AMPK links cognitive decline reversal in Alzheimer’s disease with Progeria, HIV-1 latency, & Oocyte activation: The "Shock and Live" approach

Hey Blogger Fam, take a look at my most recent post on LinkedIn when you have a minute.  Basically, a recent human study showed that Alzheimer's disease is likely reversible, contradicting what's commonly accepted in the medical profession (i.e. that it's irreversible). The super interesting thing about this study was that the protocol that the patients adhered to is just a contextual redefinition of the “Shock and Live” approach, consisting of changes in diet, exercise, and vitamin and herbal supplementation that synergistically causes the induction of cellular stress (i.e. "shock"), leading to a beneficial response in brain cells (i.e. "live”) mediated by the activation of the master metabolic regulator AMPK.  Indeed, most of the components of the protocol, including a low carb/sugar diet, fasting, caloric restriction, exercise, EPA/DHA (fish oil), melatonin, folic acid, selenium, alpha-lipoic acid, acetyl-l-carnitine, resveratrol, curcumin, CoQ10, probiotics, and prebiotics all induce a cellular stress response, activating AMPK and bringing “life” back into cells of the brain. AMPK activation has been shown to promote neurogenesis (birth of new brain cells), increase the growth of new neurons and synapses, promote the viability of neural stem cells (which produce new brain cells), and promote learning and memory formation.

Two things that really caught my eye were the use of brain stimulation and vitamin D.  Although the patients used learning-based simulations to stimulate the brain, electrical pulses/stimulation (“shock”) have been shown to activate AMPK and produce beneficial effects in many people with neurological disorders (Parkinson’s, depression, tremor, obsessive-compulsive disorder).  The cool connection is that electrical pulses have also been shown to activate oocytes, leading to the birth of healthy children.  Thus, AMPK activation via a “shock” likely explains how invasive procedures like deep-brain stimulation produce efficacious results as well as how all human life is created.  The other REALLY cool thing about the study is vitamin D.  Not only does vitamin D promote the activation of T cells (enhances the immune response), but it also induces a slight “shock” (in the form of an increase in calcium), bringing back to life (i.e. “live”) cells taken from Progeria kids that are rapidly aging and nearing death. This slight “shock” (calcium) is also what activates oocytes, bringing forth new life.  Interestingly, vitamin D has also been shown to induce the acrosome reaction in human sperm, a process that allows sperm to penetrate the egg, and without which no human being could be created via “the old fashion way”:-).  That “shock”, in the form of calcium, also ignites the acrosome reaction in sperm.  Unsurprisingly, vitamin D as well as compounds that increase calcium (“shock”) and have been used to activate both human oocytes and induce the acrosome reaction in human sperm activate AMPK.  And here’s a little extra nugget from something I’m working on.  AMPK activation is critical during a  “shock” to embryonic stem cells, forcing those cells to develop into different cells (liver, pancreas, gut, urethra, etc.) that will eventually form the baby, another rendition of the “Shock and Live” approach.  Let’s see….a “shock” induces oocyte activation, the sperm acrosome reaction, neural stem cell viability, progeria cell growth, and development of a baby from an embryo.  It looks as if the “Shock and Live” approach may be a universal theme in human biology.  If this turns out to be true…………..:-)  

AMPK links cognitive decline reversal in Alzheimer’s disease with Progeria, HIV-1 latency, & Oocyte activation: The "Shock and Live" approach 


https://www.linkedin.com/pulse/ampk-links-cognitive-decline-reversal-alzheimers-disease-finley?trk=prof-post  

In a small but highly publicized study conducted by researchers from the University of California, Los Angeles and the Buck Institute for Research on Aging, the use of a personalized therapeutic program to improve or reverse cognitive decline in 10 patients with early Alzheimer’s disease led to a clear improvement in mild and subjective cognitive impairment (MCI and SCI, respectively--Alzheimer’s precursors) for all ten patients, with a dramatic and unprecedented reversal of cognitive decline for two patients with well documented Alzheimer’s disease [32].  The therapeutic approach, known as metabolic enhancement for neurodegeneration (MEND), is a multi-pronged protocol designed to promote metabolic enhancement that consists of dietary modifications, exercise, vitamin and herbal supplementation, stress reduction incorporation, exercise, and hormonal balancing [33].       

After 10 months on the program, patient 1 (a 66-year-old professional male with documented Alzheimer’s disease, a strongly positive amyloid PET scan, and abnormal neuropsychological studies) experienced an increase in hippocampal volume from the 17^th to the 75^th percentile and an absolute increase in hippocampal volume of 11.7% as measured via volumetric analyses by Neuroquant [32].  Interestingly, interruption of compliance to the protocol led to an episode of memory loss for the patient (failed to remember that he left the car in the driveway idling).  However, re-adherence to the protocol led to marked symptomatic improvement (memory improvement and “work came more easily to him”) in addition to a significant increase in hippocampal volume [32].

Patient 2, a 69-year-old professional male and entrepreneur, also had well-documented Alzheimer’s disease, with an FDG PET scan showing reduced glucose utilization in the parietotemporal cortices and the temporal lobes, a reduction in CVLT (California Verbal Learning Test), auditory delayed memory at the 13^th percentile, and a Stroop color test at the 16^th percentile. The patient also reported subjective instances of memory loss (difficulty recognizing faces at work) and declines in cognitive capacity (loss the ability to rapidly add columns of numbers in his head) [32]. After 6 months on the MEND program, the patient’s ability to recognize faces at work and to rapidly add columns of numbers in his head returned. Strikingly, quantitative neuropsychological testing after 22 months on the program revealed an increase from the 3^rd to the 84^th percentile for CVLT-IIB, reverse digit span from the 24^th to the 74^th percentile, auditory delayed memory from the 13^th to the 79^th percentile, and CVLT-II from the 54^th to the 96^th percentile [32].

Interestingly, the authors also noted that the improvement experienced by all 10 patients had been sustained and no patient exhibited the cognitive decline characteristic of Alzheimer’s disease, despite claims that Alzheimer’s disease can not be prevented, delayed, or reversed [32,34].

The details of the MEND protocol (reproduced and adapted below) clearly indicate that a significant number of disparate modalities and compounds that synergistically modulated metabolism, leading to a striking reversal of cognitive decline in patients diagnosed with early Alzheimer’s disease, are in fact inducers of a cellular stress response. The induction of cellular stress leads to the activation of the master metabolic regulator AMPK (see red notations for references).  Synergistic AMPK activation by this protocol is essentially a contextual redefinition of the “Shock and Live” approach that defines the process of oocyte activation, a mechanism that is responsible for the creation of all human life.

(Adapted from: Bredesen DE. Reversal of cognitive decline: a novel therapeutic program. Aging (Albany NY). 2014 Sep;6(9):707-17.

Therapeutic System 1.0

Goal	Approach	Rationale and References
Optimize diet: minimize simple CHO, minimize inflammation.	Patients given choice of several low glycemic, low inflammatory, low grain diets.  Glucose deprivation activates AMPK [1,2].	Minimize inflammation, minimize insulin resistance.
Enhance autophagy, ketogenesis	Fast 12 hr each night, including 3 hr prior to bedtime. Fasting and caloric restriction activates AMPK.  AMPK stimulates brain ketogenesis and authophagy [3-6].	Reduce insulin levels, reduce Aβ.
Reduce stress	Personalized—yoga or meditation or music, etc.  AMPK reduces cortiol levels [7].	Reduction of cortisol, CRF, stress axis.
Optimize sleep	8 hr sleep per night; melatonin 0.5mg po qhs; Trp 500mg po 3x/wk if awakening. Exclude sleep apnea.  Melatonin activates AMPK [8].	[36]
Exercise	30-60′ per day, 4-6 days/wk.  Aerobic exercise activates AMPK [9].	[37, 38]
Brain stimulation	Posit or related.  Brain stimulation via high frequency stimulation (HFS) activates AMPK [10].	[39]
Homocysteine <7	Me-B12, MTHF, P5P; TMG if necessary.  MTHF (Folic acid) activates AMPK [11].	[40]
Serum B12 >500	Me-B12	[41]
CRP <1.0; A/G >1.5	Anti-inflammatory diet; curcumin; DHA/EPA; optimize hygiene. EPA, DHA, and curcumin activate AMPK [12-14].	Critical role of inflammation in AD
Fasting insulin <7; HgbA1c <5.5	Diet as above	Type II diabetes-AD relationship
Hormone balance	Optimize fT3, fT4, E2, T, progesterone, pregnenolone, cortisol. fT3, E2, and T activate AMPK [15-17].	[5, 42]
GI health	Repair if needed; prebiotics and probiotics. Prebiotics and probiotics activate AMPK [18,19].	Avoid inflammation, autoimmunity
Reduction of A-beta	Curcumin, Ashwagandha.  Curcumin activates AMPK.  Ashwagandha likely activates AMPK [12,20].	43-45
Cognitive enhancement	Bacopa monniera, MgT.  MgT likely activates AMPK [21].	[46, 47]
25OH-D3 = 50-100ng/ml	Vitamins D3, K2.  Vitamin D3 activates AMPK [22].	[48]
Increase NGF	H. erinaceus or ALCAR.  ALCAR activates AMPK [23].	[49, 50]
Provide synaptic structural components	Citicoline, DHA. DHA activates AMPK [14].	[51].
Optimize antioxidants	Mixed tocopherols and tocotrienols, Se, blueberries, NAC, ascorbate, α-lipoic acid.  Tocotrienols, Se, blueberries, and α-lipoic acid activate AMPK [24-27].	[52]
Optimize Zn:fCu ratio	Depends on values obtained	[53]
Ensure nocturnal oxygenation	Exclude or treat sleep apnea	[54]
Optimize mitochondrial function	CoQ or ubiquinol, α-lipoic acid, PQQ, NAC, ALCAR, Se, Zn, resveratrol, ascorbate, thiamine.  CoQ10, α-lipoic acid, ALCAR, Se, and resvertrol activate AMPK.  PQQ likely activates AMPK [23,25-30].	[55]
Increase focus	Pantothenic acid	Acetylcholine synthesis requirement
Increase SirT1 function	Resveratrol. Resveratrol activates AMPK [26,29].	[32]
Exclude heavy metal toxicity	Evaluate Hg, Pb, Cd; chelate if indicated	CNS effects of heavy metals
MCT effects	Coconut oil or Axona.  MCT/Coconut oil likely activates AMPK [31].	[56]

CHO, carbohydrates; Hg, mercury; Pb, lead; Cd, cadmium; MCT, medium chain triglycerides; PQQ, polyquinoline quinone; NAC, N-acetyl cysteine; CoQ, coenzyme Q; ALCAR, acetyl-L-carnitine; DHA, docosahexaenoic acid; MgT, magnesium threonate; fT3, free triiodothyronine; fT4, free thyroxine; E2, estradiol; T, testosterone; Me-B12, methylcobalamin; MTHF, methyltetrahydrofolate; P5P, pyridoxal-5-phosphate; TMG, trimethylglycine; Trp, tryptophan

As initially proposed in my most recent publication, AMPK activation is likely critical, if not indispensable, for oocyte activation via the induction of a cellular stress response (mediated by an increase in intracellular calcium levels, an AMP/ATP ratio increase, or reactive oxygen species (ROS) generation) [35].  AMPK has been shown to be critical for the resumption and maturation of meiosis in oocytes, two processes that are critical for efficient sperm- or artificially-induced oocyte activation [35].  Interestingly, several distinct compounds and modalities that induce a cellular stress response have been used to artificially activate oocytes during in vitro fertilization procedures to produce healthy children, including electrical stimulation and the calcium (Ca²⁺) ionophores ionomycin and A23187 (increases intracellular Ca²⁺ levels similar to sperm-induced oocyte activation) [35-37]. 

The “Shock and Live” approach is also analogous to the “Shock and Kill” approach, a method currently being pursued by HIV-1 cure researchers to potentially eradicate HIV-1 [35].  The “shock and kill” approach involves reactivating (i.e. “shock”) a T cell that harbors dormant HIV-1, hence reactivating the virus itself and thus inducing destruction of the T cell along with the virus or enhancing recognition and destruction of the virus-infected T cell by the immune system (i.e. “kill”) [35].  AMPK is also critical for the activation of T cells and the mounting of an effective immune response to eliminate viruses, bacteria, and cancer cells [35,38,39].  Interestingly, the same compounds that have been used to induce a “shock” to initiate the creation of human life (i.e. ionomycin and A23187) have also been used in combination with other compounds to initiate a “shock” to facilitate T cell activation and thus reactivate dormant HIV-1  [35,40]. Ca²⁺ ionophores including ionomycin have also been used to induce a “shock” to activate cytotoxic CD8⁺ T cells, a T cell subset that is critical for the destruction of viruses such as HIV-1 and cancer cells. AMPK activation has also been shown to promote the formation of long-lived cytotoxic CD8⁺ memory T cells [38,39,41].

Unsurprisingly, AMPK activation has been shown to promote neurogenesis, increase the levels of BDNF (a protein essential for the growth and differentiation of new neurons and synapses), promote viability of neural stem cells, and enhance intracellular mechanisms that promote learning and memory formation [42-44].  As AMPK is activated as a result of the induction of cellular stress, many of the components of the MEND protocol likely activate AMPK by inducing a “shock”, thus enhancing and sustaining the viability of higher level brain functioning (i.e. “live”).  Indeed, as evident by the chart above and the associated references, glucose deprivation, fasting, caloric restriction, and exercise all induce varying levels of “shock” (mediated by increased levels of ROS, intracellular Ca^2+, or AMP/ATP ratio), leading to the activation of AMPK and AMPK-induced autophagy (“live”).  Several naturally-occurring compounds, including EPA, DHA, melatonin, folic acid, selenium, alpha-lipoic acid, and acetyl-l-carnitine also activate AMPK, likely via the induction of a cellular stress response.  Additionally, as also noted from the chart, brain stimulation via electrical pulses also leads to the activation of AMPK (and long-term potentiation—a process that underlies learning and memory), similar to oocyte activation induced by electrical stimulation.   

Interestingly, resveratrol, a polyphenol produced by several plants that activates AMPK, has been shown to not only promote long-term potentiation but also enhance reactivation of latent HIV-1 while an analog of resveratrol has been shown to promote meiotic resumption in mouse oocytes (a model for human oocytes), indicating that an appropriate “shock” or cellular stress induction characterizes beneficial physiological responses in oocytes, T cells, and brain cells [45-47]. Curcumin (a compound derived from the plant Curcuma longa) and butyrate (a short chain fatty acid produced by certain gut bacteria/probiotics) have also been shown to increase neural stem cell differentiation and enhance latent HIV-1 reactivation [48-51].

A particularly striking example of an AMPK-orchestrated “Shock and Live” approach is exemplified by 1α,25-dihydroxyvitamin D3 (vitamin D).  In addition to activating AMPK in vivo, vitamin D has recently been shown to profoundly improve nuclear morphology, significantly reduce DNA damage, improve cellular proliferation, delay premature cellular senescence, and dramatically reduce progerin production in patients diagnosed with the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) [52].  As vitamin D has been shown to increase Ca²⁺ levels in the cell, HGPS cells treated with vitamin D initially experienced a slight decrease in proliferation (i.e. “shock”) followed by an increased improvement in growth rate compared to control cells, effectively delaying premature entry into cellular senescence (i.e. “live”) [52]. 

Vitamin D administered to HIV-1 patients also led to an increase in activated CD4⁺ T cells in a placebo-controlled randomized study [53].  The phorbol ester PMA/TPA (which has been shown to induce mouse oocyte activation when combined with Ca²⁺ ionophores) and vitamin D have also been shown to stimulate HIV-1 proviral activation to similar levels in a cell line latently-infected with a monocytotropic strain of HIV-1JR-FL [54,55].

Startlingly, vitamin D has also been shown to induce the acrosome reaction in human sperm, a process that is indispensable for sperm-induced fertilization by facilitating penetration of the oocyte plasma membrane [56].  Interestingly, similar to oocyte activation, an increase in intracellular Ca²⁺ levels (i.e. “shock”) is also critical for the induction of the acrosome reaction in sperm and the same compounds that have been used to activate human oocytes to produce healthy children (e.g. A23187) (i.e. “live”) have also been used to induce the acrosome reaction in human sperm (i.e. “live”), indicating that the acrosome reaction is also likely dependent on AMPK activation [57].

The induction of cellular stress and the activation of AMPK thus appear to be a central biological theme that is likely responsible for the creation of all human life, the promotion of lifespan and healthspan, and the potential amelioration or eradication of disease.  Just as the “Shock and Live” approach defines the beginnings of life, the activation of T cells, and the improvement or reversal of accelerated aging symptoms in HGPS, AMPK activation, via application of the “Shock and Live” approach, also likely represents a common mechanism of action through which distinct compounds and methodologies are able to ameliorate or reverse neurodegeneration.     

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New NIH study shows that the drug Oltipraz, an AMPK and Nrf2 activator that inhibits HIV replication, reverses nuclear aging defects in Progeria cells

"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.

Hey Blogger fam, I’m actually really starting to believe I might be on to something:-) Check out my recent LinkedIn post below about a new study published by researchers at the National Institutes of Health (NIH) that provides yet more evidence that a common pathway connects normal aging, Progeria, and HIV-1. What's really interesting about this study is that the compound used (oltipraz) to reverse aging defects in cells taken from Progeria kids works just like the broccoli sprout compound sulforaphane, which was also shown back in 2013 to reverse aging defects in Progeria cells. What the researchers don't realize (and I'm not sure that I should be letting them know) is that oltipraz, just like vitamin D, methylene blue, sulforaphane, metformin, the Nobel-prize winning drug artemisinin, and rapamycin also activates the master metabolic regulator AMPK.  As I outlined in my recent publication, AMPK activation is critical for oocyte activation and the creation of all human beings alive today and any human being that has ever lived. AMPK activation is also absolutely essential for the activation of your T cells, allowing your immune system to fend off foreign invaders (viruses, bacteria, etc) as well as to target and kill cancer cells.  Indeed, oltipraz and sulforaphane (not sure if I should be telling this either) have both been shown to inhibit HIV-1 replication in actively infected cells.  This is absolutely HUGE in that AMPK activation will not only "wake up" HIV-1 that's asleep in T cells so that the immune system can kill it, but AMPK activation will also shut down HIV-1 replication in actively infected cells, basically doing what many combo HIV-1 medications were designed to do but without the toxicity.  EGCG, a compound found in green tea, tanshinone (an herb also known as red sage), resveratrol (from grapes and red wine), and curcumin (from the spice turmeric) have each been shown to potently inhibit active HIV-1 replication, with resveratrol also reactivating dormant HIV-1.  When you step back and take a look at the big picture, it almost leaves you breathless: The activation of a single pathway, AMPK, can reverse accelerated aging defects in Progeria cells, extend lifespan and healthspan in almost every organism its been tested in, reactivates dormant viruses to be recognized by the immune system, activates other cells of the immune system to target and kill those viruses as well as cancer cells, and is responsible for the creation of every human being that is alive or that has every lived. Ok, my brain is throbbing, time for a break:-) 

New NIH study shows that the drug Oltipraz, an AMPK and Nrf2 activator that inhibits HIV replication, reverses nuclear aging defects in Progeria cells

In line with recent findings (see prior posts) that a vitamin A metabolite (or its derivatives), methylene blue, vitamin D/vitamin D receptor (VDR) signaling, and sulforaphane each 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 Cell in June of 2016 strikingly demonstrated that the transcriptional activity of Nrf2, a transcription factor implicated in longevity and a master regulator of the antioxidant response, is impaired in cells derived from HGPS patient cells, leading to an increase in chronic oxidative stress [1].  Interestingly, the reactivation of Nrf2 in HGPS patient cells by the Nrf2 activator oltipraz reversed nuclear aging defects and also restored the in vivo viability of HGPS patient-derived mesenchymal stem cells (MSCs) that were implanted into animal models.  As oltipraz has been shown to activate AMPK (similar to metformin) and inhibit HIV-1 replication and AMPK has been shown to activate and increase the transcriptional activity of Nrf2, this study contributes to mounting evidence that further substantiates the novel observation that AMPK activation represents a common mechanism that leads to the amelioration or reversal of accelerated aging defects in HGPS, inhibition of HIV-1 replication in acutely infected cells, and reactivation of HIV-1 in latently infected cells (see below).

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 via the same faulty gene splicing mechanism) 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, the authors showed that the mutant protein progerin impaired the transcriptional activity (i.e. the ability to induce gene expression) and function of Nrf2 in HGPS fibroblasts, as indicated by a 51% reduction in the antioxidant-responsive element (ARE)-Luc activity [1].  Many of the beneficial effects of increased Nrf2 activity stems from the activation of antioxidant genes by Nrf2 via binding of Nrf2 to AREs located in antioxidant genes.  Inhibiting Nrf2 via RNA interference (RNAi) further exacerbated nuclear defects in HGPS cells and the application of hydrogen peroxide to HGPS cells to increase oxidative stress increased the levels of progerin and worsened markers of cellular defects, including 53BP1, LAP2, H3K27m3, γH2AX, and HP1γ [1].

Most importantly, however, the authors showed that oltipraz ameliorated lamin B1 and HP1γ levels in HGPS fibroblasts by 73% and 53%, respectively, and also partially reduced LAP2 and H3K27me3 defects [1]. Moreover, as the ability of mesenchymal stem cells (MSCs) to respond to oxidative stress is inhibited in HGPS, the authors also generated MSCs derived from HGPS cells (i.e. HGPS-iPSC-MSCs) to determine the effects of Nrf2 activation on MSC survival and viability in vivo when implanted into an animal model.  Expectedly, the HGPS-iPSC-MSCs expressed progerin which led to stem cell attrition. However, oltipraz-mediated Nrf2 activation rescued HGPS nuclear defects (e.g. increased levels of LAP2 and lamin B1), reduced the number senescence-associated β-galactosidase positive cells (a marker of cellular senescence), and restored the in vivo viability of HGPS-MSCs, indicating that activation of Nrf2 may play a key role in ameliorating or reversing the pathology associated with HGPS [1].

Just as other chemically distinct compounds, including methylene blue, retinoic acid and/or its derivatives, vitamin D/VDR signaling, and the Nrf2 activator sulforaphane have each been shown to ameliorate or reverse accelerated aging defects and activate the master metabolic regulator AMPK, the Nrf2 activator oltipraz would also be expected to activate AMPK. Indeed, several independent studies have shown that oltipraz and/or its metabolites induce activation of AMPK, increase expression of genes that encode proteins involved in mitochondrial fuel oxidation, increase mitochondria DNA content and oxygen consumption rate, reduce cellular reactive oxygen species (ROS) production, activate LKB1 (an upstream activator of AMPK), and increase the AMP/ATP ratio (an indication of cellular stress induction) [2-6].  Interestingly, AMPK has also been shown to increase the transcriptional activity of Nrf2 and a recent study has also demonstrated that AMPK phosphorylates Nrf2, leading to retention of Nrf2 in the nucleus [7,8].

Interestingly, as the Nrf2 activator sulforaphane has been shown to stimulate the immune response by enhancing the cytotoxicity of natural killer cells and dendritic cells, promote the infiltration of T cells in neoplastic regions, and 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), the Nrf2 activator oltipraz would also be expected to possess immune-modulating effects that would impact the life cycle of HIV-1 [9-14].  Indeed, several studies have shown that oltipraz and/or its metabolites inhibit replication of HIV-1 and a recent study demonstrated that sulforaphane inhibited HIV-1 replication in macrophages through Nrf2 mobilization [15-20].

Although, the inhibition of HIV-1 replication and the reactivation of latent HIV-1 via enhancing T cell activation are seemingly at odds, the activation of AMPK as a common mechanism by sulforaphane and oltipraz provides an explanation for this dichotomy.  AMPK is critical for the activation of T cells and compounds that have been shown to potently reactivate latent HIV-1 activate AMPK and also induce selective low-level T cell activation during HIV-1 latency reversal [21].  Both AMPK and Nrf2 have also been shown to inhibit HIV-1 replication in productively infected cells (i.e. active), indicating that AMPK activation likely exerts dually beneficial roles via inhibition of active HIV-1 replication and reactivation of latent HIV-1 reservoirs [22,23].  This duality was recently demonstrated in a study in which the proteasome inhibitor Velcade (bortezomib) reactivated latent HIV-1 but also inhibited HIV-1 infectivity, as evidenced by reduced viral output and virion infectivity [24].

In light of the AMPK-activating and HIV-1 inhibitory effects exerted by oltipraz, the Cell study by NIH researchers demonstrating that oltipraz reverses nuclear aging defects and restores the in vivo viability of HGPS patient-derived MSCs is indicative of a common mechanism of AMPK activation that links diseases as seemingly disparate as HGPS, HIV1-1 latency and replication, and potentially many others (see Figure below).  As supporting evidence of this connection continues to mount, a paradigm shift recognizing the interconnectedness of disease pathology is inevitable.

https://www.linkedin.com/pulse/new-nih-study-shows-drug-oltipraz-ampk-nrf2-activator-finley?trk=pulse_spock-articles

References:

1. Kubben N, Zhang W, Wang L, et al. Repression of the Antioxidant NRF2 Pathway in Premature Aging. Cell. 2016 Jun 2;165(6):1361-74. doi: 10.1016/j.cell.2016.05.017.
2. Kim TH, Eom JS, Lee CG, Yang YM, Lee YS, Kim SG. An active metabolite of oltipraz (M2) increases mitochondrial fuel oxidation and inhibits lipogenesis in the liver by dually activating AMPK. Br J Pharmacol. 2013 Apr;168(7):1647-61. doi: 10.1111/bph.12057.
3. Bae EJ, Yang YM, Kim JW, Kim SG. Identification of a novel class of dithiolethiones that prevent hepatic insulin resistance via the adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway. Hepatology. 2007 Sep;46(3):730-9.
4. Shin SM, Kim SG. Inhibition of arachidonic acid and iron-induced mitochondrial dysfunction and apoptosis by oltipraz and novel 1,2-dithiole-3-thione congeners. Mol Pharmacol. 2009 Jan;75(1):242-53. doi: 10.1124/mol.108.051128.
5. Kwon YN, Shin SM, Cho IJ, Kim SG. Oxidized metabolites of oltipraz exert cytoprotective effects against arachidonic acid through AMP-activated protein kinase-dependent cellular antioxidant effect and mitochondrial protection. Drug Metab Dispos. 2009 Jun;37(6):1187-97.
6. Hwahng SH, Ki SH, Bae EJ, Kim HE, Kim SG. Role of adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway in repression of liver X receptor-alpha-dependent lipogenic gene induction and hepatic steatosis by a novel class of dithiolethiones. Hepatology. 2009 Jun;49(6):1913-25.
7. Mo C1, Wang L, Zhang J, et al. The crosstalk between Nrf2 and AMPK signal pathways is important for the anti-inflammatory effect of berberine in LPS-stimulated macrophages and endotoxin-shocked mice. Antioxid Redox Signal. 2014 Feb 1;20(4):574-88.
8. Joo MS, Kim WD, Lee KY, Kim JH, Koo JH, Kim SG. AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at serine 550. Mol Cell Biol. 2016 May 9. pii: MCB.00118-16. [Epub ahead of print].
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. 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.
15. Prochaska HJ, Fernandes CL, Pantoja RM, Chavan SJ. Inhibition of human immunodeficiency virus type 1 long terminal repeat-driven transcription by an in vivo metabolite of oltipraz: implications for antiretroviral therapy. Biochem Biophys Res Commun. 1996 Apr 25;221(3):548-53.
16. Chavan SJ, Bornmann WG, Flexner C, Prochaska HJ. Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: evidence for the formation of a stable adduct. Arch Biochem Biophys. 1995 Dec 1;324(1):143-52.
17. Prochaska HJ, Bornmann WG, Baron P, Polsky B. Inhibition of human immunodeficiency virus type 1 replication by 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine, an in vivo metabolite of oltipraz. Mol Pharmacol. 1995 Jul;48(1):15-20.
18. Prochaska HJ, Chavan SJ, Baron P, Polsky B. Oltipraz, a novel inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. J Cell Biochem Suppl. 1995;22:117-25.
19. Prochaska HJ, Yeh Y, Baron P, Polsky B. Oltipraz, an inhibitor of human immunodeficiency virus type 1 replication. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3953-7.
20. Furuya AK, Sharifi HJ, Jellinger RM, Cristofano P, Shi B, de Noronha CM. Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2. PLoS Pathog. 2016 Apr 19;12(4):e1005581.
21. 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.
22. Zhang HS, Chen XY, Wu TC, Zhang FJ. Tanshinone II A inhibits tat-induced HIV-1 transactivation through redox-regulated AMPK/Nampt pathway. J Cell Physiol. 2014 Sep;229(9):1193-201.
23. Zhang HS, Li HY, Zhou Y, Wu MR, Zhou HS. Nrf2 is involved in inhibiting Tat-induced HIV-1 long terminal repeat transactivation. Free Radic Biol Med. 2009 Aug 1;47(3):261-8.
24. 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.

Vitamin D significantly improves accelerated aging defects in cells from Progeria patients: 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.

Ok Blogger family, this is an absolutely HUGE study that makes the proposals in my publications come scarily close to being true:-) My LinkedIn post below describes an astounding study (just indexed on Pubmed yesterday) that shows that none other than our good (and popular) friend vitamin D actually decreased DNA damage, improved the shape of the nucleus, dramatically decreased the levels of the toxic protein progerin, and increased the lifespan of cells taken from Progeria patients. What's really interesting is that vitamin D signaling was also decreased in older cells taken from the Progeria kid's parents. Because normal unaffected people (you and I) make the same toxic protein (progerin) as Progeria kids make, just at a lower level that increases with age, it looks like good doses of vitamin D may also help to slow the aging process in us as well. And, of course, vitamin D is an activator of AMPK, just like metformin, the Nobel Prize-winning drug artemisinin, and all the rest of the compounds that reverse aging defects in Progeria (e.g. rapamycin, sulforaphane, retinoic acid, methylene blue). Vitamin D signaling is also vital for the activation of your T cells and to mount a healthy immune response to bacteria, viruses, and other pathogens. Indeed, vitamin D was shown in a study to enhance the reactivation of dormant HIV-1. If evidence continues to support the notion that diseases such as HIV-1, Progeria, and even cancer are connected by a similar pathway and that many natural compounds/drugs ward off disease and increase lifespan by activating this pathway,.........Man-oh-Man-oh-Man:-)

Vitamin D significantly improves accelerated aging defects in cells from Progeria patients: Connection between AMPK, aging, and HIV-1 reactivation

In line with recent evidence demonstrating that the AMPK activators rapamycin, sulforaphane, retinoic acid, and methylene blue ameliorated or reversed accelerating aging defects in cells derived from Hutchinson-Gilford progeria syndrome (HGPS) patients, a recent study published online in the journal Oncotarget in May of 2016 provided astonishing evidence that 1α,25-dihydroxyvitamin D3 (1,25D), the most potent metabolite of Vitamin D, profoundly improved nuclear morphology, significantly reduced DNA damage, improved cellular proliferation, delayed premature cellular senescence, and dramatically reduced progerin production in HGPS patient cells [1].  Because vitamin D has also been shown to significantly induce AMPK activation in vivo in mice, alter gene splicing, facilitate T cell activation, and enhance reactivation of latent HIV-1 in certain cell types (see below), the Oncotarget study further strengthens the connection between latent HIV-1 reactivation and HGPS, with AMPK activation representing an “indirect yet common mechanism of action” linking chemically distinct compounds.

1,25D, also known as calcitriol, is the hormonally activate metabolite of vitamin D and exerts many of its calcium regulatory activities via binding to the vitamin D receptor (VDR) [2]. The VDR is a member of the nuclear receptor family of transcription factors that also includes the retinoic acid receptor (RAR), the thyroid hormone receptor (THR), and the retinoid X receptor (RXR) [2]. Upon vitamin D binding to the VDR, the vitamin D/VDR complex heterodimerizes (i.e. forms a pair) with the RXR, and the vitamin D/VDR/RXR complex then binds to sections of DNA to promote gene transcription and expression of gene products [2].  Interestingly, the VDR is also expressed in tissues that are not associated with bone or calcium regulation, indicating that vitamin D/VDR signaling exerts pleiotropic effects [1].

Indeed, the authors in the Oncotarget study initially demonstrated that depletion of lamin A/C (the protein adversely affected in HGPS) markedly decreased VDR levels in normal fibroblasts derived from parents of HGPS patients and ectopic expression of progerin (the toxic protein product that causes accelerated aging phenotypes in HGPS) also leads to a down-regulation of VDR in other fibroblast cell types [1].  Interestingly, VDR levels were decreased during proliferation of both HGPS patient fibroblasts and normal fibroblasts isolated from the parent of the patient, implicating decreased VDR expression as a common biomarker in both HGPS and in normal aging [1].

The authors next observed that depletion of VDR in two separate lines of normal fibroblasts isolated from the parents of HGPS patients led to a decrease in the levels of the DNA repair protein BRCA1, accumulation of DNA damage (e.g. elevated γH2AX levels), and eventual growth arrest and senescence (e.g. β-galactosidase positive cells) [1].  VDR loss in HGPS patient fibroblasts of early passage also led to BRCA1 reduction and accumulation of DNA damage, suggesting that VDR signaling is critical for maintaining genomic integrity in both normal and HGPS cells [1].

Most importantly, however, the authors showed that prolonged treatment of HGPS cells with 1,25D, compared to control cells, led to an increase in the levels of VDR, BRCA1, and 53BP1 (another protein essential for DNA repair) [1].  Prolonged treatment of HGPS cells with 1,25D also substantially reduced both progerin transcript and protein levels, indicating that 1,25D may beneficially alter disease pathology in HGPS.  As accumulation of progerin causes hallmark distortions of nuclear shape in HGPS cells, the authors also showed that treatment of HGPS cells with 1,25D led to a profound improvement in nuclear morphology, as evidenced by a marked decrease in the percentage of cells with abnormally shaped nuclei in an analysis of over 500 cells per condition [1].  Lastly, compared to vehicle-treated cells, 1,25D-treated HGPS cells (two separate HGPS cell lines) grew at a higher rate with passage in culture, effectively delaying premature entry into cellular senescence [1].

Similar to the beneficial effects demonstrated by rapamycin, sulforaphane, retinoic acid, and methylene blue in HGPS cells, 1,25D’s ability to reduce DNA damage, improve nuclear morphology, and delay premature senescence strongly suggests that each of these compounds (and likely many others) share a common mechanism of action that likely involves activation of the master metabolic regulator AMPK.  Independent studies have shown that AMPK activation improves lifespan, is critical for T cell activation, and beneficially alters gene splicing in genetic disorders [3-5]. Metformin, an AMPK activator, also beneficially altered splicing of the insulin receptor gene in normal human diabetics that were not affected with a genetic disorder [5].

Indeed, 1,25D has been shown to activate AMPK in vivo in mice as well as alter gene splicing in cancer cells [6,7,13].  Interestingly, 1,25D also plays a critical role in immune system regulation, as evidenced by an increase in activated CD4+ T cells in HIV-1 patients administered 1,25D in a placebo-controlled randomized study [8].  VDR signaling plays an integral role in T cell activation, with T cell receptor triggering inducing an upregulation of PLC-γ1 (a protein critical for T cell activation) that is dependent on 1,25D and expression of the VDR [9,10].  Interestingly, as PMA (a positive control extensively used in latent HIV-1 reactivation studies) has been demonstrated to enhance 1,25D-induced promoter binding activity of the VDR, Kitano et al. demonstrated that 1,25D, PMA/TPA, and tumor necrosis factor (TNF) stimulated HIV-1 proviral activation to similar levels in a cell line latently-infected with a monocytotropic strain of HIV-1JR-FL [10-12].

Collectively, the Oncotarget study combined with the aforementioned studies suggests a compelling yet provocative connection between diseases as seemingly as disparate as HGPS and HIV-1 latency, with AMPK activation by chemically distinct compounds including sulforaphane, methylene blue, rapamycin, and retinoic acid (and likely many others including artemisinin and metformin) orchestrating the beneficial effects observed in each disease state effectuated by each compound (see Figure below). The observation of such a connection is unprecedented and, if continued to be substantiated, would necessitate a paradigm shift in the assessment of disease pathology.

https://www.linkedin.com/pulse/vitamin-d-significantly-improves-accelerated-aging-defects-finley?published=u

References:

1. 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.
2. Plum LA, DeLuca HF. Vitamin D, disease and therapeutic opportunities. Nat Rev Drug Discov. 2010 Dec;9(12):941-55.
3. Ulgherait M, Rana A, Rera M, Graniel J, Walker DW. AMPK modulates tissue and organismal aging in a non-cell-autonomous manner. Cell Rep. 2014 Sep 25;8(6):1767-80.
4. 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;203(7):1665–70.
5. 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.
6. Jang W, Kim HJ, Li H, et al. 1,25-Dyhydroxyvitamin D attenuates rotenone-induced neurotoxicity in SH-SY5Y cells through induction of autophagy. Biochem Biophys Res Commun. 2014 Aug 15;451(1):142-7.
7. Cristobo I, Larriba MJ, de los Ríos V, García F, Muñoz A, Casal JI. Proteomic analysis of 1α,25-dihydroxyvitamin D3 action on human colon cancer cells reveals a link to splicing regulation. J Proteomics. 2011 Dec 21;75(2):384-97.
8. Bang U, Kolte L, Hitz M, et al. Correlation of increases in 1,25-dihydroxyvitamin D during vitamin D therapy with activation of CD4+ T lymphocytes in HIV-1-infected males. HIV Clin Trials. 2012 May-Jun;13(3):162-70.
9. von Essen MR, Kongsbak M, Schjerling P, Olgaard K, Odum N, Geisler C. Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nat Immunol. 2010 Apr;11(4):344-9.
10. 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.
11. Jiang Y, Fleet JC. Effect of phorbol 12-myristate 13-acetate activated signaling pathways on 1α, 25 dihydroxyvitamin D3 regulated human 25-hydroxyvitamin D3 24-hydroxylase gene expression in differentiated Caco-2 cells. J Cell Biochem. 2012 May;113(5):1599-607.
12. Kitano K, Rivas CI, Baldwin GC, Vera JC, Golde DW. Tumor necrosis factor-dependent production of human immunodeficiency virus 1 in chronically infected HL-60 cells. Blood. 1993 Nov 1;82(9):2742-8.
13. 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 Jan 27. pii: ERC-15-0557.
     

Popular cancer drug Velcade shares common mechanism of action with AMPK activator Metformin: 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.

Here's another post I just authored on LinkedIn with a surprising finding that a really popular injectable cancer drug called Velcade (made well over a billion dollars for J&J in 2014) and other drugs that are in Velcade's drug class (called proteasome inhibitors) actually activates the same pathway (AMPK) as does metformin derived from the Galega plant, sulforaphane derived from broccoli sprouts, the vitamin A metabolite retinoic acid, rapamycin derived from bacteria, and the Nobel Prize-winning drug Artemisinin derived from the Artemisia plant. Velcade not only reactivated the dormant HIV-1 virus but also inhibited HIV-1 infectivity and viral output in cells that were actively infected, effectively "pulling a double shift". Velcade has also been shown to be a potent AMPK activator and beneficially alter gene splicing in cancer cells. MG132, another drug that works like and is in the same drug class as Velcade, also activates AMPK, reactivated latent HIV-1, alters gene splicing, and enhanced the removal of the toxic protein progerin in cells from children with Progeria when combined with retinoic acid. Considering that AMPK is critical for T cell activation, beneficially alters gene splicing, and extends lifespan, this looks like yet another example of the interconnectedness of diseases including cancer, HIV-1, Progeria, and even normal aging itself. Man, what on earth have I unearthed????? Time will soon tell:-)

Popular cancer drug Velcade shares common mechanism of action with AMPK activator Metformin: Connection between AMPK, aging, and HIV-1 reactivation

A recent study published in the journal Retrovirology in October of 2013 provided unexpected evidence that the commonly-used cancer drug Velcade (an FDA approved drug for the treatment of multiple myelomas, leukemias, and lymphoma) as well as two other members of the proteasome inhibitor drug class (MG132 and clasto-Lactacystin β-lactone) reactivated latent HIV-1 in two primary human CD4+ T cell model systems as well as in several other in vitro model systems [1].  Strikingly, Velcade (also known as bortezomib) not only reactivated the latent virus but also inhibited HIV-1 infectivity, as evidenced by reduced viral output and virion infectivity [1].  Interestingly, Velcade has been shown to beneficially alter gene splicing in cancer, MG132 has been shown to alter gene splicing and reduce progerin production in fibroblasts derived from Hutchinson-Gilford progeria (HGPS) patients, and both Velcade and MG132 have been shown to activate AMPK in separate studies (see below).  Such evidence again suggests (see prior posts) that chemically distinct compounds, including compounds comprising the proteasome inhibitor class, share an “indirect yet common mechanism of action” of AMPK activation that is likely induced by the induction of a cellular stress response.

The proteasome is a protein complex present in all eukaryotes that functions to degrade or remove damaged proteins by adding a consecutive series of tags to these proteins (called a poly-ubiquitin chain), facilitating proteasome binding and protein degradation [2].  Inhibition of the catalytic site of the 26S proteasome, leading to the accumulation of pro-apoptotic (i.e. self-destruct) factors and subsequent cancer cell death is widely accepted as the mechanism of action by which Velcade acts [3].  However, as noted below, other mechanisms are almost undoubtedly involved.

In the Retrovirology study, the authors initially observed that when OM-10.1 cells (a clonal population of HL-60 promyelocytes) that were latently infected with a replication-competent HIV-1 strain were exposed to all three proteasome inhibitors (i.e. Velcade, MG132, and clasto-Lactacystin β-lactone (CLBL)), proteasome inhibition occurred within two hours, along with a significant increase in the induction of HIV-1 RNAs, indicative of the initiation of latent viral transcription [1].  The authors further showed, using a clonal population of HeLa cells and a human CD4+ T cell line, that all three proteasome inhibitors induced the activation of proviral gene expression in both cell lines [1]. Interestingly, all three proteasome inhibitors as well as the HDAC inhibitor vorinostat also induced viral particle production (as evidenced by a significant increase in the HIV-1 p24 antigen) in OM-10.1 cells [1].  To produce the p24 antigen, the splicing of the HIV-1 genome must be inhibited to produce long unspliced HIV-1 mRNA.  Splicing of the viral genome is reduced on latent HIV-1 reactivation through reduction of the activities of the splicing factor SRSF1 by the splicing-associated factor p32.  SRSF1 is also implicated in the faulty splicing mechanism that causes symptoms of accelerated aging in HGPS as well as in aberrant gene splicing in vascular tissues of normal humans [25].

More importantly, however, the authors demonstrated that both Velcade and MG132 significantly induced HIV-1 proviral gene expression in two latent HIV-1 primary human CD4+ T cell models that mimic central and effector memory T cells (two memory T cell subsets that are known reservoirs for latent HIV-1) [1].  Also, Velcade and MG132 did not activate uninfected primary human resting CD4+ T cells, indicating that both proteasome inhibitors selectively activate only CD4+ memory T cells that are latently infected with HIV-1 [1].

Perhaps most startling, however, was the finding that Velcade was able to significantly reduce viral output in activated primary human CD4+ T cells isolated from PBMCs from healthy donors, as evidenced by a 30% increase in HIV-1 p24 antigen in untreated cells compared to Velcade-treated cells [1].  Velcade was also able to decrease the infectivity of viruses produced from infected CD4+ T cells, with viruses from Velcade-treated cells only able to infect one forth the number of HeLaT4 cells compared to viruses collected from untreated primary CD4+ T cells [1]. Hence, Velcade appears to able to selectively reactivate latent HIV-1 without activating uninfected resting CD4+ T cells, but inhibit HIV-1 infectivity and viral output in productively infected cells.

This seemingly perplexing, multi-pronged antiviral mechanism demonstrated by Velcade and other proteasome inhibitors can be explained by the activation of AMPK.  T cell activation (and thus latent HIV-1 reactivation) is critically dependent on the activation of AMPK and both Velcade and MG132 have been independently shown to activate AMPK, possibly via a stress-induced signal generated by the accumulation of ubiquitinated and damaged proteins [4-6].  Indeed, both PMA and ionomycin, a combination positive control for numerous latent HIV-1 reactivation studies, have been shown to activate AMPK and AMPK activation is essential to mount an effective immune response to viral and bacterial challenges [7-11].  Also, vorinostat, an HDAC inhibitor used in the Retrovirology study as a positive control to gauge latent HIV-1 reactivation, also activates AMPK, and bryostatin (PKC activator) and JQ1 (BET bromodomain inhibitor) each have been shown to both activate AMPK and potently induce latent HIV-1 reactivation without global T cell activation in independent studies [12-15].

Furthermore, low dose proteasome inhibition using MG132 has recently been shown to alter gene splicing and induce a protective antioxidative defense response dependent on the transcription factor Nrf2, a master regulator of the cellular antioxidant response [16,17].  Interestingly, AMPK activation has recently been shown to beneficially alter gene splicing in cells derived from patients with the genetic disorder myotonic dystrophy type I (DM1) and metformin (an AMPK activator) has been shown to beneficially alter gene splicing in normal humans and increase the expression of Nrf2 [18,19].  Also, both Velcade and vorinostat have been shown to alter gene splicing in human hepatoma HepG2 and Huh6 cells by altering the splicing of the Bcl-X transcript, thus preferentially increasing the levels of Bcl-xS (the short pro-apoptotic isoform) compared to Bcl-xL (the pro-survival isoform) [20].

Proteasome inhibition has also been shown to play a role in HGPS, a disease characterized by faulty alternative gene splicing leading to an accelerated aging phenotype. Interestingly, MG132, either alone or in combination with the vitamin A metabolite all-trans retinoic acid, led to a decrease in progerin levels in HGPS cells via a proteasome inhibitor-mediated induction of autophagy, implicating both autophagic and proteasomal pathways in progerin reduction [21].  Indeed, both rapamycin (derived from bacteria) and sulforaphane (from broccoli sprouts) have each been shown to activate AMPK in vivo and induce the autophagic degradation of progerin (toxic protein product that causes accelerated aging) in HGPS fibroblasts [21-24].

Cumulatively, the foregoing studies provide convincing evidence that activation of AMPK by chemically distinct compounds (as indicated in the figure below), including the proteasome inhibitors Velcade and MG132, represents an “indirect yet common mechanism of action” that links pathological diseases states associated with gene splicing, viral latency and replication, and likely many others.

https://www.linkedin.com/pulse/popular-cancer-drug-velcade-shares-common-mechanism-action-finley?trk=prof-post

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