
10. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One. 2010 Jun 16;5(6):e11160.
11. Asby DJ, Cuda F, Beyaert M, Houghton FD, Cagampang FR, Tavassoli A. AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization. Chem Biol. 2015 Jul 23;22(7):838-48.
12. Chiao YA, Kolwicz SC, Basisty N, et al. Rapamycin transiently induces mitochondrial remodeling to reprogram energy metabolism in old hearts. Aging (Albany NY). 2016 Feb;8(2):314-27.
13. Sarfstein R, Bruchim I, Fishman A, Werner H. The mechanism of action of the histone deacetylase inhibitor vorinostat involves interaction with the insulin-like growth factor signaling pathway. PLoS One. 2011;6(9):e24468.
    

Nobel Prize Winning drug Artemisinin shares common mechanism of action with AMPK activator Metformin: Connection between aging and HIV-1 reactivation

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

Another recent LinkedIn post I authored (see below) showing that a naturally occurring compound that is used as a powerful first line treatment for malaria activates the same pathway as the broccoli sprout compound sulforaphane, the vitamin A metabolite retinoic acid, metformin derived from the French Lilac, and methylene blue. This compound, called artemisinin, won the 2015 Nobel prize in Medicine for a Chinese scientist and has been used for over 2000 years to treat malaria dating back to 200 BC. This same compound has been shown to inhibit cancer by activating AMPK and mimic calorie restriction by increasing energy levels (ATP) and mitochondrial biogenesis. Just as sulforaphane, retinoic acid, and methylene blue also inhibit cancer and malaria and reverse aging in progeria cells, artemisinin (derived from the plant Artemisia annua) will likely also reverse symptoms of accelerated aging in progeria cells and in normal humans and reactivate latent HIV-1. Again, if further evidence proves this to be true............:-)

Nobel Prize Winning drug Artemisinin shares common mechanism of action with AMPK activator Metformin: Connection between aging and HIV-1 reactivation

A recent study published online in the journal Oncotarget in June of 2015 provided incredible evidence that a common metabolite of artemisinin and its analogs, a powerful anti-malarial drug derived from the plant Artemisia annua (a plant that has been used by Chinese herbalists for more than 2000 years to treat malaria, with the earliest recording dating back to 200 BC) shares a common mechanism of action with several other chemically distinct compounds (including a compound that has shown powerful effects in reactivating latent HIV-1) that is dependent on the activation of the master metabolic regulator AMPK [1,2].

Artemisinin or its semi-synthetic derivatives (which are all metabolized into dihydroartemisinin) are considered the standard first-line treatment for malaria caused by the protozoan parasite Plasmodium falciparum [3]. Half of the 2015 Nobel Prize in Medicine was awarded to the Chinese scientist Tu Youyou for her discovery of artemisinin [4]. Although the mechanism of action of artemisinin has been vigorously debated for decades and is currently unknown, the generation of free radicals leading to death of the parasite is a widely-accepted theory [5].

In addition to its anti-malarial effects, dihydroartemisinin has also been shown to exert potent anti-cancer effects in a range of malignancies. In cancer research, artemisinin is characterized as a synthetic lethal c-Myc inhibitor [1]. c-Myc is one of the most frequently deregulated oncoproteins (a protein that could contribute to oncogenesis due to mutations or increased expression) in human cancers and is also upregulated during HIV-1 latency [6,7]. Interestingly, in the Oncotarget study, the authors demonstrated that regardless of the class to which each studied drug had been placed (i.e., direct, indirect, or synthetic lethal c-Myc inhibitors), the therapeutic action of each drug converges on a single overriding mechanism that involves the activation of AMPK due to depletion of ATP stores induced by mitochondrial dysfunction, resulting in cell cycle arrest and differentiation or apoptosis (i.e. cell death) of several cancer cell lines [1].

Although structurally dissimilar and considered an indirect c-Myc inhibitor in cancer research, the BRD4 inhibitor JQ1 was also shown to act via the same mechanism of action as dihydroartemisinin in this study via induction of mitochondrial dysfunction, reduced ATP levels, activation of AMPK, and cell cycle arrest and differentiation of leukemia cells [1].

The authors also reasoned that if a common mechanism of action of mitochondrial dysfunction and compensatory AMPK activation links dihydroartemisinin and JQ1, then the use of well-characterized mitochondrial inhibitors that also activate AMPK should produce the same or similar results as dihydroartemisinin and JQ1. Indeed, the use of metformin (inhibits complex I of the mitochondrial electron transport chain) or oligomycin (inhibits complex V of the mitochondrial electron transport chain) also lead to a decrease in Myc protein levels, reduction in the levels of ATP, activation of AMPK, and cancer cell differentiation, providing convincing evidence that compounds as chemically distinct as metformin, dihydroartemisinin, JQ1, and oligomycin share a common mechanism of action that centers on AMPK activation [1]. Moreover, artemisinin has been shown in another recent study to inhibit neuroblastoma proliferation (using SH-SY5Y cells) via the activation of AMPK, as AMPK inhibition by compound C or siRNA abrogated artemisinin’s effects [8].

Interestingly, also in the Oncotarget study, the use of the phorbol ester TPA/PMA, produced similar results as metformin, dihydroartemisinin, and JQ1 on cancer cell differentiation [1]. As noted above, JQ1 has also been shown to be a powerful re-activator of latent HIV-1, facilitating its eventual detection and destruction by the immune system, and PMA combined with ionomycin (a chemical that increases the levels of calcium in a cell) is such a powerful reactivator of latent HIV-1 in T cells that it is used as a benchmark positive control in latent HIV-1 reactivation studies [9,10].

Bryostatin-1, a protein kinase C modulator, was shown to activate AMPK and, when combined with metformin, reactivate latent HIV-1 in the THP-p89 monocytic cell line in an AMPK-dependent manner [11]. As AMPK is essential for T cell activation, bryostatin-1 combined with JQ-1 increased surface expression of T cell activation markers that reactivate latent HIV-1 to levels comparable with the levels of positive controls [12]. Furthermore, the phorbol ester PMA was also shown to induce differentiation of SH-SY5Y human neuroblastoma cells (similar to retinoic acid and dihydroartemisinin) in an AMPK-dependent manner [1,13,14]. Given that c-Myc (which is inhibited by metformin and dihydroartemisinin) is upregulated during HIV-1 latency and directly activates transcription of the splicing factor SRSF1 (a splicing factor that is deregulated in Hutchinson-Gilford progeria syndrome and also promotes HIV-1 latency), a common mechanism of action potentially links HIV-1 latency and progeria with chemically distinct compounds [7,15].

More remarkable, however, is a study recently published online in the journal PeerJ in March of 2015 demonstrating that artesunate (in doses much lower than those used to treat malaria) mimics certain aspects of caloric restriction when given to mice, triggers mitochondrial biogenesis, and attenuates telomere attrition [16]. Administration of artesunate, a semi-synthetic derivative of artemisinin that is metabolized to dihydroartemisinin, to mice lead to an increase in endothelial nitric oxide synthase (eNOS) with an accompanying increase in the levels of nitric oxide and COX4 (a component of the mitochondrial respiratory chain) that was mirrored by the application of hydrogen peroxide (H2O2), indicating that an oxidative burst is responsible for enhancement of mitochondrial structure and function by artesunate [16].

Indeed, skeletal muscle cells from artesunate treated mice possessed more mitochondrial layers than control cells and the levels of mitochondrial localized SIRT3, the antioxidant enzymes Mn-SOD (primarily localized to the mitochondria), Cu-ZN/SOD, and catalase, as well as glutathione were upregulated in response to both artesunate and H2O2, indicative again of increased mitochondrial functionality [16]. Most importantly, however, artesunate increased the phosphorylation and activation of both AMPK and eNOS (which is activated by AMPK), upregulated SIRT1 levels (increases life- and healthspan), increased PGC1-alpha levels (master transcriptional regulator of mitochondrial biogenesis), and increased the levels of the mitochondrial biomarkers CYT C and MNF2. Increased telomere length was also observed compared to control mice [16].

Collectively, the Oncotarget and PeerJ studies paint an astonishing molecular portrait in which artemisinin’s mechanism of action likely converges with that of metformin, JQ1, bryostatin-1, oligomycin, and many others via a single overriding mechanism that is orchestrated by stress-induced (e.g. free radical generation, increase in intracellular calcium levels, increase in the AMP/ATP ratio) activation of AMPK. Indeed, compounds that generate oxidative stress (H2O2, etc.) are well-characterized activators of AMPK and have also been shown to reactivate latent HIV-1 [17,18].

Enhanced mitochondrial functionality is also essential for T cell activation/latent HIV-1 reactivation as well as in the amelioration or correction of accelerated aging defects in progeria [19,20]. Interestingly, just as artemisinin has been shown to activate AMPK, increase PGC-1 alpha levels, and enhance mitochondrial biogenesis and functionality, methylene blue has also been shown to activate AMPK and correct nuclear morphology and reverse accelerated aging defects in progeria cells by increasing PGC-1a levels and enhancing mitochondrial functionality, implying that artemisinin may also act to slow or reverse aging defects in progeria cells [19,21].

Additionally, all-trans retinoic/retinoic acid signaling also activates AMPK, is essential for T cell activation, promotes the differentiation of SH-SY5Y cells similar to PMA (artemisinin also inhibits SH-SY5Y proliferation), reduces the levels of the splicing factor SRSF1 (a splicing factor that is activated by c-Myc, upregulated in HIV-1 latency, and deregulated in progeria), and decreases progerin mRNA levels in progeria cells, leading to amelioration of accelerated aging defects [see prior post on retinoic acid for references]. Also, the broccoli sprout compound sulforaphane has been shown to activate AMPK, stimulate the immune response, decrease epigenetic factors that promote HIV-1 latency, and reverse accelerated aging symptoms in progeria cells by inducing autophagy of the mutant progerin protein [see prior post on sulforaphane for references]. Interestingly, AMPK activation is essential for the induction of autophagy and artemisinin activates AMPK and upregulates autophagy, giving further indication that artemisinin may correct accelerating aging defects in progeria cells, reactivate latent HIV-1, and potentially ameliorate symptoms of aging in normal humans as well [22,23].

Interestingly, artesunate has been shown to increase the suicidal death of erythrocytes (i.e. eryptosis) via modulation of intracellular calcium or oxidative stress, which may potentially accelerate elimination of infected erythrocytes (i.e. red blood cells) prior to exit by Plasmodium falciparium, thus reducing parasitemia and positively influencing the course of malaria. Surprisingly, in addition to an increase in intracellular calcium or oxidative stress, both of which activate AMPK, several naturally-occurring compounds that are known activators of AMPK also induce suicidal death in erythrocytes, including curcumin (from the spice turmeric), ursolic acid (found in many fruits and herbs including apples, basil, bilberries, etc.), retinoic acid, and sulforaphane [27-29].

Lastly, as an example of the existence of a common mechanism of AMPK activation among distinct compounds, just as artemisinin is not only a powerful anti-malarial drug but also activates AMPK, PGC-1a, Sirtuins, and inhibits telomere attrition, methylene blue, sulforaphane, and retinoic acid have each been shown to have additional anti-malarial effects as evidenced by phagocytosis (i.e. cell “eating” or engulfment) of an erythrocyte infected with Plasmodium falciparium, providing additional indications that artemisinin may prove efficacious in the treatment of progeria and the reversal of HIV-1 latency [24-26]. If further evidence substantiates this interconnectedness, a paradigm shift in the treatment and prevention of diseases once thought incurable will be inevitable.

https://www.linkedin.com/pulse/nobel-prize-winning-drug-artemisinin-shares-common-mechanism-finley?trk=mp-reader-card

References:

1. Wang H, Sharma L, Lu J, Finch P, Fletcher S, Prochownik EV. Structurally diverse c-Myc inhibitors share a common mechanism of action involving ATP depletion. Oncotarget. 2015 Jun 30;6(18):15857-70.


2. http://wwwnc.cdc.gov/eid/article/20/7/ET-2007_article


3. Woodrow CJ, Haynes RK, Krishna S. Artemisinins. Postgrad Med J. 2005 Feb;81(952):71-8.


4. http://www.nobelprize.org/nobel_prizes/medicine/laureates/2015/press.pdf


5. Meshnick SR. Artemisinin: mechanisms of action, resistance and toxicity. Int J Parasitol. 2002 Dec 4;32(13):1655-60.


6. Morton JP, Sansom OJ. MYC-y mice: from tumour initiation to therapeutic targeting of endogenous MYC. Mol Oncol. 2013 Apr;7(2):248-58


7. Jiang G, Espeseth A, Hazuda DJ, Margolis DM. C-Myc and Sp1 contribute to proviral latency by recruiting histone deacetylase 1 to the human immunodeficiency virus type 1 promoter. J Virol 2007;81(20):10914–23.


8. Tan WQ, Chen G, Jia B, Ye M. Artemisinin inhibits neuroblastoma proliferation through activation of AHP-activated protein kinase (AMPK) signaling. Pharmazie. 2014 Jun;69(6):468-72.


9. Darcis G, Kula A, Bouchat S, et al. An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression. PLoS Pathog. 2015 Jul 30;11(7):e1005063.


10. Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog. 2013;9(12):e1003834.


11. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One. 2010 Jun 16;5(6):e11160.


12. Laird GM, Bullen CK, Rosenbloom DI, et al. Ex vivo analysis identifies effective HIV-1 latency–reversing drug combinations. J Clin Invest. 2015 May;125(5):1901-12.


13. Zogovic N, Tovilovic-Kovacevic G, Misirkic-Marjanovic M, et al. Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells. J Neurochem. 2015 Apr;133(2):223-32.


14. Meseguer S, Mudduluru G, Escamilla JM, Allgayer H, Barettino D. MicroRNAs-10a and -10b contribute to retinoic acid-induced differentiation of neuroblastoma cells and target the alternative splicing regulatory factor SFRS1 (SF2/ASF). J Biol Chem. 2011 Feb 11;286(6):4150-64.


15. Das S, Anczuków O, Akerman M, Krainer AR. Oncogenic splicing factor SRSF1 is a critical transcriptional target of MYC. Cell Rep 2012;1(2):110–7.


16. Wang DT, He J, Wu M, Li SM, Gao Q, Zeng QP. Artemisinin mimics calorie restriction to trigger mitochondrial biogenesis and compromise telomere shortening in mice. PeerJ. 2015 Mar 5;3:e822.


17. Auciello FR, Ross FA, Ikematsu N, Hardie DG. Oxidative stress activates AMPK in cultured cells primarily by increasing cellular AMP and/or ADP. FEBS Lett. 2014 Sep 17;588(18):3361-6.


18. Piette J, Legrand-Poels S. HIV-1 reactivation after an oxidative stress mediated by different reactive oxygen species. Chem Biol Interact. 1994 Jun;91(2-3):79-89.


19. Xiong ZM, Choi JY, Wang K, et al. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell. 2015 Dec 14. doi: 10.1111/acel.12434.


20. Ron-Harel N, Sharpe AH, Haigis MC. Mitochondrial metabolism in T cell activation and senescence: a mini-review. Gerontology. 2015;61(2):131-8.


21. Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM. Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue. Redox Biol. 2015 Dec;6:426-35. doi: 10.1016/j.redox.2015.09.004.


22. Egan DF, Shackelford DB, Mihaylova MM, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011 Jan 28;331(6016):456-61.


23. Zhang ZS, Wang J, Shen YB, et al. Dihydroartemisinin increases temozolomide efficacy in glioma cells by inducing autophagy. Oncol Lett. 2015 Jul;10(1):379-383.


24. Ohrt C, Li Q, Obaldia N, Im-Erbsin R, Xie L, Berman J. Efficacy of intravenous methylene blue, intravenous artesunate, and their combination in preclinical models of malaria. Malar J. 2014 Oct 21;13:415.

25. Olagnier D, Lavergne RA, Meunier E, et al. Nrf2, a PPARγ alternative pathway to promote CD36 expression on inflammatory macrophages: implication for malaria. PLoS Pathog. 2011 Sep;7(9):e1002254.


26. Serghides L, Kain KC. Mechanism of protection induced by vitamin A in falciparum malaria. Lancet. 2002 Apr 20;359(9315):1404-6.


27. Alzoubi K, Calabrò S, Bissinger R, Abed M, Faggio C, Lang F. Stimulation of suicidal erythrocyte death by artesunate. Cell Physiol Biochem. 2014;34(6):2232-44.


28. Föller M, Bobbala D, Koka S, Huber SM, Gulbins E, Lang F. Suicide for survival--death of infected erythrocytes as a host mechanism to survive malaria. Cell Physiol Biochem. 2009;24(3-4):133-40.


29. Alzoubi K, Calabrò S, Faggio C, Lang F. Stimulation of suicidal erythrocyte death by sulforaphane. Basic Clin Pharmacol Toxicol. 2015 Mar;116(3):229-35.

Anti-aging drug Rapamycin shares common mechanism of action with Metformin and Artemisinin: Connection between AMPK, Progeria, and HIV-1 reactivation

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

Here's another LinkedIn post (see below) I just published about a recent article that adds even more evidence to the notion that diseases that we think are completely unconnected are actually linked by a common mechanism and many natural products and some drugs are able to effectively treat or eradicate diseases including Hutchinson-Gilford progeria syndrome, HIV-1 latency, malaria, autoimmune disorders, cancer, and even slow the aging process itself by beneficially activating a single p...athway. The drug, called rapamycin, is well known throughout the aging research community and has been shown to extend the lifespan and healthspan of basically every organism it’s been tested in. It's also been shown to inhibit accelerated cellular aging defects in progeria, similar to the broccoli compound sulforaphane, the vitamin A metabolite retinoic acid, and methylene blue. Just like the aforementioned compounds, rapamycin, in this study, was shown to be a potent activator of the master metabolic regulator AMPK, very much similar to the diabetes drug metformin and the anti-malarial and Noble Prize-winning drug artemisinin. Interestingly, rapamycin and methylene blue also inhibits the growth of the parasite that causes malaria. Again, the connections among these diseases are striking, especially considering that most of these compounds exert their beneficial effects by indirectly activating one pathway (in which I theorized about and published in a few of my papers). If this turns out to be true......:-)

Anti-aging drug Rapamycin shares common mechanism of action with Metformin and Artemisinin: Connection between AMPK, Progeria, and HIV-1 reactivation

Just as a recent study demonstrated that the anti-malarial drug artemisinin (see last post) shares a common mechanism of action of AMPK activation with the diabetes drug metformin, a recent study published online in February of 2016 by researchers at the University of Washington in the journal Aging (Albany NY) clearly demonstrated that rapamycin, a drug that is used to prevent rejection in organ transplantation that also extends the median and maximal lifespan of several animal models even when fed late in life (e.g. in male and female mice), significantly activated AMPK in vivo (i.e. in a living organism) in hearts from normal elderly mice, indicating that AMPK activation may indeed link pleiotropic effects on aging, HIV-1, and gene splicing exerted by compounds as chemically distinct as rapamycin, metformin, artemisinin, methylene blue, sulforaphane, retinoic acid, and possibly many others [1-3].

Rapamycin (also known as sirolimus) is a macrolide drug originally derived from the bacterium Streptomyces hygroscopicus found on Easter Island (Chilean island in the southeastern Pacific Ocean) that is often used as an immunosuppressant to prevent rejection in organ transplantation [4].  However, rapamycin  has also demonstrated immune enhancing/stimulating properties (discussed below) as well as anti-proliferative properties within the context of viral challenges and cancer metastasis, respectively, indicating that the beneficial effects of rapamycin are likely disease- and cell-type specific [4,5].  The efficacy of rapamycin administration is often attributed to its inhibition of a cellular protein complex known as mammalian target of rapamycin complex 1 (mTORC1).  In response to environmental or nutritional stimuli (e.g. amino acids, growth factors, cellular/energy stress), mTORC1 plays a critical role in the promotion of cellular growth by inhibiting catabolic processes such as autophagy (intracellular degradation system) and promoting anabolic processes such as mRNA translation and lipid biosynthesis [6 ].

Interestingly, in the Aging (Albany NY) study, the authors found that within just two weeks of treatment of a dose of rapamycin (2.24 mg/kg/day) initially described by the National Institute on Aging Interventions Testing Program (a multi-institutional study designed to test the effectiveness of various compounds to extend lifespan in mice) lead to a reversal of cardiac aging phenotypes in normal aged mice compared to control mice, as evidenced by an improvement in diastolic function, a lower heart weight normalized to body weight (HW/BW), and a reversal of cardiac hypertrophy at all time points.  Expectedly, rapamycin also inhibited the phosphorylation of a downstream target of mTORC1, ribosomal protein S6, a protein thought to be involved in pressure-overload induced cardiac hypertrophy [1].

More importantly, however, was the rapamycin-induced induction of autophagy, mitochondrial biogenesis, and phosphorylation and activation of AMPK in elderly mice hearts. As phosphorylation of ULK1 (a kinase critical for autophagic induction) at serine-757 by mTORC1 inhibits autophagic induction by ULK1, rapamycin transiently increased autophagic flux after 1 week of treatment (returned to control levels at weeks 2 and 10), as evidenced by an increased LC3II/LC3I ratio, an increase in ATG5, and a decrease in ULK1 phosphorylation at serine-757 [1].  Using real-time PCR, the authors also showed that rapamycin treatment transiently increased the expression of the mitochondrial biogenesis marker PCG-1α in the first 2 weeks as well as the protein expression of mitochondrial transcriptional factor A (TFAM) (a transcription factor downstream of PCG-1α) in the 1st and 2nd weeks, with both returning to control levels by week 10 [1].  Because global proteomic analysis revealed a mixture of increased and decreased levels of mitochondrial proteins during autophagic induction and initial mitochondrial biogenesis after the 1st week of treatment, but a general increase in mitochondrial proteins after the 2nd week of treatment (autophagy returned to normal but mitochondrial biogenesis still elevated), rapamycin appears to initially enhance the removal of old mitochondrial components via ULK1-initiated autophagy and replace those components with newly synthesized mitochondria [1].

Most strikingly, however, was the phosphorylation and activation of AMPK in vivo in elderly mice hearts by rapamycin.  Interestingly, the authors demonstrated that rapamycin treatment significantly increased phosphorylation and activation of AMPK during the entire 10 week period at all time points (1, 2, and 10 weeks), with peak levels at week 2 [1].  Because rapamycin-induced phosphorylation and activation of AMPK coincides with and possibly precedes the initial induction of ULK1-mediated autophagy, upregulation of PCG-1α- and TFAM-induced mitochondrial biogenesis, and the reversal of fatty acid oxidation reduction (see study), the inhibition of mTORC1 by rapamycin likely represents a cellular stress response, leading to the activation of AMPK and generating the observed responses in elderly mice hearts.  Indeed, AMPK has been shown in separate studies to phosphorylate and activate ULK1 at serine 317 and serine 777, inhibit mTORC1, increase the levels of PCG-1α and TFAM, enhance mitochondrial biogenesis, and promote fatty acid oxidation [7-10].

The Aging (Albany NY) study provides compelling evidence that the beneficial pleiotropic and lifespan-extending effects of rapamycin are orchestrated by the phosphorylation and activation of AMPK via inhibition of mTORC1.  This mechanism of action is also shared by other chemically distinct compounds that have been shown to activate AMPK, increase mitochondrial biogenesis, and induce autophagy, leading to beneficial effects in diseases as seemingly as disparate as Hutchinson-Gilford progeria syndrome (HGPS), malaria, and HIV-1 latency. 

With respect to HGPS, an accelerated aging disease caused by a dominant negative, gene splicing-mediated mutation leading to the overexpression of the toxic protein progerin, rapamycin has been shown in studies to enhance autophagic degradation of progerin in HGPS fibroblasts, leading to an improvement in nuclear morphology [11,12].  As noted above, AMPK inhibits mTORC1 and phosphorylates and activates ULK1, a critical initiator of autophagic flux.  Because rapamycin also activates AMPK and enhances autophagic flux, autophagic degradation of progerin production in HGPS is likely mediated by rapamycin-induced AMPK activation.  Indeed, sulforaphane, an isothiocyanate derived from cruciferous vegetables, also activates AMPK and has been recently shown to increase autophagic degradation of progerin, decrease levels of ROS, enhance cellular proliferation, improve nuclear morphology, and increase intracellular ATP levels in HGPS fibroblasts, implying a provocative common mechanism of action of AMPK activation between sulforaphane and rapamycin [13,14].  This common mechanism of action appears to extend to the recent findings that both methylene blue and retinoic acid, each shown independently to activate AMPK, also improves nuclear morphology and, in the case of methylene blue, corrects mitochondrial functioning in HGPS fibroblasts [12,15-17].  Interestingly, similar to rapamycin in the Aging (Albany NY) study, methylene blue was shown to not only increase the levels of  PCG-1α in HGPS fibroblasts, but also correct damage to mitochondria in healthy cells [15].  Also, similar to both rapamycin and sulforaphane, methylene blue, in an AMPK-dependent manner, has been shown to induce macroautophagy in vivo in the hippocampus (area of the brain essential for learning and memory) of mice and in mouse hippocampal HT22 cells [18].

Additionally, methylene blue increased the levels of LMNA mRNA (gene involved in the pathology of HGPS) and retinoic acid induced a down regulation in progerin mRNA transcripts, implying that methylene blue and retinoic acid beneficially altered the splicing of the LMNA gene [12,15].  Moreover, metformin, a prototypical AMPK activator that has been shown to exert beneficial effects in numerous disease states as well as improve lifespan and healthspan, has been shown to correct alternative splicing defects in cells derived from myotonic dystrophy type I patients in an AMPK-dependent manner and beneficially alter splicing of the insulin receptor gene in diabetic patients who were taking metformin but who did not have DM1, in typical therapeutic dosages [19].

The interplay of alternative splicing correction and autophagic induction by AMPK activators is also substantiated by the actions of the splicing-associated factor p32.  p32, often noted as a molecular chaperone, is essential for mitochondrial bioenergetics and oxidative phosphorylation, both of which are enhanced by AMPK activation [20].  A recent study also demonstrated that p32 is essential for the induction of autophagic flux by acting as a chaperone for the autophagic initiator ULK1 [21].  Indeed, silencing or depletion of p32 significantly inhibited autophagic flux induced by both rapamycin and starvation, implying a provocative connection between p32, ULK1, AMPK, and rapamycin [21].  Interestingly, SRSF1 (also known as ASF/SF2), a splicing factor that has been implicated in the aberrant splicing of the LMNA gene in HGPS as well as in faulty splicing of genes in normal humans that lead to endothelial senescence, activates mTORC1 but is inhibited by p32 [22-25].  UV radiation has also been shown, in separate studies, to upregulate SRSF1 and induce the production of progerin, while mutation of splice sites in genes have been shown to increase SRSF1 recruitment [26-28].  Strikingly, increased SRSF1 activity has been shown to prevent the reactivation of latent HIV-1 reservoirs while p32 activity has been shown to be increased in latent HIV-1 reactivation, thus inhibiting SRSF1 activity and providing an astounding parallel between HGPS, autophagy, normal aging, and HIV-1 latency [29] (see below).

Interestingly, rapamycin also shares a common mechanism of action of AMPK activation with artemisinin, a powerful anti-malarial drug that has been in use for more than 2000 years. Dihydroartemisinin (a metabolite of an artemisinin derivative) was shown to induce cancer cell differentiation via activation of AMPK, a result that was mimicked by metformin administration in the same study [3].  Dihydroartemisinin has also been shown to inhibit phosphorylation of mTORC1 in rhabdomyosarcoma cells as well as induce autophagy in glioma cells in vitro and in vivo [30,31].   Additionally, artesunate (artemisinin derivative metabolized to dihydroartemisinin) mimicked certain aspects of caloric restriction when given to mice (increased SIRT1, MnSOD, CuZnSOD catalase, glutathione), trigged mitochondrial biogenesis (increased AMPK activation, PCG-1α, SIRT3, CYTC and MNF2), and attenuated telomere attrition [32]. Because the AMPK activators methylene blue, sulforaphane, and retinoic acid have also been shown to be effective in the treatment of malaria (see prior Artemisinin post for references), rapamycin would also be expected to exert at least partial anti-malarial effects. Indeed, in a recent in vitro study, both rapamycin and everolimus (mTOR inhibitor) were associated with complete inhibition of Plasmodium falciparum growth while other studies demonstrated that inhibition of mTOC1 with rapamycin increases parasite clearance in the spleen, reduces parasite accumulation in peripheral tissues (e.g. brain), and prevents experimental cerebral malaria, indicating a common mechanism of action between artemisinin and rapamycin [33,34].

Lastly, activation of AMPK in normal elderly mice demonstrated in the Aging (Albany NY) study implicates the potential utility of rapamycin administration to enhance reactivation of latent HIV-1 reservoirs, facilitating its detection and destruction by the immune system. Interestingly, studies have shown that T cell activation (and thus latent HIV-1 reactivation) is critically dependent on AMPK activation and mitochondrial ATP production, as knockdown of a subunit of AMPK (AMPKα1) leads to a significant reduction in mitochondrial bioenergetics and an inhibition of primary T cell responses in vivo to viral and bacterial infections [35-37].  Additionally, autophagy has also been shown to be essential for and upregulated on T cell activation, as evidenced by a decrease in ATP generation, defective IL-2 and IFN-γ production, and reduced proliferation in Th cells after deletion of Atg7 (a gene critical for macroautophagy) specifically in T cells [38].  Also, the activity of the splicing factor SRSF1, which activates mTOC1, is upregulated during HIV-1 latency, causing excessive HIV-1 mRNA gene splicing and thus reducing the unspliced mRNA necessary for reactivation of the virus [29]. However, viral reactivation was indeed shown to reduce SRSF1 activity and increase the activity of p32, an endogenous inhibitor of SRSF1 that also increases mitochondrial functionality and oxidative phosphorylation [20,29].  Interestingly, rapamycin has also been shown to antagonize the effects of SRSF1 in several independent studies [24,39].

Based on these studies, a common mechanism of AMPK activation as a prerequisite for efficient latent HIV-1 reactivation is clear. Although rapamycin acts as an immunosuppressant in the context of organ transplantation, several studies have shown that both rapamycin and metformin enhance the immune response to viral challenges in both humans and mice, indicating a temporally appropriate facilitation of CD4+ and CD8+ T cell activation and formation of memory CD8+ T cells, thus enhancing the detection and destruction of viruses, intracellular pathogens, and cancer cells by functioning as immunotherapeutics [40-43].  Interestingly, metformin may also act as an immunosuppressant in autoimmune-specific disease contexts, but when combined with bryostatin (a PKC activator that also activates AMPK), enhances the reactivation of cells latently infected with HIV-1 [44,45]. Interestingly, a recent study also demonstrated that when combined with bryostatin, the compound JQ1 fully reactivated latently infected HIV-1 viral reservoirs to the level of positive controls [46].  Although the compound JQ1 is used in latency reversal studies to reactivate latent HIV-1, JQ1 is also being studied as an anti-cancer agent due to its indirect inhibition of the oncoprotein c-Myc.  However, a recent study has demonstrated that JQ1, dihydroartemisinin (metabolite of an artemisinin derivative), and other compounds labeled as c-Myc inhibitors, share a common mechanism of action that involves the induction of cellular stress, followed by the activation of AMPK and subsequent differentiation and/or apoptosis of cancer cells [3].

Again, data from the Aging (Albany NY) combined with the foregoing studies paints an astonishing picture of the interconnectedness of diseases as seemingly disparate as HGPS, HIV-1 latency, normal aging, and malaria. Even more shocking is that AMPK, which is activated by rapamycin, metformin, artemisinin, methylene blue, sulforaphane, and retinoic acid, is critical for T cell activation, enhances ULK1-initated autophagy, beneficially alters gene splicing, inhibits mTORC1, stimulates mitochondrial biogenesis, and improves lifespan and healthspan in several animal model organisms.  If further studies continue to affirm AMPK activation by chemically distinct compounds as a central mechanism of action for the amelioration or eradication of disease, a paradigm shift in the practice of medicine must ensue.

https://www.linkedin.com/pulse/anti-aging-drug-rapamycin-shares-common-mechanism-action-finley?trk=mp-reader-card

References:

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17. Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM. Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue. Redox Biol. 2015 Dec;6:426-35.
18. Xie L, Li W, Winters A, Yuan F, Jin K, Yang S. Methylene blue induces macroautophagy through 5' adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation. Front Cell Neurosci. 2013 May 3;7:56.
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40. Keating R, Hertz T, Wehenkel M, et al. The kinase mTOR modulates the antibody response to provide cross-protective immunity to lethal infection with influenza virus. Nat Immunol. 2013 Dec;14(12):1266-76.
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46. Darcis G, Kula A, Bouchat S, et al. An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression. PLoS Pathog. 2015 Jul 30;11(7):e1005063.

A Common Mechanism linking AMPK, Progeria, Aging, & HIV-1 reactivation: A picture is worth a thousand words

Here's a quick post I recently put up on LinkedIn (see below) along with a picture I drafted that helps bring to life the interconnectedness of normal aging, Progeria, and HIV-1 reactivation. If you notice, a lot of compounds are converging on the blue circle called AMPK. It's known as the master metabolic regulator and its basically present in every cell in your body, from head to toe. AMPK is significantly activated by activities and compounds that are known to extend your lifespan as well as your healthspan (i.e. lifespan without disease), including exercise and an enormous array of plant-based compounds including metformin (from Galega), sulforaphane (from broccoli sprouts), and resveratrol (from grapes). The activation of AMPK is necessary to activate your T cells in order to mount an effective immune response to viruses, bacteria, and cancer cells. The activation of AMPK has also been shown to beneficially alter gene splicing in inherited diseases as well as in normal humans (metformin beneficially alters gene splicing in diabetics). Interestingly, this picture shows that AMPK can activate T cells infected with HIV-1, thus pushing the virus out of its hiding spot and making it easier to detect and be destroyed by the immune system. AMPK activation can also activate your cytotoxic T cells to help destroy the HIV-1 virus and AMPK also alters the same gene splicing factors that characterize HIV-1 reactivation, normal aging, and Progeria. What other disease states could be ameliorated or even reversed by AMPK activators? Could Alzheimer’s, Parkinson’s, cancer, autoimmune disorders, heart disease, etc. all be connected by a centralized pathway? More to come;-)

A Common Mechanism linking AMPK, Progeria, Aging, & HIV-1 reactivation: A picture is worth a thousand words

Here’s a graphic view that gives a pretty good layout of the overall interconnectedness of Hutchinson-Gilford progeria syndrome (HGPS), HIV-1 latency, and normal aging (pointed arrow = activation; blocked/blunted arrow = inhibition).  What follows is sort of a condensed explanation of how these diseases are connected and how one compound that looks completely different from another (say, methylene blue and rapamycin) can exert the same or similar effects in the same disease states by primarily activating a particular pathway. To keep this post relatively short, references can be found by accessing the links below to separate posts I’ve done previously on each compound.As you can see from the figure, the activation of AMPK is the focal point that is essential for efficient T cell activation that ultimately reactivates the latent HIV-1 virus (leading to either detection and destruction by the immune system or a self-destruct mechanism by the T cell itself) and the beneficial alteration of gene splicing in the LMNA gene, the gene that’s mutated in HGPS.

As the figure shows, ASF/SF2 (also known as SRSF1) is involved in both aberrant gene splicing in HGPS and gene splicing in normal T cells and in T cells latently infected with HIV-1. Indeed, excessive splicing of the latent HIV-1 genome by ASF/SF2 prevents the reactivation of the virus. ASF/SF2 also has been shown to both promote aberrant gene splicing of LMNA via excessive use of a cryptic splice site in the LMNA gene in HGPS and promote aberrant splicing of vascular genes in normal humans, promoting endothelial cell senescence (i.e. loss in ability of cells to divide).

Interestingly, normal humans also possess this same cryptic splice site in their LMNA genes and produce the same toxic protein progerin (the protein that is responsible for symptoms of HGPS) in small amounts that increase with age, whereas HGPS patients produce extremely large amounts of the protein early on in life. Not surprisingly, UV radiation (which promotes accelerating aging in various cells) increases the activity of ASF/SF2 in normal cells and increases the production of progerin in HGPS cells, indicating that ASF/SF2 promotes the use of the LMNA cryptic splice site in both normal humans and HGPS patients, generating symptoms of aging and accelerated aging.

Comparatively, when T cells that are latently (i.e. dormant) infected with HIV-1 are reactivated with various compounds, the levels of ASF/SF2 decrease, preventing excessive splicing of the HIV-1 genome and thus potentiating elimination of the virus by the immune system, possibly leading to a cure.  When normal T cells are activated, the splicing activities of ASF/SF2 are also decreased, indicating that downregulation of ASF/SF2 is necessary for T cell activation generally.

Incidentally, studies have shown that selective activation of only T cells latently infected with HIV-1 may be the only successful way to reactivate latent HIV-1.  Interestingly, the splicing-associated factor p32 is an endogenous and natural inhibitor of ASF/SF2.  As such, p32 activity is indeed upregulated on latent HIV-1 reactivation that is induced by T cell activation.  Interestingly, p32 is also absolutely critical for mitochondrial biogenesis and functionality, as it serves as a chaperone necessary for the production of mitochondrial proteins that are indispensable for oxidative phosphorylation.  Mitochondria are known as the “powerhouses” of the cell and are a vital source of energy in the form of a molecule known as ATP that drives countless cellular processes in nearly every cell in the body.

Indeed, mitochondria are critical for T cell activation and congregate around the connection that forms between a T cell and a cell that presents a piece of a foreign invader in order to activate the T cell.  Similarly, ATP levels, mitochondrial functionality, and mitochondrial biogenesis are significantly decreased in HGPS cells, providing a provocative indication that increased p32 activity may inhibit excessive splicing activities of ASF/SF2 and enhance mitochondrial functionality, thus driving efficient latent HIV-1 reactivation.  Moreover, increased p32 activity may inhibit excessive splicing activities of ASF/SF2 in HGPS and in normal cells as well as increase mitochondrial biogenesis, thus decreasing the use of the LMNA cryptic splice site and thus ameliorating symptoms of normal and accelerated aging.

Interestingly, p32 has also been shown to be essential for a process known as autophagy, a process in which a cell degrades and recycles worn or dysfunctional intracellular components (i.e. organelles including mitochondria). Again, the similarities are striking between HGPS and HIV-1 latency.  On T cell activation, autophagy is significantly increased and inhibition of autophagy prevents efficient T cell activation.  The activation of autophagy by rapamycin and sulforaphane in HGPS cells also leads to the elimination of the toxic protein progerin by autophagic degradation.  Perhaps most tellingly, however, is that the induction of autophagy by rapamycin has been shown to be dependent on the stabilization of the autophagic inducer ULK1 by the splicing factor p32, the same splicing factor that is indispensable for mitochondrial function and inhibits the excessive splicing activities of ASF/SF2, both of which have been shown to be associated with HIV-1 latency and HGPS.

Mitochondrial biogenesis, autophagy, T cell activation, and gene splicing are all controlled and influenced by cellular metabolism, which implicates the master metabolic regulator AMPK as a key orchestrator in the connection between HGPS, aging, and HIV-1 latency.  Indeed, AMPK activation is critical for T cell activation (inhibition of AMPK leads to decreased T cell responses to viral and bacterial challenges), significantly increases mitochondrial biogenesis, beneficially alters gene splicing, and promotes the induction of autophagy by activating ULK1 (the same protein that is stabilized by p32, characterizing autophagy induced by rapamycin).

As such, it would be expected that chemically distinct compounds that have been shown to both enhance the activation of T cells and correct cellular defects associated with accelerated aging in HGPS would activate AMPK. Indeed, the compounds methylene blue, retinoic acid, rapamycin, and sulforaphane have each been shown to enhance T cell activation and/or latent HIV-1 reactivation, reverse cellular aging defects in HGPS cells, and activate AMPK.  As metformin (diabetes drug), a prototypical AMPK activator, has been shown to improve lifespan and healthspan, beneficially alter gene splicing in the disease Myotonic Dystrophy type I, and enhance the reactivation of latent HIV-1, metformin will also likely drive mitochondrial biogenesis, beneficially alter gene splicing, and improve accelerating aging defects in HGPS.  Curiously, although the Noble Prize-winning drug artemisinin is more commonly known as an anti-malarial drug, it has recently been shown (along with another compound, JQ1, that reactivates latent HIV-1) to activate AMPK in cancer cells (killing them or inducing differentiation), activate AMPK and induce mitochondrial biogenesis in mice (mimicking caloric restriction), induce autophagy, and enhance T cell functionality, indicating that artemisinin will also likely reverse cellular aging defects in HGPS and in normal human cells as well as enhance latent HIV-1 reactivation.

Most importantly, however, was the recent finding that rapamycin, a compound derived from a bacterium, potently activates AMPK in normal elderly mice.  The importance of this finding is instrumental, considering that rapamycin has been found to increase maximal lifespan in vivo in normal mice even when administered late in life.  Although it is widely thought that the effects of rapamycin stem from the inhibition of a protein called mTOR (mTOR activation drives anabolic processes in the cell and its over-activation is implicated in many disease states), the recent finding that rapamycin potently activated AMPK in vivo in normal elderly mice during the entire 10 week study provides strong and convincing evidence that rapamycin, very much similar to other compounds, indirectly activates AMPK by inhibiting mTOR.  Indeed, in that study, autophagy was increased in the first week and mitochondrial biogenesis was increased in the first and second weeks but both fell back to control levels by the 10th week of the study.  However, AMPK was significantly activated for the entire duration of the study (all 10 weeks), indicating that AMPK activation preceded and induced the activation of autophagy and mitochondrial biogenesis.

Indeed, as the induction of autophagy and mitochondrial biogenesis leads to the correction of accelerated cellular aging defects in HGPS cells and efficient T cell activation-induced latent HIV-1 reactivation, and because p32 is critical for mitochondrial function and the induction of ULK1-mediated autophagy by rapamycin, AMPK activation by rapamycin likely represents (similar to the other aforementioned compounds) an “indirect yet primary mechanism of action” of rapamycin. The potential clinical implications of AMPK activation representing a common mechanism of action for compounds as seemingly as distinct as artemisinin, rapamycin, metformin, resveratrol, sulforaphane, retinoic acid, methylene blue, and potentially many others is beyond mind-boggling and may represent a truly novel insight into the interconnectedness of disease pathology and cellular metabolism.

https://www.linkedin.com/pulse/common-mechanism-linking-ampk-progeria-aging-hiv-1-latency-finley


References:

1. Anti-aging drug Rapamycin shares common mechanism of action with Metformin and Artemisinin: Connection between AMPK, Progeria, and HIV-1 reactivation, https://www.linkedin.com/pulse/anti-aging-drug-rapamycin-shares-common-mechanism-action-finley?trk=prof-post
2. Nobel Prize Winning drug Artemisinin shares common mechanism of action with AMPK activator Metformin: Connection between aging and HIV-1 reactivation, https://www.linkedin.com/pulse/nobel-prize-winning-drug-artemisinin-shares-common-mechanism-finley?trk=prof-post
3. AMPK activator Metformin alters gene splicing in humans: Potential connection between AMPK, accelerated aging, and HIV-1 reactivation, https://www.linkedin.com/pulse/ampk-activator-metformin-alters-gene-splicing-humans-potential?trk=prof-post
4. Broccoli Sprout compound reverses aging defects in Progeria cells: Potential connection between AMPK, accelerated aging, and HIV-1 reactivation, https://www.linkedin.com/pulse/broccoli-sprout-compound-reverses-aging-defects-progeria-finley?trk=mp-reader-card
5. Vitamin A metabolite reverses aging defects in Progeria cells: Potential connection between AMPK, accelerated aging, and HIV-1 reactivation, https://www.linkedin.com/pulse/vitamin-metabolite-reverses-aging-defects-progeria-cells-finley?trk=mp-reader-card
6. Symptoms of Progeria reversed by safe, inexpensive compound: Connection between AMPK, accelerated aging, and HIV-1 reactivation, https://www.linkedin.com/pulse/symptoms-progeria-reversed-safe-inexpensive-compound-between-finley?trk=mp-reader-card
7. Common Mechanism Linking HIV-1 and Progeria, https://www.linkedin.com/pulse/common-mechanism-linking-hiv-1-progeria-jahahreeh-finley?trk=mp-reader-card
8.  Potential Correction of Gene Splicing in Progeria via Naturally Occurring Compounds, https://www.linkedin.com/pulse/potential-correction-gene-splicing-progeria-via-naturally-finley?trk=mp-reader-card

AMPK activator Metformin alters gene splicing in humans: Potential connection between AMPK, accelerated aging, and HIV-1 reactivation

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

Another recent post of mine (see below) from LinkedIn on a study that is definitely a HUGE plus in making my hypothesis on treating/reversing progeria that much more real. Metformin, a widely-prescribed anti-diabetic drug derived from the plant Galega officinalis (a plant that has been used to treat symptoms of diabetes mellitus dating back to the Middle Ages) that has been shown to increase the lifespan and healthspan of several organisms, corrects gene splicing in cells from patients with the autosomal-dominant muscle wasting disease myotonic dystrophy type I (DM1). Progeria is an autosomal-dominant disease like DM1 and both are characterized by faulty gene splicing. What's super interesting about this study is that metformin also beneficially altered gene splicing in human diabetic patients taking metformin who didn't have DM1. Just as methylene blue, retinoic acid (vitamin A metabolite), and sulforaphane (broccoli sprout compound) all activate AMPK and reverse aging defects in progeria cells, I think I was the first one to predict via my publications that metformin (and other AMPK activators) may correct gene splicing and reverse aging defects in progeria cells by activating AMPK. Because this study shows that metformin can do just that (alter gene splicing) in the disease DM1 and in normal diabetics and because metformin also enhances the reactivation of dormant HIV-1 infected T cells, it seems that maybe my hypothesis of disease interconnectedness may turn out to be true. IF IT DOES......................:-)

AMPK activator Metformin alters gene splicing in humans: Potential connection between AMPK, accelerated aging, and HIV-1 reactivation

A recent study published online in the Journal Molecular Therapy Nucleic Acids in November of 2015 provided startling evidence that metformin, a widely-prescribed anti-diabetic drug derived from the plant Galega officinalis (a plant that has been used to treat symptoms of diabetes mellitus dating back to the Middle Ages) that has been shown to increase the lifespan and healthspan of several organisms, corrects alternative splicing defects in primary myoblasts derived from patients with myotonic dystrophy type I (DM1) as well as in derivatives of embryonic stem cells that carry the DM1 mutation in an AMPK-dependent manner.  Strikingly, metformin also beneficially altered the splicing of the insulin receptor gene in diabetic patients who were taking metformin but who did not have DM1, in typical therapeutic dosages [1].
DM1, a genetic disorder inherited in an autosomal dominant fashion, is characterized by muscle weakness and wasting, cardiac conduction abnormalities, and myotonia (prolonged muscle contraction) [2].  According to the NIH, “ ‘Autosomal’ means that the gene in question is located on one of the numbered, or non-sex, chromosomes. ‘Dominant’ means that a single copy of the disease-associated mutation is enough to cause the disease.” [3].  DM1 is known as a trinucelotide repeat disorder, a condition in which there is an expansion or repeat of two or three nucleotides in a particular gene that can lead to manifestation of disease if the sequence of repeats extends beyond a certain number.  For example, the affected gene in DM1, called DMPK, codes for a protein that is primarily expressed in skeletal muscle.  A repeat expansion consisting of the cytosine-thymine-guanine (CTG) triplet between 5 and 37 repeats is considered normal while repeats exceeding 50 nearly always results in symptomatic disease [4].

Interestingly, DM1 is also known as a model of spliceopathy (i.e. misregulated alternative splicing), and is characterized by defects in the alternative RNA splicing machinery, generating alternatively spliced isoforms that contribute to the severity of the disease, including insulin resistance [1]. Using mesodermal precursor cells (MPCs) that were derived from embryonic stem cells from an embryo that was a DM1-gene carrier, the authors initially showed that metformin corrected splicing defects in the insulin receptor (INSR) gene by increasing the proportion of INSR with exon 11 inclusion in both DM1 MPCs and in wild-type MPCs, indicating that metformin has the ability to positively regulate alternative splicing in normal cells as well as in mutated cells [1].  Metformin also corrected alternative splicing defects in the DM1-associated genes TNNT2 and Cln1 by lowering the percentage of exon 5 inclusion in TNNT2 and lowering the percentage of exon 7a inclusion in Clcn1 to levels similar to wild-type MPCs [1].  Additionally, using deep RNA sequencing, the authors also demonstrated that metformin altered gene expression and alternative splicing of a large number of genes in DM1 MPCs, with metformin affecting the expression of a total of 1,171 genes and regulating eighty-nine splicing events and 416 exons above 10% [1].

Metformin’s mechanism of action has been shown to be dependent on its ability to activate the master metabolic regulator AMPK by increasing the intracellular AMP/ATP ratio, thus inducing a compensatory cellular stress response [5]. Indeed, the authors showed that metformin specifically inhibited complex I of the mitochondrial electron transport chain in DM1 MPCs, leading to an increase in the AMP/ATP ratio [1]. Additionally, by using AICAR, a pharmacological AMPK activator widely used in studies to asses the effects of AMPK activation, the authors showed that treatment of DM1 MPCs with AICAR altered exon inclusion on five of the splicing events most regulated by metformin (MDM4 exon 7 [decreased exon inclusion], GPCPD1 exon 5 [decreased exon inclusion], CCNL2 exon 7 [increased exon inclusion], RAGE exon 3 [decreased exon inclusion], and ZFAND1 exon 3 [decreased exon inclusion]) [1].  Such data provides compelling evidence that metformin alters alternative splicing via the activation of AMPK.

The authors however extended the analysis one step further, testing the effects of metformin treatment on alternative splicing events associated with muscle strength by using myoblasts from two different patients diagnosed with DM1 and two healthy patients. Interestingly, metformin beneficially altered the splicing of 6 genes (INSR exon 11, TNNT2 exon 5, ATP2A1 exon 22, DMD exon 71, DMD exon 78, and KIF13A exon 32), as evidenced by a shift in the isoform ratio towards control values [1].

Perhaps most importantly, however, was the verification of metformin’s effect on alternative splicing in vivo via the use of human subjects.  A clinical trial was performed wherein 15 diabetic patients, who did not have DM1 but were currently taking metformin for more than a year (between 2.1 and 3 g/day), were recruited and the effects of metformin on alternative splicing were conducted on peripheral blood lymphocytes (PBLs).  Strikingly, using quantitative PCR, the authors showed that metformin triggered the inclusion of INSR exon 11 in a clinical setting. However, when metformin was temporarily replaced by another diabetic drug that was shown in other experiments not to effect INSR exon 11 alternative splicing, INSR exon 11 inclusion was decreased.  However, when the patients were placed back on metformin, INSR exon 11 inclusion increased, indicating that metformin also alters alternative splicing in vivo in human subjects.  Curiously, metformin also decreased FAS (CD95) exon 6 exclusion in PBLs from treated patients, generating a shift from the anti-apoptotic to the pro-apoptotic protein isoform [1].

The results from this study represents an incredible example of how compounds as chemically distinct as metformin, retinoic acid, sulforaphane, and likely many others share a common indirect mechanism of action of AMPK activation, leading to correction of gene splicing in genetic disorders characterized by autosomal dominance (e.g. progeria and DM1), reactivation of latent viral infections to facilitate immune system eradication (e.g. HIV-1 latency), and regulation of cellular processes that are almost universally associated with both normal and accelerated aging (e.g. mitochondrial dysfunction and telomere erosion).

Indeed, methylene blue has recently been shown to reverse accelerated aging defects, correct mitochondrial function, increase PGC1a levels, and increase lamin A mRNA (evidence of alteration of alternative splicing) in fibroblasts derived from patients with Hutchinson-Gilford progeria syndrome, an accelerated aging disease caused by aberrant alternative splicing of the LMNA gene [6].  Methylene blue has also shown been shown to delay cellular senescence in lung cells via AMPK activation, decrease telomere erosion, and reactivate latent HIV-1 via photosensitization (see prior post for references). Similarly, metformin has also been shown to enhance mitochondrial function through enhancing an AMPK-mediated PGC1a upregulation, correct gene splicing in an AMPK-dependent manner in DM1, increase lifespan and healthspan in several organisms, and synergize with bryostatin to enhance the reactivation of latent HIV-1 [1,7,8].  Interestingly, bryostatin, a protein kinase C activator, was also shown to phosphorylate and activate AMPK, implying that bryostatin is an indirect AMPK activator as well [8].

This common theme of AMPK activation also extends to sulforaphane, the broccoli sprout compound that was recently found to also reverse aging defects in progeria cells [9].  Sulforaphane was shown to enhance the autophagic degradation of progerin (mutant protein) in cells derived from progeria patients, increase the proportions of lamin A (normal protein) yet decrease those of progerin in progeria cells, stimulate the immune response against cancer cells, inhibit scrapie prion protein accumulation by activating AMPK, and inhibit two epigenetic factors (EZH2 and SUV39H1) that keep HIV-1 dormant in infected T cells that are also dysregulated in progeria cells (see prior post for references).  As AMPK is essential for T cell activation, metformin has been shown to enhance latent HIV-1 reactivation, increase the formation of cytotoxic memory CD8+  T cells (targets and kills viruses and cancer cells), induce autophagy by activating the AMPK-ULK1 pathway, and modulate the repressive epigenetic factors EZH2 and SUV39H1 [8,10-13].

The commonality of AMPK activation between metformin and sulforaphane also extends to retinoic acid (and/or its derivatives), a vitamin A metabolite that activates AMPK and was recently shown to reverse aging defects in progeria cells, decrease progerin mRNA in progeria cells (evidence of alteration of alternative splicing), regulate the same gene splicing factors that play a role in both HGPS and latent HIV-1 reactivation, enhance T cell activation, and act synergistically with other agents to reactivate latent HIV-1 [14, see prior post for other references].  Again, AMPK activation is likely playing a critical role in each of these processes, as AMPK activation beneficially alters alternative splicing and is essential for T cell activation and thus latent HIV-1 reactivation.

Furthermore, in the 2015 Molecular Therapy Nucleic Acids study referenced above, metformin decreased FAS (CD95) exon 6 exclusion in PBLs from treated patients, generating a shift from the anti- to the pro-apoptotic isoform of the protein.  T cell activation and latent HIV-1 reactivation induced by the positive control PHA also leads to an increase in the pro-apoptotic isoform of the protein (i.e. FAS exon 6 inclusion), providing further evidence that metformin indeed alters alternative splicing and likely facilitates latent HIV-1 reactivation [15].

Cumulatively, the results from these studies dramatically indicate that compounds such as methylene blue, sulforaphane, and retinoic that have been shown to not only correct aging defects in progeria cells but enhance T cell activation or latent HIV-1 reactivation do so via a common mechanism of AMPK activation that likely also involves correction of alternative splicing.  Although no studies have been conducted to date, the application of metformin to progeria cells will likely beneficially alter alternative splicing, enhance mitochondrial function, and reverse accelerated aging defects in progeria cells, in addition to enhancing latent HIV-1 reactivation.  That diseases such as progeria and the reversal of HIV-1 latency may be connected by a common pathway and that the activation of this pathway (via AMPK) by chemically distinct compounds represents a common mechanism of action would be unprecedented and undoubtedly warrant a reevaluation of disease interconnectedness.  

https://www.linkedin.com/pulse/ampk-activator-metformin-alters-gene-splicing-humans-potential?trk=mp-reader-card

References:

1. Laustriat D, Gide J, Barrault L, et al. In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin. Mol Ther Nucleic Acids. 2015 Nov 3;4:e262.
2. http://www.ncbi.nlm.nih.gov/books/NBK1165/
3. http://ghr.nlm.nih.gov/glossary=autosomaldominant
4. Turner C, Hilton-Jones D. The myotonic dystrophies: diagnosis and management. J Neurol Neurosurg Psychiatry. 2010 Apr;81(4):358-67.
5. Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol. 2011 Sep 2;13(9):1016-23.
6. Xiong ZM, Choi JY, Wang K, et al. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell. 2015 Dec 14. doi: 10.1111/acel.12434.
7. Salminen A, Kaarniranta K. AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network. Ageing Res Rev. 2012 Apr;11(2):230-41.
8. Mehla R, Bivalkar-Mehla S, Zhang R, et al. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One. 2010 Jun 16;5(6):e11160.  
9. Gabriel D, Roedl D, Gordon LB, Djabali K. Sulforaphane enhances progerin clearance in Hutchinson-Gilford progeria fibroblasts. Aging Cell. 2015 Feb;14(1):78-91.
10. Pearce EL, Walsh MC, Cejas PJ, et al. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature. 2009 Jul 2;460(7251):103-7.
11. Egan DF, Shackelford DB, Mihaylova MM, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011 Jan 28;331(6016):456-61.
12. Bao B, Wang Z, Ali S, et al. Metformin inhibits cell proliferation, migration and invasion by attenuating CSC function mediated by deregulating miRNAs in pancreatic cancer cells. Cancer Prev Res (Phila). 2012 Mar;5(3):355-64.
13. Xin Li, Juncheng Wei, Satoko Matsumura, Yuqi Guo, Huawei Yuan. Epigenetic alterations in the multifaceted inhibitory effects of metformin on castration resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2014-4033.
14. Pellegrini C, Columbaro M, Capanni C, et al. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype. Oncotarget. 2015 Oct 6;6(30):29914-28.
15. C Liu, J Cheng, and J D Mountz. Differential expression of human Fas mRNA species upon peripheral blood mononuclear cell activation. Biochem J. 1995 Sep 15; 310(Pt 3): 957–963.

Broccoli Sprout compound reverses aging defects in Progeria cells: Potential connection between AMPK, accelerated aging, and HIV-1 reactivation

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

Here's another REALLY interesting recent post of mine from LinkedIn (see below) : Sulforaphane, a compound that is found heavily concentrated in cruciferous vegetables, particularly broccoli sprouts, actually reversed many of the symptoms of accelerated aging in cells taken from children diagnosed with the accelerated aging disorder Hutchinson-Gilford progeria syndrome. Interestingly, sulforaphane corrected the shape of the nucleus, increased the growth and proliferation of progeria cells, decreased the levels of the mutant progerin protein, and also increased the levels of ATP produced by the cells. What's really jaw-dropping is that sulforaphane also did the same thing to normal cells (corrected nuclear shape, increased growth and proliferation, decreased mutant progerin levels). Because normal people (you and I) produce the same mutant protein (progerin) that progeria kids produce that causes accelerated aging, just at lower levels that increase as we age, sulforaphane will likely have a positive effect on slowing the aging process in normal humans. And just as methylene blue and retinoic acid reactivate dormant HIV-1 to facilitate removal by the immune system, sulforaphane also boosts the immune response and inhibits factors that keep HIV-1 dormant. Each one of these compounds are likely acting via a common mechanism of AMPK activation that links diseases that most people (including experts) think are completely unrelated (e.g. progeria, HIV-1, fertility, cancer, etc). If this turns out to be true............:-)

Broccoli Sprout compound reverses aging defects in Progeria cells: Potential connection between AMPK, accelerated aging, and HIV-1 reactivation 

In line with recent findings (see prior posts) that a vitamin A metabolite (or its derivatives) and the safe, inexpensive compound methylene blue significantly ameliorated or reversed accelerated aging defects in cells derived from patients diagnosed with Hutchinson-Gilford progeria syndrome (HGPS), a recent study published in the Journal Aging Cell in February of 2015 strikingly demonstrated that sulforaphane, a compound found in broccoli sprouts and other cruciferous vegetables, enhances the disposal of the mutant protein progerin (the protein responsible for accelerated aging) in HGPS cells, leading to a reduction in DNA damage and improved nuclear morphology (i.e. shape) in HGPS fibroblasts [1].

HGPS is a rare genetic disorder caused by the faulty splicing of a gene called the LMNA gene, producing large amounts of a mutant protein known as progerin.  Progerin accumulation (which also accumulates gradually in normal humans) at a very early age in HGPS patients leads to distortions in the shape of the nucleus, aberrations in the signaling mechanisms that occur in the nucleus, as well as mitochondrial dysfunction (the primary energy-producing organelle of the cell), leading to characteristic symptoms of accelerating aging such as thinning of the hair, wrinkling of the skin, and eventual cardiovascular disease.
Interestingly, in this study researchers showed that in HGPS fibroblasts, both autophagy and proteasome activity, two cellular processes that are essential for the removal of damaged or mutated proteins, were significantly decreased [1]. Surprisingly, sulforaphane treatment increased autophagy and proteasome activity in both HGPS cells and in normal cells, leading to a decrease/clearance of mutant progerin in HGPS cells by nearly 40% by day 9 compared to mock-treated control cells.

Moreover, sulforaphane also significantly increased the growth rate of both HGPS and normal cells by day 3 and ameliorated proliferation (i.e. increase in cell number) defects by day 9 in HGPS cells [1]. Additionally, sulforaphane treatment decreased the levels of reactive oxygen species (reactive molecules that can damage DNA, lipids, and proteins) that were elevated in HGPS cells and also increased intracellular ATP levels (indispensable energy molecule produced by mitochondria) in both normal and HGPS cells, implying a similar mechanism of action in HGPS cells as methylene blue (e.g. enhancing mitochondrial biogenesis/functionality) [1,2].

Sulforaphane administration also improved nuclear morphology in both normal and HGPS cells, as evidenced by a reduction in the frequency of nuclear blebbing after 4 days of treatment that was further enhanced after 9 days.  The levels of DNA damage in HGPS cells was also reduced by sulforaphane through improving DNA repair.  Perhaps most importantly, however, is that both control and HGPS cells that underwent long-term administration of sulforaphane for several months experienced sustained increases in growth, an increase in the proportions of normal lamin A, and a decrease in the levels of mutant progerin levels during an 85-day period, dramatically demonstrating that sulforaphane is capable of inducing an increase in the levels of normal lamin A but a decrease in mutant progerin levels in both normal and HGPS cells [1].

Because methylene blue was shown to increase lamin A mRNA ratios and retinoic acid or its derivatives was shown to decrease progerin mRNA levels in HGPS cells, it is likely that sulforaphane, methylene blue, and retinoic acid are capable of positively influencing splicing of the LMNA gene in both normal persons and children affected with progeria though activation of the master metabolic regulator AMPK [2-4]. Indeed, normal humans possess the same cryptic splice site in the LMNA gene that is responsible for mutant progerin production in HGPS and normal humans gradually accumulate, over many decades, the same mutant progerin protein that causes accelerated aging in HGPS (see prior posts).  Sulforaphane has been shown to alter gene splicing in pre-malignant prostate tissue as well as increase the levels of PGC-1a, the same transcription factor that was induced by methylene blue to facilitate correction of mitochondrial abnormalities in progeria cells [2,5,6].

Additionally, just as methylene blue and retinoic acid have been shown to enhance T cell activation and reactivate latent/dormant HIV-1 infected T cells to facilitate viral destruction and removal by the immune system (see prior posts for references), sulforaphane has also been shown to stimulate the immune response by enhancing the cytotoxicity of natural killer cells and dendritic cells against prostate cancer cells and promote the infiltration of T cells in neoplastic regions [7]. Strikingly, sulforaphane has also been shown to decrease several epigenetic markers that promote the latency/dormancy of memory T cells infected with HIV-1, including EZH2 and SUV39H1, both of which are also dysregulated in progeria cells [8-12].

Such pleiotropic effects in seemingly disparate disease states may be explained, provocatively, by a common mechanism of action that methylene blue, retinoic acid, and sulforaphane (and many other compounds) possess:  the activation of AMPK. Because AMPK activation has been shown to beneficially alter gene splicing, increase lifespan and healthspan in several organisms, and is essential for activation of T cells (see prior posts for references), a transient induction of cellular stress, leading to a compensatory activation of AMPK, quite possibly explains how such chemically distinct compounds can have similar therapeutic effects in infectious diseases, disorders associated with gene splicing, accelerated aging, and likely many others (cancer, neurodegeneration, etc).

Indeed, the activation of AMPK by sulforaphane was shown to prevent neurotoxicity induced by scrapie prion protein (PrpSc), a misfolded protein that causes the neurological disorder Creutzfeldt-Jakob Disease in humans, scrapie in sheep, and “mad cow disease” in cattle [13]. An unimaginable shift in the practice of medicine would be inevitable if a common mechanism of AMPK activation actually links several distinct compounds with their efficacy in several distinct disease states.  

https://www.linkedin.com/pulse/broccoli-sprout-compound-reverses-aging-defects-progeria-finley?trk=mp-reader-card 


References:

1. Gabriel D, Roedl D, Gordon LB, Djabali K. Sulforaphane enhances progerin clearance in Hutchinson-Gilford progeria fibroblasts. Aging Cell. 2015 Feb;14(1):78-91.
2. Xiong ZM, Choi JY, Wang K, et al. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell. 2015 Dec 14. doi: 10.1111/acel.12434.
3. Pellegrini C, Columbaro M, Capanni C, et al. All-trans retinoic acid and rapamycin normalize Hutchinson Gilford progeria fibroblast phenotype. Oncotarget. 2015 Oct 6;6(30):29914-28.
4. Kubben N, Brimacombe KR, Donegan M, Li Z, Misteli T. A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds. Methods. 2015 Sep 1. pii: S1046-2023(15)30070-0.
5. Traka MH, Spinks CA, Doleman JF, et al. The dietary isothiocyanate sulforaphane modulates gene expression and alternative gene splicing in a PTEN null preclinical murine model of prostate cancer. Mol Cancer. 2010 Jul 13;9:189.
6. Fernandes RO, Bonetto JH, Baregzay B, et al. Modulation of apoptosis by sulforaphane is associated with PGC-1α stimulation and decreased oxidative stress in cardiac myoblasts.
7. Singh SV, Warin R, Xiao D, et al. Sulforaphane inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice in association with increased cytotoxicity of natural killer cells. Cancer Res. 2009 Mar 1;69(5):2117-25.
8. Balasubramanian S, Chew YC, Eckert RL. Sulforaphane suppresses polycomb group protein level via a proteasome-dependent mechanism in skin cancer cells. Mol Pharmacol. 2011 Nov;80(5):870-8.
9. Watson GW, Wickramasekara S, Palomera-Sanchez Z, et al. SUV39H1/H3K9me3 attenuates sulforaphane-induced apoptotic signaling in PC3 prostate cancer cells. Oncogenesis. 2014 Dec 8;3:e131.
10. McCord RP, Nazario-Toole A, Zhang H, et al. Correlated alterations in genome organization, histone methylation, and DNA-lamin A/C interactions in Hutchinson-Gilford progeria syndrome. Genome Res. 2013 Feb;23(2):260-9.
11. Liu B, Wang Z, Zhang L, Ghosh S, Zheng H, Zhou Z. Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model. Nat Commun. 2013;4:1868.
12. Das B, Dobrowolski C, Shahir AM, et al. Short chain fatty acids potently induce latent HIV-1 in T-cells by activating P-TEFb and multiple histone modifications. Virology. 2015 Jan 1;474:65-81.
13. Lee JH, Jeong JK, Park SY. Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway. Neuroscience. 2014 Oct 10;278:31-9.