AMPK identified for the first time in Human Sperm: The “Shock and Live” approach links the creation of all human life with Progeria & HIV-1

AMPK identified for the first time in Human Sperm: The “Shock and Live” approach links the creation of all human life with Progeria & HIV-1

https://www.linkedin.com/pulse/ampk-identified-first-time-human-sperm-shock-live-approach-finley?articleId=6199709017560928257#comments-6199709017560928257&trk=prof-post 

Sperm cell showing AMPK, highlighted in green, localized across the sperm tail and at the entire acrosome in the sperm head. Adapted from: See reference [1].

In line with recent findings demonstrating that cellular stress-induced activation of the master metabolic regulator AMPK is critical for oocyte meiotic resumption and maturation (processes that are essential for subsequent oocyte activation), T cell activation, and extension of lifespan, a recent study published in September of 2016 identified for the first time that AMPK is present in the whole human spermatozoa (i.e. sperm). AMPK localized along the tail, the midpiece, and at the entire acrosome, with inhibition of AMPK significantly decreasing sperm motility in all motility parameters analyzed [1].  Given that an increase in intracellular calcium levels, which activate AMPK, is critical for oocyte activation, sperm acrosome reaction, and T cell activation, and that several compounds that improve accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome also promote oocyte meiotic induction, induce the acrosome reaction in human sperm, and activate AMPK, the results from this ground-breaking study provide compelling evidence that AMPK likely occupies a central node facilitating the creation of all human life, the potential eradication of HIV-1, and the amelioration of symptoms associated with accelerated aging.          

Similar to oocyte activation, which is indispensable for the creation of all human life, the sperm acrosome reaction is also indispensable for the creation of all human life outside of a clinical setting (human oocyte fertilization and embryonic development can be achieved with sperm that have not undergone the acrosome reaction via in vitro fertilization techniques such as ICSI) [2]. The sperm acrosome reaction, an exocytotic process that is indispensable for oocyte penetration and fertilization, is characterized by the release of contents (e.g. proteolytic enzymes) of the acrosome (a cap-like structure residing in the anterior portion of the sperm head) that facilitates penetration of the outer protective coat of the egg, thus allowing fusion of sperm and egg cell membranes [3].  Interestingly, as an increase in intracellular calcium, similar to oocyte activation, is critical for induction of the acrosome reaction, physiological induction of the acrosome reaction by compounds found in the vicinity of the oocyte (e.g. progesterone) or pharmacological inducers (e.g. the calcium ionophore A23187) of the acrosome reaction each share a common mechanism of promoting intracellular calcium increases in sperm [3,4].

Because an intracellular calcium increase plays a pivotal role in both oocyte activation and induction of the acrosome reaction and because AMPK activation has also been shown to promote oocyte meiotic resumption, maturation, and likely oocyte activation, the acrosome reaction in sperm would also be predicted to be critically dependent on AMPK activation [4].  Indeed, Calle-Guisado et al. identified AMPK for the first time in human spermatozoa (i.e. mature motile sperm), with localization of AMPK at the entire acrosome, midpiece, and tail [5].  The authors used semen obtained from healthy human donors by masturbation and spermatozoa motility was analyzed using motility parameters including curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), percent of motile and progressive spermatozoa, amplitude of lateral head movement (ALH), beat cross of flagellum frequency (BCF), and linearity (LIN) [5].  Using both immunoflourescence and Western blotting techniques, the authors initially demonstrated that AMPK protein levels were highly expressed in freshly ejaculated human sperm, with localization of AMPK at the entire acrosome, the midpiece, and along the tail sperm.  Strikingly, using the same techniques along with antibodies to detect the phosphorylated/activated form of AMPK (pThr172-AMPK), the authors also showed that activated AMPK is also located along human spermatozoa, with prominent staining at the most apical portion of the acrosome and at the sperm tail, providing compelling evidence that activation of AMPK likely plays a critical role in the induction of the acrosome reaction and in facilitation of sperm motility [5].  Indeed, separation of sperm fraction into low and high motility sperm for each donor revealed that activated AMPK is predominant and present with higher intensity in highly motile sperm compared to low motility sperm. Additionally, after 20 hours of treatment with compound C, a pharmacological inhibitor of AMPK, the authors noted a significant decrease (65%) in both the percent of motile sperm present and the percentage of sperm with progressive motility (67% decrease).  20 hours of treatment with compound C also led to a significant decrease in the coefficients of BCF (56% reduction), ALH (45% reduction), and LIN (60% reduction) as well as a statistically significant reduction (approximately 50%) in all sperm velocity parameters tested (VCL, VAP, and VSL) [5].

Interestingly, published at around the same time as the study by Calle-Guisado et al., a study published by Aparicio et al. also identified AMPK in human spermatozoa as well as several functionally active autophagy-related proteins, indicating that autophagy may play a crucial role in cell survival and motility [6].  Interestingly, AMPK has been shown to be a critical regulator of autophagy via phosphorylating and activating ULK1, a key initiator of autophagic induction [7].  Autophagy is an evolutionarily conserved process that maintains cellular homeostasis through targeted degradation of damaged or long-lived intracellular proteins and organelles. Aparicio et al. showed that the autophagic proteins LC3, Atg5, Atg16, Beclin 1, and p62 were present in human spermatozoa as well as AMPKα 1/2 and mTOR, two critical regulators of cellular metabolism [6].  Activation of autophagy significantly increased spermatozoa viability and motility whereas autophagy inhibition decreased viability, motility, and the intracellular concentrations of calcium and ATP.  A majority (72%) of freshly incubated sperm cells were stained for LC3 (protein widely used to study autophagic induction) in the acrosome, further indicating that activated AMPK, which also demonstrates prominent staining in the acrosome, is likely critical for the induction of the acrosome reaction [5,6].  Curiously, the ratio of LC3-II/LC3-I (indicator of autophagic induction) was increased by both chloroquine, an autophagy inhibitor that activated AMPK in sperm, and rapamycin, an autophagy activator and mTOR inhibitor that has also been shown to increase the lifespan of several model organisms [6,8]. As the authors also demonstrated that rapamycin increased progressive motility and the percentage of rapid spermatozoa, whereas Calle-Guisado et al. showed that activated AMPK showed prominent staining at the sperm acrosome and tail, it would be expected that rapamycin would also activate AMPK [5,6]. Indeed, two recent studies published in 2016 revealed for the first time that rapamycin potently induced AMPK activation in normal elderly mice in vivo [9,10].  Because rapamycin and AMPK activation have been shown to promote autophagy by activating ULK1, the results from these two studies detecting AMPK for the first time in human spermatozoa provides compelling evidence that AMPK activation is likely a critical determinant for enhancing motility and promoting the acrosome reaction in sperm [7,9].   

As noted above, the induction of the acrosome reaction and oocyte activation are both critically dependent on an increase in intracellular calcium levels.  Interestingly, the acrosome reaction and oocyte activation also share many of the same intracellular signaling mediators, including phospholipase C (PLC), diacylglycerol (DAG), and  protein kinase C (PKC) [3,4].  Because AMPK activation is essential for oocyte meiotic induction, maturation, and likely oocyte activation, and because activated AMPK displays prominent staining at the sperm acrosome and is critical for sperm motility, it would be expected that compounds that induce the acrosome reaction in sperm will also induce oocyte activation and activate AMPK.  Indeed, calcium ionophores including A23187 have been shown to induce the acrosome reaction in human spermatozoa, activate AMPK, and induce human oocyte activation, leading to the birth of normal healthy children [11-13]. Interestingly, reactive oxygen species (ROS)/oxidative stress, which also activates AMPK, has been shown to induce the acrosome reaction in human sperm and the free radical-generating agent menadione has been shown to induce AMPK-dependent meiotic resumption in cumulus-enclosed and denuded mouse oocytes via promotion of oxidative stress, indicating that induction of cellular stress (i.e. “Shock”) via increases in the levels of ROS or intracellular calcium likely induces AMPK-mediated oocyte meiotic induction/maturation and oocyte activation as well as induction of the acrosome reaction in sperm (i.e. “Live”) [11,14,15].  

Furthermore, as the induction of cellular stress (e.g. intracellular calcium increases, ROS, etc.) leads to oocyte activation and the acrosome reaction in sperm, the exposure of HIV-1 latently infected monocyte or lymphocyte cell lines to hydrogen peroxide (H2O2)/ROS has also been shown to reactivate HIV-1 and the calcium ionophore A23187 has been shown reactivate latent HIV-1 as well [16,17]. Additionally, because AMPK plays a critical role in T cell activation (and thus reactivation of latent HIV-1 that resides in T cells) and because knockdown of AMPK or the upstream AMPK kinase CaMKK2 significantly inhibits HIV-1 replication, cellular stress-induced AMPK activation appears to also link oocyte activation, sperm acrosome reaction, and reactivation of latent HIV-1 (facilitating immune system detection and destruction of the virus) [18,19].

Lastly, cellular stress-induced AMPK activation likely also links oocyte meiotic induction, oocyte activation, sperm acrosome reaction, and amelioration of accelerated cellular aging defects associated with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS).  Strikingly, 1α,25-dihydroxyvitamin D3, the most potent metabolite of vitamin D, has been shown to delay premature senescence and significantly improve accelerated aging in patient-derived HGPS cells, activate AMPK, and induce the acrosome reaction in human sperm [20-22].  MG132, a proteasome inhibitor, has also been shown to improve symptoms of accelerated aging in HGPS fibroblasts, activate AMPK, reactivate latent HIV-1, and induce oocyte meiotic resumption [23-26].  Additionally, methylene blue and retinoic acid, both of which activate AMPK, have been shown to ameliorate accelerated cellular aging defects in HGPS fibroblasts, with methylene blue also inducing oocyte meiotic induction and retinoic acid enhancing maturation and activation of both mouse and human oocytes [23,27-34].

The results from the aforementioned studies engenders a provocative and novel proposition that oocyte activation, a prerequisite for the creation of all human life, and the acrosome reaction in sperm share a common mechanism of AMPK activation that is promoted by the induction of cellular stress (e.g. ROS, intracellular calcium increase, AMP/ADP:ATP ratio increase, etc).  As first proposed in my most recent publication, this cellular stress-induced activation of AMPK also links oocyte activation and reactivation of latently infected HIV-1 reservoirs, facilitating detection and destruction of the virus by the immune system [4].  Additionally, AMPK-activating compounds (e.g. vitamin D, MG132, methylene blue) that have been shown to induce oocyte meiotic resumption and the acrosome reaction in sperm also ameliorate accelerated cellular aging defects in HGPS, a genetic disorder characterized by excessive accumulation of a toxic protein (called progerin) that is also produced in much smaller quantities by normal humans [35].  Also, as noted in my most previous LinkedIn Post, H2O2/ROS induced by the bacterium Wolbachia in a mosquito strain that serves as a vector for dengue and Zika viruses has also been shown to enhance antiviral immune responses in those mosquitoes, decreasing dengue virus replication and transmission [36].  Because AMPK activation in that mosquito strain by compounds that increase cellular stress leads to an increase in lifespan and enhancement of immune responses and because H2O2, ROS, or intracellular calcium increases have been shown to induce oocyte activation, the acrosome reaction in sperm, and reactivate latent HIV-1, the beneficial effects of cellular stress-induced AMPK activation may not only link the creation of all human life with HGPS and latent HIV-1 reactivation, but may indeed represent a unifying theme that crosses species boundaries.        

  References:

1. Calle-Guisado V, de Llera AH, Martin-Hidalgo D, et al. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility. Asian J Androl. 2016 Sep 27. doi: 10.4103/1008-682X.185848. [Epub ahead of print]

2. Gianaroli L, Magli MC, Ferraretti AP, et al. Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection. Fertil Steril. 2010 Feb;93(3):807-13.

3. Darszon A, Nishigaki T, Beltran C, Treviño CL. Calcium channels in the development, maturation, and function of spermatozoa. Physiol Rev. 2011 Oct;91(4):1305-55.

4. Finley J. Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1. Med Hypotheses. 2016 Aug;93:34-47.

5. Calle-Guisado V, de Llera AH, Martin-Hidalgo D, et al. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility. Asian J Androl. 2016 Sep 27. doi: 10.4103/1008-682X.185848. [Epub ahead of print].

6. Aparicio IM, Espino J, Bejarano I, et al. Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility. Sci Rep. 2016 Sep 16;6:33647.

7. Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011 Feb;13(2):132-41.

8. Harrison DE, Strong R, Sharp ZD, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.  Nature. 2009 Jul 16;460(7253):392-5.

9. 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.

10. Lesniewski LA, Seals DR, Walker AE, et al. Dietary rapamycin supplementation reverses age-related vascular dysfunction and oxidative stress, while modulating nutrient-sensing, cell cycle, and senescence pathways. Aging Cell. 2016 Sep 22. doi: 10.1111/acel.12524. [Epub ahead of print].

11. de Lamirande E, Tsai C, Harakat A, Gagnon C. Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. J Androl. 1998 Sep-Oct;19(5):585-94.

12. Fogarty S, Hawley SA, Green KA, Saner N, Mustard KJ, Hardie DG. Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP. Biochem J. 2010 Jan 27;426(1):109-18.

13. Ebner T, Montag M, Oocyte Activation Study Group, et al. Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study. Reprod Biomed Online. 2015 Apr;30(4):359-65.

14. Sundararaman A, Amirtham U, Rangarajan A. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation. J Biol Chem. 2016 Jul 8;291(28):14410-29.

15. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.

16. 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.

17. Fujinaga K, Zhong Q, Nakaya T, et al. Extracellular Nef protein regulates productive HIV-1 infection from latency. J Immunol. 1995 Dec 1;155(11):5289-98.

18. Tamás P, Hawley SA, Clarke RG, et al. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med. 2006 Jul 10;203(7):1665-70.

19. Zhou H, Xu M, Huang Q, Gates AT, et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe. 2008 Nov 13;4(5):495-504.

20. Kreienkamp R, Croke M, Neumann MA, et al. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes. Oncotarget. 2016 Apr 27. doi: 10.18632/oncotarget.9065.

21. Swami S, Krishnan AV, Williams J, et al.. Vitamin D mitigates the adverse effects of obesity on breast cancer in mice. Endocr Relat Cancer. 2016 Apr;23(4):251-64.

22. Blomberg Jensen M, Bjerrum PJ, Jessen TE, et al. Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa. Hum Reprod. 2011 Jun;26(6):1307-17.
23. 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.

24. Jiang S, Park DW, Gao Y, et al. Participation of proteasome-ubiquitin protein degradation in autophagy and the activation of AMP-activated protein kinase. Cell Signal. 2015 Jun;27(6):1186-97.

25. Huo LJ, Fan HY, Zhong ZS, Chen DY, Schatten H, Sun QY. Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation. Mech Dev. 2004 Oct;121(10):1275-87.

26. Miller LK, Kobayashi Y, Chen CC, Russnak TA, Ron Y, Dougherty JP. Proteasome inhibitors act as bifunctional antagonists of human immunodeficiency virus type 1 latency and replication. Retrovirology. 2013 Oct 24;10:120.

27. Xiong ZM, Choi JY, Wang K, et al. Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria. Aging Cell. 2016 Apr;15(2):279-90.

28. Shin SY, Kim TH, Wu H, Choi YH, Kim SG. SIRT1 activation by methylene blue, a repurposed drug, leads to AMPK-mediated inhibition of steatosis and steatohepatitis. Eur J Pharmacol. 2014 Mar 15;727:115-24.

29. Downs SM, Humpherson PG, Leese HJ. Meiotic induction in cumulus cell-enclosed mouse oocytes: involvement of the pentose phosphate pathway. Biol Reprod. 1998 Apr;58(4):1084-94.

30. Kim YM, Kim JH, Park SW, Kim HJ, Chang KC. Retinoic acid inhibits tissue factor and HMGB1 via modulation of AMPK activity in TNF-α activated endothelial cells and LPS-injected mice. Atherosclerosis. 2015 Aug;241(2):615-23.

31. Nasiri E, Mahmoudi R, Bahadori MH, Amiri I. The Effect of Retinoic Acid on in vitro Maturation and Fertilization Rate of Mouse Germinal Vesicle Stage Oocytes. Cell J. 2011 Spring;13(1):19-24.

32. Tahaei LS, Eimani H, Yazdi PE, Ebrahimi B, Fathi R. Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system. J Assist Reprod Genet. 2011 Jun;28(6):553-8.

33. Pauli SA, Session DR, Shang W, et al. Analysis of follicular fluid retinoids in women undergoing in vitro fertilization: retinoic acid influences embryo quality and is reduced in women with endometriosis. Reprod Sci. 2013 Sep;20(9):1116-24.

34. Best MW, Wu J, Pauli SA, et al. A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells. Mol Hum Reprod. 2015 Jun;21(6):527-34.   

35. Finley J. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome. Med Hypotheses. 2015 Sep;85(3):320-32.

36. https://www.linkedin.com/pulse/probiotic-activates-ampk-mosquitoes-inhibiting-zika-dengue-finley?trk=pulse_spock-articles

 

“Probiotic” activates AMPK in mosquitoes, inhibiting Zika and Dengue viruses: The Shock and Live Approach links Viruses, Oocytes, Sperm, & Progeria

“Probiotic” activates AMPK in mosquitoes, inhibiting Zika and Dengue viruses: The Shock and Live Approach links Viruses, Oocytes, Sperm, & Progeria

https://www.linkedin.com/pulse/probiotic-activates-ampk-mosquitoes-inhibiting-zika-dengue-finley?trk=pulse_spock-articles

  

Reports last week from the Australia-based non-profit organization Eliminate Dengue provided details of an ongoing initiative to decrease the spread of dengue and Zika viruses through the coordinated release of male and female mosquitoes (called Aedes aegypti) that were purposely infected with a bacterium that inhibits the mosquito’s ability to transmit the two viruses to humans [1].  Set to launch in Colombia and Brazil early next year, the control campaigns will be funded with $18 million from donors including the U.S. and British governments as well as the Bill & Melinda Gates Foundation.  Interestingly, the Google health spin-off Verily also recently announced a similar initiative to prevent the spread of dengue and Zika virus transmission by using the same bacterium to infect male mosquitoes only. Infected male mosquitoes are unable to reproduce with normal/uninfected female mosquitoes [1,2].    

Interestingly, in addition to significantly inhibiting the transmission of the two viruses by Aedes aegypti to humans, the bacterium, called Wolbachia, also occurs naturally in many insects and is transmitted successfully to offspring by females after an infected female mates with an infected male, or an infected female mates with a normal/uninfected male [3].  However, a normal/uninfected female is only able to produce viable offspring with a normal/uninfected male, leading to a significant reproductive advantage for Wolbachia-infected females and thus propagation of Wolbachia throughout mosquitoes in the wild. Additionally, only female mosquitoes bite and require a blood meal to promote egg production, further highlighting the potential of reducing dengue and Zika virus transmission to humans via Wolbachia-infected female mosquitoes.

Although the method through which Wolbachia is transmitted to mosquito offspring has been well studied, how Wolbachia infection leads to a decrease in dengue and Zika virus replication in, and transmission by, mosquitoes is heavily debated.  Interestingly, although Wolbachia does not naturally occur in Aedes aegypti, Wolbachia appears to impart beneficial immunomodulatory effects in these mosquitoes not unlike that of certain beneficial bacteria that normally colonize the human gut.  Such bacteria, also known as “probiotics”, are typified by the genera Lactobacillus and Bifidobacterium and have been shown to significantly enhance natural and acquired immunity, induce reactive oxygen species (ROS) generation and the upregulation of Nrf2-dependent cytoprotective genes, produce short chain fatty acids (SCFAs) that promote T cell differentiation, and produce antimicrobial peptides that exhibit bactericidal activity [4-7].  Interestingly, the induction of cellular stress via ROS generation has been shown to activate the master metabolic regulator AMPK and AMPK activation has been shown to be critical for T cell activation and the mounting of an effective immune response in vivo to viral and bacterial infections [8-10].  Indeed, acetate, propionate, and butyrate, three SCFAs abundantly produced by probiotic bacteria, each activate AMPK, indicating that the immunomodulatory effects of probiotic bacteria are dependant on cellular stress-induced activation of AMPK [11]. Furthermore, AMPK has recently been discovered in Aedes aegypti and activation of AMPK in Aedes aegypti by compounds that induce cellular stress leads to an enhanced immune response and increased longevity in Aedes aegypti (see below), implicating the provocative assertion that inhibition of dengue and Zika virus replication via Wolbachia infection occurs as result of cellular stress-induced activation of AMPK [12].

As noted above, Wolbachia, which are found in reproductive tissues of arthropods, often engage in a mutualistic relationship with its host. Indeed, Wolbachia has been shown to induce resistance to RNA viral infections in the model organism Drosophila melanogaster [13]. D. melanogaster (also known as the fruit fly) has been studied extensively in biological research and activation of AMPK has been shown to extend lifespan as well as slow aging in D. melanogaster through autophagic induction via upregulation of Atg1 (ULK1 in mammals), reduction of insulin-like peptide levels in the brain, and an increase in 4E-BP [14,15]. 

Interestingly, insulin-like peptides and hyperphosphorylation of 4E-BP (i.e. inactivation) is positively associated with target of rapamycin (TOR) activation and egg development in Aedes aegypti (Ae. Aegypti) [16]. Mammalian target of rapamycin (mTORC1/TORC1) is a protein kinase that plays a critical role in promoting mRNA translation and is the primary target of rapamycin, a macrolide drug that extends lifespan in several model organisms including normal genetically heterogeneous mice and D. melanogaster [17,18].  Rapamycin was shown to delay yolk deposition in control Ae. aegypti eggs and inhibit insulin-mediated 4E-BP (a negative regulator of translation) phosphorylation. Silencing of 4E-BP also led to a reduction in lifespan of adult female mosquitoes, mirroring results obtained via AMPK activation in D. melanogaster [15,16,19]. Strikingly, in addition to delaying egg maturation in Ae. aegypti, rapamycin has also been shown to dramatically increase the number of Wolbachia in D. melanogaster oocytes via TORC1 inhibition, whereas somatic hyperactivation of TORC1 decreases Wolbachia titer in oocytes [20]. Interestingly, ablation of insulin-producing cells in the D. melanogaster brain abolished the yeast-induced reduction of Wolbachia in oocytes, mirroring previous results showing that AMPK-induced lifespan extension in D. melanogaster is associated with a decrease in insulin-like peptides in the brain [15,20].    

Because rapamycin, similar to AMPK activation by diverse compounds, induces autophagy, inhibits mTOR/TORC1, promotes mitochondrial biogenesis, and extends  lifespan in several model organisms, it would expected that rapamycin would also induce activation of AMPK, likely via the induction of cellular stress (i.e. increase in the levels of ROS, intracellular calcium increase, increase in the AMP:ADP/ATP ratio, etc). Indeed, two recent publications clearly demonstrated that rapamycin potently induced the activation of AMPK in vivo in normal elderly mice, along with an upregulation of ULK1 (mammalian orthologue of Atg1 critical for the induction of autophagy) and PGC-1a (a transcription factor essential for promoting mitochondrial biogenesis) [21,22].  As rapamycin has been shown to increase the number of Wolbachia in D. melanogaster oocytes, delay egg maturation and inhibit insulin-mediated 4E-BP phosphorylation in Ae. aegypti, and increase lifespan in D. melanogaster, the recent finding that activation of AMPK in Ae. aegypti also increases lifespan and improves the immune response (see below) further bolsters the notion that activation of AMPK via the induction of cellular stress likely promotes lifespan extension, immune system enhancement, and Wolbachia  propagation in oocytes in both D. melanogaster and Ae. aegypti.  Moreover, because certain bacteria (e.g. Lactobacillus) that colonize the human gut induces beneficial immune responses by inducing cellular stress (e.g. increased ROS levels) and also produce compounds that activate AMPK, the inhibition of dengue and Zika virus replication in, and transmission by, Ae. aegypti is likely the result of a Wolbachia-induced cellular stress response, leading to the up regulation of anti-viral mechanisms that are likely modulated by AMPK.

Indeed, a recent study by Wong et al. demonstrated that ROS/oxidative stress is positively correlated with Wolbachia-mediated antiviral protection in D. melanogaster [23]. The authors of the study observed that H2O2 (hydrogen peroxide) was increased 1.25- to 2- fold in flies that harbored protective strains of Wolbachia (e.g. wMelCS, wRi, wAu) compared to Wolbachia-free controls. Interestingly, flies with a null mutation in Cu/Zn SOD (an antioxidant enzyme) exhibited elevated endogenous levels of oxidative stress that mimicked Wolbachia-induced oxidative stress.  In these Wolbachia-free Cu/Zn SOD mutant flies, a 70% survival rate was observed at 4 days post-infection after Drosophila C virus infection compared to a less than 10% survival rate for non-mutant flies, indicating that elevated levels of oxidative stress induced by protective Wolbachia strains confers a survival advantage by decreasing susceptibility to viral infection via activation of signaling pathways that potentiate anti-viral immune responses [23].

As noted above, ROS/oxidative stress has been shown to activate AMPK and AMPK activation in both D. melanogaster and Ae. Aegypti leads to an increase in lifespan [8,12,15].  Interestingly, Sykiotis et al. showed that oxidants/oxidative stress promote lifespan extension in D. melanogaster males by activating the Nrf2 (CncC) pathway, a master antioxidant transcription factor that induces the expression of several antioxidant genes including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) [24].  Curiously, Sykiotis et al. also observed that the synthetic dithiolthione oltipraz, which has been shown to activate AMPK, inhibit HIV-1 replication, and reverse accelerating aging defects in Hutchinson-Gilford progeria syndrome (see below), also induced Nrf2 signaling in flies [24-27].  AMPK has also been shown to increase the transcriptional activity of Nrf2 and increase Nrf2 nuclear retention via phosphorylation, suggesting that AMPK may represent a central node in ROS/oxidative stress-induced immune and anti-viral responses [28,29].

Indeed, a recent study by Pan et al. showed that infection of Ae. Aegypti with Wolbachia led to a ROS/oxidative stress-induced upregulation of genes associated with immunity and reduction-oxidation, thus enhancing inhibition of dengue virus replication [30]. Several gene transcripts related to the immunity and redox/stress/mitochondrion group were upregulated in Wolbachia-infected Ae. aegypti females before blood feeding and after infection with dengue virus (DENV) serotype 2, including the antimicrobial peptides cecropin D (CECD) and defensin C (DEFC) as well as the antioxidants glutathione peroxidase, CuZnSOD, MnSOD, and glutathione peroxidase [30].  A significant increase in the levels of H2O2 and in the transcript abundance of both NADPH oxidase M (NOXM) and dual oxidase 2 (DUOX2), two enzymes that generate ROS, were also observed in Wolbachia-infected mosquitoes compared to control mosquitoes.  Interestingly, the expression of several Toll pathway (a pathway that mediates the production of antioxidants and antimicrobial peptides) marker genes as well as the antimicrobial peptides CECD and DEFC were increased by the addition of H2O2 in a sugar solution given to female control/uninfected mosquitoes [30].  This effect was also mirrored in Wolbachia-infected female mosquitoes, wherein silencing of NOXM and DUOX2 deactivated the Toll pathway and suppressed the expression of CECD and DEFC, indicating that Wolbachia-induced ROS is required for activation of the Toll pathway and induction of antimicrobial peptides.  Importantly, individual or double RNAi-induced knockdown of DEFC and CECD significantly increased viral titers of DENV serotype 2 in Wolbachia-infected mosquitoes compared to controls, providing further evidence that Wolbachia-induced ROS leads to a beneficial cellular response characterized by antimicrobial-mediated inhibition of viral replication [30].

Because Wolbachia-induced ROS/oxidative stress leads to beneficial anti-viral cellular responses in both D. melanogaster and Ae. aegypti and because AMPK activation, which increases Nrf2 transcriptional activity, leads to increased lifespan in D. melanogaster, it would be expected that cellular stress-induced AMPK activation in Ae. aegypti would also lead to an increase in lifespan as well as an enhanced immune response.  Indeed, Nunes et al. showed that autophagy, immune system activation, and the average lifespan of Ae. Aegypti (a vector for dengue as well as chikungunya and Zika virus) is increased by feeding mosquitoes several plant-based polyphenols [31].  Interestingly, administration of resveratrol (derived from grapes), quercetin (found in many fruits and vegetables), epigallocathechin-3-gallate (EGCG, found in green tea), and genistein (found in soybeans) each increased average lifespan for male and female mosquitoes compared to controls.  Insects fed resveratrol also displayed higher levels of AMPK phosphorylation/activation compared to controls and compound C, a pharmacological inhibitor of AMPK, blocked the suppression of triglyceride (TG) content induced by resveratrol [31]. Resveratrol treatment also led to immunomodulatory effects, with reduced bacterial populations for female mosquitoes compared to controls, an effect that was mimicked by AICAR (a prototypical AMPK activator) but inhibited by compound C.  Interestingly, resveratrol only slightly decreased bacterial populations in vitro, indicating that the effects of resveratrol are indirect and likely associated with activation of the immune response in mosquitoes. Autophagy was also stimulated in resveratrol- and AICAR-fed mosquitoes which was abolished by silencing of AMPK [31].  

Interestingly, each of the polyphenols used in that study (resveratrol, quercetin, epigallocathechin-3-gallate, and genistein) have each been shown to induce cellular stress (e.g. ROS generation), similar to Wolbachia-induced ROS generation, in a number of mammalian cells in addition to activating AMPK [32-35].  As AMPK is activated by a number of different compounds (polyphenols, bacterial metabolites, etc.) and methodologies (e.g. electrical stimulation) that induce cellular stress, it is likely that Wolbachia-induced inhibition of dengue and Zika virus replication in and transmission by Ae. Aegypti is also modulated by AMPK, a master metabolic regulator that increases lifespan and improves immune and antioxidant responses in another Wolbachia-infected insect, D. melanogaster [36,37].  Although Nunes et al. did not determine if the polyphenols tested inhibited dengue virus replication in or transmission by Ae. Aegypti, recent studies have shown that in mammalian cells quercetin and analogs of resveratrol exhibit significant inhibitory activities against dengue virus type 2 [38,39].  EGCG has also recently been shown to inhibit Zika virus entry in Vero E6 cells (albeit at higher concentrations), indicating that cellular stress-induced activation of AMPK may represent a common mechanism of action that spans species boundaries to prevent the replication and/or transmission of dengue and Zika viruses [40].  Additionally, salidroside (derived from the plant Rhodiola rosea) and curcumin (derived from the plant Curcuma longa) have both been shown to exhibit anti-dengue virus activity in vitro and induce AMPK activation in vivo, providing further evidence that many structurally diverse compounds likely share a common mechanism of stress-induced AMPK activation to effectuate antiviral responses [41-44]. 

Remarkably, cellular stress-induced activation of AMPK (i.e. “Shock”) appears to link the antiviral effects mediated by a Wolbachia-induced increase in ROS production in A. aegypti with the amelioration of accelerated cellular aging defects in the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS), the reactivation of latent HIV-1 in infected T cells (to facilitate immune system detection and viral destruction), oocyte meiotic resumption, and the acrosome reaction in sperm (i.e. “Live”).

Indeed, the proteasome inhibitor MG132 has recently been shown to inhibit progerin (the toxic protein that causes accelerated cellular aging defects) in fibroblasts derived from HGPS patients, inhibit dengue, West Nile, and Yellow fever viruses, and activate AMPK [45-47]. 1α,25-dihydroxyvitamin D3, the most potent metabolite of vitamin D, has also been shown to delay premature senescence and significantly improve accelerated aging in patient-derived HGPS cells, reduce dengue virus infection in human myelomonocyte and hepatic cell lines, and activate AMPK [48-50].  Interestingly, nuclear aging defects in HGPS patient-derived cells were also reversed via activation of the antioxidant transcription factor Nrf2 by oltipraz. Oltipraz has also been shown to activate AMPK, inhibit HIV-1 replication, and induced Nrf2 signaling in D. melanogaster [24-27]. 

Furthermore, just as Wolbachia-induced ROS/hydrogen peroxide (H2O2) generation (i.e. “Shock”) leads to a compensatory upregulation of antimicrobial peptides and an enhanced antiviral immune response in Ae. Aegypti (i.e. “Live”), the exposure of HIV-1 latently infected monocyte or lymphocyte cell lines to H2O2 has also been shown to reactivate HIV-1 [51]. Additionally, ROS induces activation of AMPK and AMPK plays a critical role in T cell activation (and thus reactivation of latent HIV-1 that resides in T cell cells) [8,52].

AMPK activation has also been shown to play a critical role in the initiation of oocyte meiotic resumption and maturation (in preparation for oocyte activation) and the free radical-generating agent menadione (i.e. “Shock”) has been shown to induce AMPK-dependent meiotic resumption in cumulus-enclosed and denuded mouse oocytes (i.e. “Live”) via promotion of oxidative stress [53].  Lastly, ROS also plays a critical role in the induction of the acrosome reaction in sperm, a process that facilitates oocyte penetration through release of hydrolytic enzymes and is indispensable, along with oocyte activation, for the creation of all human life outside of a clinical setting [54].  Strikingly, AMPK has recently been found for the first time to be localized across the entire acrosome in human sperm and both H2O2/ROS and vitamin D (i.e. “Shock) have been shown to induce the acrosome reaction in human sperm (i.e. “Live”), providing compelling evidence that cellular stress-induced AMPK activation is critical for the induction of the acrosome reaction in human sperm and the creation of human life [55-57].

The antiviral effects mediated by Wolbachia-induced ROS generation paints a clear yet provocative portrait that cellular-stress mediated activation of AMPK represents a common mechanism of action that spans species boundaries, beneficially modulating cellular processes that are involved in the immune response, fertilization, and aging itself.  Indeed, structurally distinct compounds including MG132 and vitamin D have been shown to exert potent antiviral effects against dengue virus and significantly improve accelerated aging defects in HGPS.  More tellingly, H2O2/ROS/oxidative stress has been shown to reactivate latent HIV-1, promote oocyte meiotic resumption, and induce the acrosome reaction in human sperm.  Because AMPK is critical for oocyte meiotic resumption (and hence oocyte activation) and AMPK has recently been found for the first time to be localized across the entire acrosome in human sperm, AMPK activation may indeed be critical for the creation of all human life, as originally proposed in my recent publication [58].  Perhaps beyond perplexing on first glance, Wolbachia, mosquitoes, and the creation of all human life are likely connected.

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AMPK links beneficial responses to the “Fever Effect” in Autism with Progeria, HIV-1 latency, & Oocyte activation: The "Shock and Live" approach

Hey Blogger Fam, check out my latest post when you have a minute. The post basically explains why some children with Autism get better temporarily (i.e. improvement in speech, decrease in repetitive behaviors) when they get a fever.  Also known as the “fever effect”, this is basically another rendition of the “shock and live” approach, in which the induction of mild stress to a cell, in this case heat stress, leads to the activation of the master metabolic regulator AMPK and a beneficial cellular response.  The child’s behavior improves because mild heat stress or a “shock” to neurons of the brain causes those brain cells to do what they were designed to do that much better.  The broccoli sprout compound sulforaphane, which activates AMPK, also activates the heat shock response and has shown efficacious results in patients with autism and also improves accelerated aging defects in skin cells taken from progeria patients.  How is this possible? Is the correct functioning of neurons related to slowing or reversing accelerated aging defects? Indeed it is.  Heat stress, which has been shown in independent studies to activate AMPK, is yet an additional term that equates to “what doesn’t kill you makes you stronger”.  Heat stress, hyperthermia, or the activation of heat shock proteins have been shown to promote T cell functionality, wake up dormant HIV-1 viruses so that the immune system can detect and kill it, promote the maturation of oocytes in preparation for artificial oocyte activation or activation by sperm, promote the induction of the acrosome reaction in sperm (a process necessary for oocyte penetration), promote “activation” and killing of cancer stem cells, promote the develop of embryonic stem cells into cells that will eventually make up the body of the baby, and enhance learning and memory in the brain.  The fancy term to describe the process that enhances learning and memory is called long-term potentiation, which basically involves a strengthening in connections between neurons if they are continuously challenged. Heat stress, an intellectually stimulating environment, exercise, and countless naturally-occurring compounds all enhance long-term potentiation, learning and memory, and activate AMPK. Interestingly, high-frequency stimulation and deep brain stimulation also activate AMPK in neurons and improve neurological symptoms, indicating that mild transient electrical pulses are another form of stress.  Lastly, a stark example of how heat stress and learning and memory in neurons are connected to the creation of human life involves a heat-sensitive channel called TRPV3. Mild heat stress and a compound from oregano (called carvacrol) activates this channel.  This channel is also present in the brain and in oocytes.  If you knock this channel out in the brain, long-term potentiation and learning and memory are impaired.  However, if you activate this channel on oocytes with the oregano compound, the oocyte will become activated as if it had been fertilized by sperm (called parthenogenesis).  Again, heat stress and compounds that induce mild stress promotes a compensatory and beneficial response from a cell. This response is orchestrated by AMPK activation and results in a “shock to live” (e.g. oocyte maturation/activation, stem cell “activation”, learning and memory in the brain, sperm capacitation/acrosome reaction) or a “shock to kill” (e.g. latent HIV-1 reactivation, cancer stem cell “activation” and/or cell death).       

AMPK links beneficial responses to the “Fever Effect” in Autism with Progeria, HIV-1 latency, & Oocyte activation: The "Shock and Live" approach

https://www.linkedin.com/pulse/ampk-links-beneficial-responses-fever-effect-autism-progeria-finley?trk=pulse_spock-articles 

In line with recent findings demonstrating that sulforaphane, an isothiocyanate heavily concentrated in broccoli sprouts, significantly ameliorated accelerated aging defects in cells derived from patients diagnosed with Hutchinson-Gilford progeria syndrome (HGPS), a recently completed placebo-controlled, double-blind, randomized trial by researchers from Harvard Medical School, The Johns Hopkins University School of Medicine, and University of Massachusetts Medical School demonstrated that participants diagnosed with moderate to severe Autism Spectrum Disorder (ASD) who received sulforaphane showed substantial and significant improvements in behavior, social interaction, and verbal communication compared to participants assigned to placebo [1,2].  Discontinuation of sulforaphane also led to a reversal of these improvements, mirroring levels obtained before initiation of treatment [2].

Interestingly, parents and clinicians, over the past few decades, have reported noticeable improvements in behavior in children with ASD during or after the onset of febrile illness (i.e. fever).  Also known as the “fever effect”, transient increases in body temperature may positively influence neuronal and synaptic function in the brain of ASD patients, an effect that appears to be at least partially mimicked by sulforaphane. As described below, because sulforaphane and other compounds regulate proteins that are critical for the induction of the heat shock response and because heat shock proteins also play critical roles in the reactivation of latent HIV-1, oocyte maturation, sperm capacitation/acrosome reaction, the promotion of learning and memory in the brain, and differentiation of adult stem cells, the “fever effect” likely represents a “shock” or the induction of cellular stress, leading to the activation of the master metabolic regulator AMPK, resulting in a beneficial cellular stress response. The response to this stressor generates a cell-specific “shock to live” (e.g. oocyte maturation/activation, stem cell differentiation, stimulation of learning and memory, sperm capacitation/acrosome reaction) or a “shock to kill” (e.g. latent HIV-1 reactivation, cancer stem cell differentiation and/or cell death).

ASD, a neuro-developmental disorder that disproportionately affects males, is characterized by repetitive or compulsive behaviors, impairments in social development, and deficiencies in verbal and non-verbal communication that are often observable within the first two years of life.  Causes underlying ASD also appear to be multi-factorial, with interactions between environmental factors and genetics leading to deleterious alterations in neuronal network functionality and immune system regulation [3-5].  As noted above, anecdotes and case reports have indicated that amelioration of symptoms associated with ASD are positively correlated with the onset of febrile illness. A particularly interesting example of the “fever effect” in ASD occurred at New York University’s Bellevue Psychiatric Hospital, in which improvement in concentration and social interactions were noted in children with temperatures between 38.9 to 40.6°C, due to an outbreak of viral upper respiratory tract infections [6-8].  Indeed, a prospective study of 30 children with ASD during and after febrile illness (body temperature greater than or equal to 38.0°C) revealed that compared to afebrile ASD patients, fewer aberrant behaviors for febrile ASD patients were recorded on the Aberrant Behavior Checklist subscales of irritability, hyperactivity, stereotypy (i.e. persistent repetition of an act), and inappropriate speech, indicating a transient enhancement or correction of neuronal functioning in response to fever onset [8].

Because sulforaphane has been shown to transiently induce cellular stress, resulting in the upregulation of the master antioxidant transcription factor Nrf2 and activation of AMPK (which activates Nrf2), febrile illness likely induces a transient induction of cellular stress (i.e. “heat shock”), resulting in a compensatory and beneficial upregulation of cellular factors that enhance or promote neuronal functioning [9,10,11]. Indeed, Singh et al. recently demonstrated in a placebo-controlled, double-blind, randomized trial that patients receiving sulforaphane showed significant behavior improvements as measured by the Aberrant Behavior Checklist and the Social Responsiveness Scale compared to patients receiving placebo [2].  Patients who received sulforaphane also experienced improvements in social interaction, abnormal behavior, and verbal communication on the Clinical Global Impression Improvement Scale [2].

Because sulforaphane induces cellular stress via an increase in stressors including reactive oxygen species (ROS) and because febrile illness represents the induction of cellular stress, the beneficial effects of sulforaphane demonstrated in ASD patients suggests that sulforaphane may also induce cellular stress and a beneficial cellular response by activating or upregulating mediators that promote the heat shock response. Heat shock consists of subjecting a cell to a higher temperature than the ideal body temperature of an organism.  The heat shock response is a cellular response to heat shock that induces the activation of heat shock factor-1 (HSF1), the major regulator of heat shock protein (HSP) transcription, and the upregulation of HSPs that aid in repairing or targeting misfolded proteins for degradation [12].  Interestingly, HSPs also respond to other forms of cellular stress, including ROS and intracellular calcium (Ca2+) increases, both of which also activate AMPK [13-15].  Interestingly, sulforaphane has been shown to induce a significant and rapid HSF1 mediated heat shock response and heat stress has also been shown to increase ROS production, promoting nuclear translocation of Nrf2 and upregulation of Nrf2 target genes [16,17]. Furthermore, as sulforaphane activates AMPK and AMPK activates Nrf2, heat stress has also been shown to activate AMPK, promoting insulin-independent glucose transport in muscle cells and the killing of breast and pancreatic cancer stem cells [18,19].  HSP90 has also been to found to interact with and maintain AMPK activity, providing further evidence that the induction of  heat shock likely leads to activation of AMPK and a beneficial cellular response [53].

Interestingly, as sulforaphane has been shown to activate both AMPK and Nrf2 and significantly ameliorate accelerated aging defects associated with Hutchinson-Gilford progeria syndrome (HGPS) (which is associated with dysfunctional Nrf2 signaling), other AMPK-activating compounds that have demonstrated efficacious results in HGPS may also be expected to induce certain mediators constituting the heat shock response [1].  Indeed, the proteasome inhibitor MG132, the macrolide rapamycin, and vitamin D have each been shown to improve symptoms of accelerated aging in HGPS fibroblasts, activate HSF1 or increase HSP expression, and activate AMPK, indicating that chemically distinct compounds including sulforaphane, MG132, rapamycin, and vitamin D likely induce cellular stress, leading to the activation of AMPK and a beneficial cellular response in diseases as disparate as HGPS and ASD [20-28].

As febrile illness and the induction of the heat shock response have been shown to mitigate irritability, hyperactivity, stereotypy, and inappropriate speech in ASD patients, it would be expected that the application of heat stress or the induction of mediators of the heat shock response would also enhance or improve certain aspects of neuronal functionality.  Indeed, heat stress alone and heat stress preconditioning before diffuse axonal injury led to higher expression levels of HSP70 and a significant improvement in the Morris Water Maze task (a behavioral procedure used to study spatial learning and memory) and long-term potentiation (LTP) in rats compared with diffuse axonal injury alone [29]. LTP is characterized by a “persistent increase in synaptic strength following high-frequency stimulation” and is often studied in pyramidal neurons of the hippocampus, an area of the brain important for learning and memory [30].  Interestingly, neuronal depolarization (i.e. activation) has been shown to activate HSF1, heat shock has been shown to improve synaptic integrity and memory consolidation, and HSF1 agonists (e.g. exercise) have been shown to improve cognition in models of dementia [31]. A recent study by Notenboom et al. also showed that prolonged hyperthermia in rats enhanced hippocampal CA1 long-term potentiation and sprouting of mossy fiber collaterals into the dentate gyrus [32].  Perhaps most proactively, however, is a recent study by Brown et al. demonstrating that high-frequency stimulation-induced LTP was attenuated at CA3-CA1 pyramidal cell synapses in hippocampal slices from Trpv1 and Trpv3 knockout (KO) mice [33].

Interestingly, carvacrol, a monoterpenoid phenol found in the essential oil of Origanum vulgare (oregano), is a potent activator of the transient receptor potential cation channel, subfamily V, member 3 (TRPV3), a nonselective cation channel that functions in vasoregulation and temperature sensation and is activated between the temperatures of 22 and 40 degrees Celsius [34,35].  The TRPV3 channel has also been shown to associate with and form heteromeric channels with the Ca2+-permeable TRPV1 channel, another nonselective cation channel that is also regulated by temperature sensation and is activated by both physical and chemical stimuli, including temperatures greater than 43 degrees Celsius as well as the natural compound capsaicin (an active component of chili peppers from the genus Capsicum) [35-37]. As both carvacrol and capsaicin have been shown to activate AMPK and heat stress has been shown to both enhance hippocampal LTP and activate AMPK, AMPK activation likely represents a common mechanism of action explaining the therapeutic actions of several chemically distinct compounds and methodologies in the promotion of synaptic plasticity and LTP [38,39].  Indeed, a recent study demonstrated that high frequency stimulation of hippocampal neurons in the dentate gyrus activates AMPK and induces early-phase long-term potentiation (E-LTP) in vivo in rats [40]. E-LTP was prevented by pharmacological inhibition of AMPK, highlighting a critical role for AMPK in learning and memory [40]. High frequency deep brain stimulation of the lateral habenula (an area of the brain that plays an important role in emotion, motivation, and reward) has also been shown to activate AMPK, facilitating antidepressant actions in an animal model of tricyclic antidepressant resistance [41].  Resveratrol, a phytoalexin found in grapes and red wine, has also been shown to rapidly increase  protein levels and synaptic accumulation of the AMPA receptor and increase the strength of excitatory synaptic transmission in rat primary neurons via AMPK activation [42]. Because AMPA receptors mediate fast excitatory transmission and are essential for the induction of LTP and synaptic plasticity, AMPK activation likely represents a central node underlying the beneficial effects of heat stress and compounds such as sulforaphane in ameliorating neuro-developmental effects associated ASD.

Interestingly, because knockdown of TRPV3 channels (channels that are activated in response to mild temperature elevations and by compounds including carvacrol) attenuates LTP in hippocampal pyramidal neurons and because TRPV3 channels are also located on oocytes, it would be expected that mild and transient heat stress would enhance or promote oocyte maturation and/or activation.  Indeed, AMPK activation has been consistently shown to play a critical role in the induction of oocyte meiotic resumption and maturation in preparation for oocyte activation and heat stress has also been shown to stimulate oocyte meiotic resumption and maturation in an AMPK-dependent manner [43, 44]. Intriguingly, the proteasome inhibitor MG132 and methylene blue have both been shown to activate AMPK, alleviate accelerated aging defects in fibroblasts derived from Hutchinson-Gilford progeria syndrome (HGPS) patients, and stimulate oocyte meiotic resumption, providing compelling evidence that AMPK activation represents a central node linking the therapeutic effects of chemically distinct compounds that facilitate the creation of human life [20,27,45-48].

Furthermore, Carvacho et al. demonstrated that the TRPV3 channel is differentially expressed in mouse oocytes during maturation and reaches peak density and activity at metaphase II, the stage at which oocytes develop the competency to initiate Ca2+ oscillations in response to fertilization [49].  The authors also showed that strontium chloride (SrCl2), a compound that induces parthenogenetic activation of mammalian oocytes and has resulted in successful term pregnancies and the birth of normal children, promotes Ca2+ oscillations and induces oocyte activation via TRPV3-mediated Sr2+ influx, as TRPV3 deletion in oocytes (TrpV3−/−) failed to respond to Sr2+-induced activation [49,50].  As the TRPV3 channel is Ca2+ permeable and SrCl2-induced oocyte activation is thought to occur via mimicking Ca2+ by sensitizing and potentiating IP3 receptors, the authors also showed that application of the TRPV3 agonist carvacrol in heterozygous oocytes (TrpV3+/−) led to a substantial increase in intracellular Ca2+ levels as well as parthenogenesis in both TrpV3+/− and wild-type oocytes (TrpV3+/+) (as measured by pronuclear formation and cleavage to the 2-cell stage), whereas TRPV3-deficient oocytes failed to respond [49]. Because TRPV3 channels are also critical for hippocampal LTP in the brain and because the TRPV3 channel agonist carvacrol activates AMPK and AMPK activation is essential for the induction of hippocampal LTP, activation of AMPK is likely critical for the creation of all human life (via oocyte activation) and for the processes of learning and memory formation [51].

Interestingly, recent studies have also demonstrated that knockout of HSF1 in oocytes leads to depletion of HSP90alpha, delayed meiotic resumption, and defective asymmetrical division [52]. As HSP90 has been shown to interact with and maintain AMPK activity and AMPK activation is essential for efficient T cell activation, heat stress or the induction of mediators of the heat shock response would be expected to promote the reactivation of latent HIV-1 in CD4+ memory T cells via low-level T cell activation, a method known as the “shock and kill” approach [51,53]. T cell activation-induced latent HIV-1 reactivation will likely facilitate destruction of the virus through immune-system detection or via virus-induced destruction of the host cell [51]. Indeed, recent studies have demonstrated that T cell activation at fever temperatures (39.5°C) activates HSF1 and HSF1 is essential for T cell proliferation in vitro [54,55]. Also, knockdown of AMPK and CaMKK2 (an upstream activator of AMPK) has been shown to significantly inhibit HIV-1 replication [56]. Strikingly, Roesch et al. showed that HIV-1 replication was increased 2 to 7 fold by culturing primary CD4+ T lymphocytes at a fever-like temperature (39.5°C) and that hyperthermia enhanced HIV-1 reactivation in a model of latently-infected cells in a HSP90-dependent manner [57].  Several recent studies have also demonstrated that HSP90 promotes HIV-1 reactivation from latency in CD4+ T cells by enhancing the activity of host cell several transcription factors (NF-κB, NFAT, and STAT5) that are critical for both T cell activation and HIV-1 reactivation and replication [58,59].  Particularly compelling is a recent study by Pan et al. showing that HSF1 participates in HIV-1 transcription and is essential for latent HIV-1 reactivation by binding to the HIV 5'-long terminal repeat (LTR) to reactivate viral transcription [60].  Overexpression of HSF1 improved HIV transcription whereas knockout of  HSF1 inhibited HIV transcription [60].  Interestingly, in this same study, resveratrol, MG132, and hemin (an iron-containing porphyrin) were also shown to reactivate latent HIV-1 in a T cell line [60]. Similar to resveratrol and MG132, hemin has also been shown to activate AMPK, again placing AMPK activation as a centerpiece facilitating the reactivation of latent HIV-1, oocyte meiotic induction and activation, and learning and memory formation induced by LTP [61].

Activation of mediators of the heat shock response may also play critical roles in other physiological processes that are essential for the creation and the beginnings of human life.  As noted above, in addition to heat stress, HSPs also respond to other forms of cellular stress, including ROS and intracellular Ca2+ increases, both of which activate AMPK [13-15].  Additionally, HSP90 has been found to localize in the neck, midpiece, and tail regions of human sperm and HSP90 inhibition significantly decreases intracellular Ca2+ concentrations during capacitation, a process that is essential for oocyte fertilization [62].  Indeed, an increase in the levels of intracellular Ca2+ is critical for the initiation of the acrosome reaction in sperm, a process that facilitates sperm penetration of the oocyte and is thus indispensable for fertilization.  Interestingly, ROS and vitamin D, both of which activate AMPK, have each been shown to induce the acrosome reaction in sperm, indicating that induction of cellular stress is critical for the promotion of the acrosome reaction in sperm, facilitating the creation of human life [15,28,63,64].  Vitamin D has also been shown to improve accelerated cellular aging defects in progeria, indicating that the creation of human life and the amelioration of symptoms of accelerated aging may depend on the induction of a cellular stress response [22].  Heat stress has also been shown to promote the differentiation of human adult stem cells and AMPK activation has also recently been shown to be critical in embryonic development by facilitating the differentiation of mouse embryonic stem cells into endoderm, again indicating that AMPK activation is critical, if not indispensable, for the creation and beginnings of life [65,66,67].

In conclusion, the evidence presented above strongly supports the provocative implication that the induction of cellular stress, mediated by heat, ROS, intracellular Ca2+ increases, an AMP/ATP ratio increase, etc. leads to a beneficial compensatory cellular response.  The induction of this cellular response and subsequent activation of AMPK by chemically distinct compounds including sulforaphane, MG132, vitamin D, rapamycin, methylene blue, carvacrol, and likely many others indicates that cellular stress-induced AMPK activation may represent a central node in the amelioration of disease and the creation of human life. Indeed, cell and context-specific AMPK activation may induce a “shock to live” (e.g. oocyte maturation/activation, stem cell differentiation, learning and memory in the brain, sperm capacitation/acrosome reaction) or a “shock to kill” (e.g. latent HIV-1 reactivation, cancer stem cell differentiation and/or cell death).

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