I wrote about blueberries back in September 2009 in the blog entry Blueberries and health – the research case, citing 11 research citations there on their positive health effects.  I have long been in the habit of eating a cupful of wild blueberries every morning together with raisin bran and walnuts.  However, my mind has been concerned with more exotic longevity things reported in this blog. At the AAAS meeting I was exposed to some interesting new research relating to those little purple critters.    In this post I review mostly-new research relating blueberries to cognition, neural inflammation,  Alzheimer’s disease, cancer, and cardiovascular health.

Blueberries and microglial activation

I have discussed emerging knowledge about the role of activated microglia in blog entries relating to inflammatory nervous system disorders including Spinal cord injury pain, and New views of Alzheimer’s disease and new approaches to treating it.  “Microglia are a type of glial cell that are the resident macrophages of the brain and spinal cord, and thus act as the first and main form of active immune defense in the central nervous system (CNS)(ref).” Microglia can become activated due to physical injury, toxic assault, the presence of pathogens, or disease conditions such as Alzheimer’s disease.  When activated, microglia release pro-inflammatory cytokine signaling molecules like TNF-alpha, NO, IL-1 and IL-6, and this process can excite neurons to further excite the microglia.  The loop of mutual excitement can feed on itself contributing to a worsening pathological inflammatory phenotype.  It appears that blueberries can help block the process.

The 2007 publication Inhibitory effects of blueberry extract on the production of inflammatory mediators in lipopolysaccharide-activated BV2 microglia was one of the first relating blueberries to microglia.  “Sustained microglial activation in the central nervous system (CNS) has been extensively investigated in age-related neurodegenerative diseases and has been postulated to lead to neuronal cell loss in these conditions. Recent studies have shown that antiinflammatory drugs may suppress microglial activation and thus protect against microglial overactivation and subsequent cell loss. Research also suggests that fruits such as berries may contain both antioxidant and antiinflammatory polyphenols that may be important in this regard. Our previous research showed that blueberry extract was effective in preventing oxidant-induced calcium response deficits in M1 (muscarinic receptor)-transfected COS-7 cells. Extrapolating from these findings, the current study investigated the effect of blueberry extract on preventing inflammation-induced activation of microglia. Results indicated that treatments with blueberry extract inhibited the production of the inflammatory mediator nitric oxide (NO) as well as the cytokines interleukin-1beta and tumor necrosis factor-alpha, in cell conditioned media from lipopolysaccharide (LPS)-activated BV2 microglia. Also, mRNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-activated BV2 cells were significantly reduced by treatments with blueberry extract. The results suggest that blueberry polyphenols attenuate inflammatory responses of brain microglia and could be potentially useful in modulation of inflammatory conditions in the CNS.”  LPS is an endotoxin often used in laboratory and mouse experiments to elicit powerful immune responses and excite microglia. 

The 2010 publication Blueberry supplementation attenuates microglial activation in hippocampal intraocular grafts to aged hosts is one of several that expands on this theme. “Transplantation of central nervous tissue has been proposed as a therapeutic intervention for age-related neurodegenerative diseases and stroke. However, survival of embryonic neuronal cells is hampered by detrimental factors in the aged host brain such as circulating inflammatory cytokines and oxidative stress. We have previously found that supplementation with 2% blueberry in the diet increases graft growth and neuronal survival in intraocular hippocampal grafts to aged hosts. In the present study we explored possible biochemical mechanisms for this increased survival, and we here report decreased microglial activation and astrogliosis in intraocular hippocampal grafts to middle-aged hosts fed a 2% blueberry diet. Markers for astrocytes and for activated microglial cells were both decreased long-term after grafting to blueberry-treated hosts compared with age-matched rats on a control diet. Similar findings were obtained in the host brain, with a reduction in OX-6 immunoreactive microglial cells in the hippocampus of those recipients treated with blueberry. In addition, immunoreactivity for the pro-inflammatory cytokine IL-6 was found to be significantly attenuated in intraocular grafts by the 2% blueberry diet. These studies demonstrate direct effects of blueberry upon microglial activation both during isolated conditions and in the aged host brain and suggest that this nutraceutical can attenuate age-induced inflammation.”

In my previous post on blueberries I mentioned a related citation: Blueberry polyphenols attenuate kainic acid-induced decrements in cognition and alter inflammatory gene expression in rat hippocampus, “These results indicate that blueberry polyphenols attenuate learning impairments following neurotoxic insult and exert anti-inflammatory actions, perhaps via alteration of gene expression.” It was observed that blueberries attenuated the expression of NF-kappaB induced by the neurotoxic kainic acid and augmented the expression of IGF-1. Blueberries can be protective of mental capability when exposed to brain toxins.”  While this 2005 document does not mention the role of microglia, their involvement can now clearly by hypothesized.

The mechanisms of action of blueberries appear to be shared by other anthocyanin-expressing flavonoids such as found in other berries.  The 2009 publication Flavonoids and brain health: multiple effects underpinned by common mechanisms outlines some of those actions that go along with inhibition of microglial activation. “The neuroprotective actions of dietary flavonoids involve a number of effects within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory, learning and cognitive function. This multiplicity of effects appears to be underpinned by two processes. Firstly, they interact with important neuronal signalling cascades leading to an inhibition of apoptosis triggered by neurotoxic species and to a promotion of neuronal survival and differentiation. These interactions include selective actions on a number of protein kinase and lipid kinase signalling cascades, most notably the PI3K/Akt and MAP kinase pathways which regulate pro-survival transcription factors and gene expression. Secondly, they induce peripheral and cerebral vascular blood flow in a manner which may lead to the induction of angiogenesis, and new nerve cell growth in the hippocampus. Therefore, the consumption of flavonoid-rich foods, such as berries and cocoa, throughout life holds a potential to limit the neurodegeneration associated with a variety of neurological disorders and to prevent or reverse normal or abnormal deteriorations in cognitive performance.”

The 2010 publication Blueberry supplementation improves memory in older adults reports on a small human study confirming what we have pretty much known all along.  “The prevalence of dementia is increasing with expansion of the older adult population. In the absence of effective therapy, preventive approaches are essential to address this public health problem. Blueberries contain polyphenolic compounds, most prominently anthocyanins, which have antioxidant and anti-inflammatory effects. In addition, anthocyanins have been associated with increased neuronal signaling in brain centers, mediating memory function as well as improved glucose disposal, benefits that would be expected to mitigate neurodegeneration. This study investigated the effects of daily consumption of wild blueberry juice in a sample of nine older adults with early memory changes. At 12 weeks, improved paired associate learning (p = 0.009) and word list recall (p = 0.04) were observed. In addition, there were trends suggesting reduced depressive symptoms (p = 0.08) and lower glucose levels (p = 0.10). We also compared the memory performances of the blueberry subjects with a demographically matched sample who consumed a berry placebo beverage in a companion trial of identical design and observed comparable results for paired associate learning. The findings of this preliminary study suggest that moderate-term blueberry supplementation can confer neurocognitive benefit and establish a basis for more comprehensive human trials to study preventive potential and neuronal mechanisms.”  Clearly, larger-scale studies are needed to confirm the results. 

Pterostilbene

Blueberries contain pterostilbene. “Pterostilbene is a stilbenoid chemically related to resveratrol. It is thought to be the key compound found predominantly in blueberries (as well as grapes) that exhibit anti-cancer, anti-hypercholesterolemia, anti-hypertriglyceridemia properties, as well as fight off and reverse cognitive decline. It is believed that the compound also has anti-diabetic properties, but so far very little has been studied on this issue. Additionally, it is also touted as a potent anti-fungal(ref).”

Pterostilbene, inflammation, glycation and diabetes

Pterostilbene is a powerful anti-inflammatory, responsible for some of the effects of blueberries in controlling a range of inflammatory disease conditions.  The 2008 publication Pterostilbene suppressed lipopolysaccharide-induced up-expression of iNOS and COX-2 in murine macrophages reports “Pterostilbene, an active constituent of blueberries, is known to possess anti-inflammatory activity and also to induce apoptosis in various types of cancer cells. Here, we investigated the inhibitory effects of pterostilbene on the induction of NO synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS).”  Continuing, “Treatment with pterostilbene resulted in the reduction of LPS-induced nuclear translocation of the nuclear factor-kappaB (NFkappaB) subunit and the dependent transcriptional activity of NFkappaB by blocking phosphorylation of inhibitor kappaB (IkappaB)alpha and p65 and subsequent degradation of IkappaB alpha. Transient transfection experiments using NFkappaB reporter constructs indicated that pterostilbene inhibits the transcriptional activity of NFkappaB in LPS-stimulated mouse macrophages. We found that pterostilbene also inhibited LPS-induced activation of PI3K/Akt, extracellular signal-regulated kinase 1/2 and p38 MAPK. Taken together, these results show that pterostilbene down regulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NFkappaB by interfering with the activation of PI3K/Akt/IKK and MAPK. These results have an important implication for using pterostilbene toward the development of an effective anti-inflammatory agent(ref).”  Inhibition of the transcriptional activity of NFkappaB is not only a useful anti-inflammatory strategy but has been proposed as a powerful anti-aging strategy as discussed several times in this blog and in my treatise(ref).  See the discussion under the subheading Increase in aberrant NF-kappaB signaling.  Thirty nine supplements in my anti-aging combined supplement firewall are inhibitors of NFkappaB.  To these can be added blueberries and a number of other phyto-substances in my normal food diet.

Pterostilbene and cancer

A number of recent studies have focused on the anti-cancer properties of pterostilbene. I cite a selected few here. 

–         The 2010 report Pterostilbene inhibits pancreatic cancer in vitro,

–         The 2010 report Dietary intake of pterostilbene, a constituent of blueberries, inhibits the β-catenin/p65 downstream signaling pathway and colon carcinogenesis in rats,

–         The 2010 report Pterostilbene inhibits breast cancer in vitro through mitochondrial depolarization and induction of caspase-dependent apoptosis,

–         The 2010 report Pterostilbene inhibits lung cancer through induction of apoptosis,

–         The 2009 publication Pterostilbene inhibited tumor invasion via suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells,

–         The 2009 publication Anti-inflammatory action of pterostilbene is mediated through the p38 mitogen-activated protein kinase pathway in colon cancer cells, and

–         The 2007 report Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats.

Pterostilbene, inflammation, glycation and diabetes

One of the theories of aging is Tissue Glycation, a process deeply implicated in diabetes.  Tissue glycation involves cross linking of tissue proteins with sugars resulting in the formation of Advanced Glycation Endproducts (AGEs).  The result of AGEs can be self-propagating systemic or “silent” tissue inflammation. AGEs are recognized by cell RAGE receptors which result in the production of cytokine chemicals that can induce unwanted and potentially deadly inflammation in blood vessels, nerve, liver and other tissues. Atherosclerosis can be a consequence. AGEs are responsible for much bodily mischief related to aging leading to deterioration of function and structure of organs. They play important roles in diabetes, atherosclerosis, vascular disease, kidney failure, and neuropathy including Alzheimer’s disease. The presence of AGEs also appears to negatively impact on immune system functioning.  Diabetes in particular appears to have its roots due to glycation and people with high blood sugar levels are particularly susceptible to glycation.

The 2006 publication Wild blueberry (Vaccinium angustifolium) consumption affects the composition and structure of glycosaminoglycans in Sprague-Dawley rat aorta is one of several that speaks to the effects of blueberries on tissue glycation.  “Our results demonstrate for the first time that a diet rich in blueberries results in structural alterations in rat aortic tissue GAGs. These changes may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment.”  Other relevant documents include the 2009 publication Seasonal phytochemical variation of anti-glycation principles in lowbush blueberry (Vaccinium angustifolium) and Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait.

The previous blog post on blueberries contains a number of additional research citations worth reviewing. One clear conclusion from reading those citations and the citations quoted here is that researchers are taking the anti-inflammatory, brain-health, cancer-fighting, diabetes-fighting and anti-aging properties of blueberries most seriously.  Eating blueberries goes far beyond being a health food fad.

Putting it all together

My daily cup of blueberries is an important dietary component of my total anti-aging firewalls regimen, taking its place along with olive oil, walnuts, green tea, garlic and many other healthy foods.  In general, I go along with the philosophy expressed in the 2009  publication Grape juice, berries, and walnuts affect brain aging and behavior.  “Numerous studies have indicated that individuals consuming a diet containing high amounts of fruits and vegetables exhibit fewer age-related diseases such as Alzheimer’s disease. Research from our laboratory has suggested that dietary supplementation with fruit or vegetable extracts high in antioxidants (e.g. blueberries, strawberries, walnuts, and Concord grape juice) can decrease the enhanced vulnerability to oxidative stress that occurs in aging and these reductions are expressed as improvements in behavior. Additional mechanisms involved in the beneficial effects of fruits and vegetables include enhancement of neuronal communication via increases in neuronal signaling and decreases in stress signals induced by oxidative/inflammatory stressors (e.g. nuclear factor kappaB). Moreover, collaborative findings indicate that blueberry or Concord grape juice supplementation in humans with mild cognitive impairment increased verbal memory performance, thus translating our animal findings to humans. Taken together, these results suggest that a greater intake of high-antioxidant foods such as berries, Concord grapes, and walnuts may increase “health span” and enhance cognitive and motor function in aging.”

I end this note with a speculation relating to my personal health.  In the blog post Spinal cord injury pain, I reported on an extremely painful injury to my spine incurred about a year ago while swimming in extremely cold water.  I reported how terrible pains were showing up in unrelated parts of my body – e.g. my knee, my back, my hips, and my rotator cuff.  A runaway inflammatory condition in my spine was sending out large quantities of cytokines creating debilitating havoc in all those places.  My personal diagnosis was microglial over-activation due to injury in my spine, though none of the medical personnel treating me had ever heard of that.   They thought my diverse pains were psychosomatic because the painful parts were not direct;y connected to my spine.  Finally, I reported how the pains went away when I started taking gabapentin, and how I came across an obscure publication indicating that gabapentin reverses spinal microglial activation in a rat model(ref).  Since then, all pains or problems associated with the spinal injury have vanished though I have kept myself on a low dose of gabapentin ever since, just-in-case.  What I have to say now is that, having read the research about blueberries and microglia cited above, perhaps blueberries get some or all of the credit for having quenching my activated microglia – instead of or in addition to the gabapentin.  About when the accident happened, I had raised my daily blueberry consumption to a small cupful and this may have helped a lot.  I will soon phase the gabapentin out completely and see what happens.

Please see the medical disclaimer for this blog.

I have written about calorie restriction (CR) a number of times in this blog. CR is the most-proven approach to life extension, involving an ancient biological pathway that works across a variety of species. This blog entry focuses on CR mimetics, substances that can presumably produce the same results as CR, and focuses particularly on mannoheptulose, a sugar in avocados that seems to do the trick.

Ample background on CR can be found in my blog entries Calorie Restriction, longevity, and waiting for proof of what works,  Calorie restriction research roundup – Part I, and Calorie restriction research roundup – Part II.  These entries discuss the molecular pathways involved in CR, and the Part II entry touches on the possible use of resveratrol as a CR mimetic.  These three blog entries ref, ref, and ref touch on the SIRT1 longevity gene and how it activates the CR pathway and on resveratrol and other drugs being developed as likely CR mimetics.  However the blog entry What does Resveratrol do? cites research that throws into question whether resveratrol really activates SIRT1 and is in fact a CR mimetic. 

Actually, a number of substances have been proposed as CR mimetics including several in the anti-aging firewalls combined supplement regimen: resveratrol, carnosine, alpha-lipoic acid, acetyl-l-carnitine, and mixed antioxidants(ref).  The proposed list also includes metformin, gymnema, 2-deoxyglucose, aminoguanidine, hydroxycitrate, thiazolidinediones, lodoacetate, modulators of NPY, exandin, PYY3-36, leptin, oxaloacetate, cinnamon and avocado extract.  I focus here on avocado extract because there seems to be some solid research relating to it.   

A 2009 publication from researchers at several collaborating institutions tells the story, Mannoheptulose: glycolytic inhibitor and novel caloric restriction mimetic. “Caloric restriction (CR) is the most robust and reproducible strategy for retarding aging. Benefits of CR have been demonstrated in multiple species, but application to human or companion animal aging represents a challenge. In 1998 the concept of CR “mimetic” (CRM) was introduced as a method to obtain “anti-aging” and health-promoting benefits of CR without reducing food intake. We hypothesized that an effective CRM would best mimic the effects of CR if it impeded initial stages of energy metabolism. We focused initially on glycolytic inhibition using 2-deoxyglucose (2DG). Upon entry into cells, this glucose analog is phosphorylated and becomes a strong competitive inhibitor of phosphohexose isomerase. 2DG effectively induces a CR-like state in rats based on metabolic effects such as reduced plasma glucose, insulin, body temperature, pulse, heart rate and inhibiting tumor growth. Results show 2DG has a narrow window between efficacy and toxicity so recently we shifted our focus to mannoheptulose (MH), a seven-carbon sugar that reduces glycolysis via hexokinase inhibition. MH appears non-toxic with negligible effects on food intake and BW, and increased insulin tolerance by 25% in mice. MH extends median and maximal lifespan (~15%) in D. melanogaster and median lifespan (~30%) in C3H/HeJ mice. These findings, coupled with simple extraction from avocados, suggest that MH may be a practical, highly effective CRM.”    

“Mannoheptulose is a hexokinase inhibitor. It is a heptose, a monosaccharide with seven carbon atoms. By blocking the enzyme hexokinase, it prevents glucose phosphorylation. As a result less dextrose units are broken down into smaller molecules in an organism. It is found as D-mannoheptulose in avocado(ref).^[1] ” In simple terms, it works to block the metabolism if glucose.
 

From ScienceNews April 20, 2009  “So Roth and his team (from P&G Pet Care, Wayne State University, Southern Illinois University and the Pennington Biomedical Research Institute) have been mining avocados for an alternative — MH (for mannoheptulose). It’s a fairly simple sugar with a 7-carbon backbone. — When fed to mice in fairly concentrated doses (roughly 300 milligrams per kilogram of an animal’s body weight), it improved insulin sensitivity and the clearance of glucose from the blood. Meaning it helped overcome diabetes-like impairments to blood-sugar control. MH supplementation also improved the ability of insulin, a hormone, to get cells throughout the body to do its bidding (and that’s a good thing). –MH revved up the burning of fats in muscle. That’s the opposite of fat deposition and something that these scientists note “would be an expected effect of a calorie restriction mimetic.”  — Treated mice also lived longer — some 30 percent longer than untreated animals. And they were happier, I’m guessing, because they didn’t have to give up most of their chow to achieve this life extension. Indeed, their food intake and weight matched that of untreated mice.”

Virtually everybody has taken acetaminophen (also known as paracetamol) from time to time; probably a lot of it over the years.  It is of course the key ingredient in the over-the-counter pain-killer Tylenol®,  is widely sold as an inexpensive over-the-counter analgesic and is included in a very large number of proprietary OTC pain formulations.  Could something so familiar and so mundane as acetaminophen have anti-aging properties?  That is the story I explore here. 

This is the first blog entry inspired by what I came across in the last few days at the American Aging Association’s 39^th annual meeting in Portland Oregon.  Wandering through the poster presentations I came across one entitled  Acetaminophen improves Protein Translational Signaling in Aged Skeletal Muscle prepared by a team including Miazong Wu and Hua Liu from Marshall University.    Scanning the poster quickly I came across an assertion that grabbed my attention, and that is that “Acetaminophen improves mTOR-related signaling in aged skeletal muscles.”  Further, in a rat study “Compared to 26 and 27 month old F344BN rats the expression of the mTOR complex proteins raptor and GbetaL and the phosphorylation of the negative regulator tuberin/TSC2 (Thr1462) were reduced in the soleus muscles of very aged animals (33 months old).” I thought “improves” the signaling meant reduces it, but I was wrong.

Background on mTOR

I will come back to this research shortly but first want to say why I so-quickly became excited when I scanned the poster.  Inhibition of mTOR signaling is one of extremely few interventions known that can extend lives across a variety of species including mammals.  I have mentioned mTOR frequently in this blog.  See the earlier blog post Longevity genes, mTOR and lifespan.  I repeat “Mammalian target of rapamycin (mTOR) is a protein encoded in humans by the FRAP1 gene.  As the name suggests, mTOR is targeted by the immunosuppressive drug rapamycin, a drug used clinically to treat graft rejection and restenosis and being tested as a treatment for autoimmune diseases.“ Inhibiting the expression of mTOR produces all kinds of health effects in experimental animals.  The blog entry Viva mTOR! Caveat mTOR! tells how feeding  rapamycin to mice late in life inhibits mTOR expression and extends their lives, so rapamycin is being considered as a longevity drug for humans.  I also  describes potential hazards of taking it.  The blog entry More mTOR links to aging theories looks at the involvement of the mTOR pathway in the hypoxic response biochemical pathway involved in aging and in the stem cell supply chain theory of aging.  Finally, I added a candidate theory of aging to my treatise Increasing mTOR signalling. 

A bottom-line conclusion of all this background is that if we could find a safe way to inhibit the expression of mTOR we might well live longer – 14% for males and 8% for females both average and maximum if we are like mice(ref).  So the possibility of being able to extend our lives using something so simple and relatively safe as acetaminophen came across as very exciting to me.  Unfortunately, looking a bit more carefully this possibility seems unlikely.   “Improving” mTOR signaling as described in the poster meant increasing it, not reducing it. So, I am afraid that taking acetaminophen is likely to work at cross-purposes and on the whole be a negative intervention.

Getting back to the current presentation at the AAAS meeting, the poster presentation appeared to document the case for acetaminophen “improving” the expression of TOR in mouse tissues and having rejuvenating effects on mouse muscle tissues fairly well, but that latest work still remains unpublished even in abstract form.  However, some of the same the authors published a paper in 2009 describing earlier stages of their work Aging-Associated Dysfunction of Akt/Protein Kinase B: S-Nitrosylation and Acetaminophen Intervention.  “Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention.  –  Principal Findings — Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, along with decreased levels of insulin receptor beta (IR-β), phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1) (Ser241). In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS). Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month) animals with acetaminophen (30 mg/kg body weight/day) for 6-months “Our results show that aging skeletal muscle exhibits impaired Akt kinase activity and that acetaminophen-induced improvements in Akt signaling are associated with increases in myocyte size and the expression of myosin and actin, along with decreases in muscle apoptosis.”   

A number of protein analyses studies described in the 2009 paper demonstrated  “Dysfunction of Akt in the very aged muscle can be corrected by acetaminophen,” “Akt dysfunction is associated with increases in iNOS and Akt S-nitrosylation,” “Akt hyper-phosphorylation is associated with a loss of PTEN protein,” “Akt dysfunction is associated with decreases of muscle fiber cross-sectional area,” and “Akt dysregulation is associated with increases in myocyte apoptosis.” 

Of relevance to our focus on mTOR, the 2009 paper reported “Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, — The abundance of phosphorylated mTOR (pmTOR) (Ser2448) and mTOR total protein in the very aged soleus were lower than that in the adult animals (−86.3% and −86.8%, respectively; P<0.05.”  In other words there was an age-related decline in mTOR expression in these muscle cells that accompanied accelerated expression of certain Akt proteins and loss of functionality.   Moreover “chronic acetaminophen treatment restored the amount of phosphorylated and total mTOR to a level equivalent to that seen in 6- and 27-month old animals (P>0.05).” 

The ability of acetaminophen to correct age-related problems in aged muscle was reported on in the above-mentioned 2009 paper, but the role of mTOR in the process is only clarified in the current 2010 yet-unpublished poster presentation.  I quote only selectively from what was on the poster or the printed abstract for it.  “Here we hypothysize that age-related impairments in Akt/mTOR function are associated with reduced translational signaling and that these age-associated alterations, if present, can be attenuated by acetaminophen treatment.” — “Compared to 6 and 27 month old F344BN rats, the expression of the mTOR-complex proteins raptor and GbetaL and the phosphorylation of negative regulator tuberin/TSC2 (Thr1462) were reduced in the soleus muscle of very aged animals (33months old).  These changes in Akt/mTOR pathway signaling proteins were in turn associated with decreased phosphorylation.” “ —  Age-associated alterations in the Akt/mTOR pathway signaling and in the phosphorylation of the stress-responsive eIF2alpha protein were attenuated by chronic acetaminophen treatment –Conclusion: Aging is associated with impairments in the regulation  of proteins thought to be important in controlling mRNA translation and acetaminophen may be useful for the treatment of age-related muscle atrophy by reducing oxidative stress.”  (Whoops, in this condensed presentation I not quite sure where the last conclusion related to oxidative stress comes from.) 

The 2009 publication Impaired overload-induced hypertrophy in Obese Zucker rat slow-twitch skeletal muscle provides independent confirmation that inadequate mTOR expression may be responsible for stress or age-related atrophy in the soleus muscle.  “Taken together, these data suggest that IR or other related co-morbidities may impair the ability of the soleus to activate mTOR signaling and undergo load-induced muscle hypertrophy.”  If you are further interested in the molecular signaling pathways that affect muscle atrophy and hypertrophy, you can start with Akt signalling through GSK-3β, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy, and then work your way through the citations. 

So the situation appears to be: 

–         The study described in the poster and its antecedent publication  suggest that age-related muscle atrophy is likely to be due to (or at least associated with) age-related decline in Akt/mTOR signaling and that this atrophy can be at least partially offset by increasing Akt/mTOR signaling using acetaminophen.  In aged muscle cells a strategy for longevity of capability is increasing mTOR signaling.

–         Other studies covered in previous blog entries suggest that median and maximal lifespan of organisms including mice and possibly people can be increased by inhibiting mTOR signaling.  

The paradox is one I have possibly encountered before.  As I recall, there are many who argue that HGH administration can increase muscle strength, vitality and several indicators of youth but there is also evidence that it does not extend and possibly shortens lifespans(ref)(ref). 

Both the poster and the earlier report record feeding the mice with acetaminophen (30 mg/kg body weight/day) for 6-months.  For a 150 pound human being this is equivalent to a 2,045mg daily dose.  Taking large chronic doses of acetaminophen is potentially risky.  “While generally safe for use at recommended doses (1,000 mg per single dose and up to 4,000 mg per day for adults, up to 2,000 mg per day if drinking alcohol^[2]), acute overdoses of paracetamol can cause potentially fatal liver damage and, in rare individuals, a normal dose can do the same; the risk is heightened by alcohol consumption. Paracetamol toxicity is the foremost cause of acute liver failure in the Western world, and accounts for most drug overdoses in the United States, the United Kingdom, Australia and New Zealand(ref).^[3][4][5][6] ” 

I have put the giant bottle of acetaminophen tablets from Costco back under the bathroom sink.

I have written a number of times in this blog about iPSCs (induced pluripotent stem cells) including the exciting possibility of closing the loop in the stem cell supply chain and thereby enabling very long lives.  See the posts IPSCs, telomerase, and closing the loop in the stem cell supply chain, Progress in closing the stem cell supply chain loop and The stem cell supply chain – closing the loop for very long lives.  I have also pointed out in a recent presentation(ref) that, for this concept to become real, a number of technical challenges must be overcome including: a) obtaining iPSCs that are free of DNA contamination, and that have long telomeres and full hESC pluripotency, b) developing reliable means for assuring differentiation into adult stem cells of various types, and c) developing reliable and safe means for introducing  those cells into their respective body niches.  I further stated that although much research is being devoted to these approaches, 10-20 years are likely to be required before the stem cell supply chain can truly be closed in humans.   I believe the main challenges that will have to be faced are of a bioengineering nature. 

There is another application for iPSCs which is likely to become very important in the immediate future: supplying large quantities of specialized body cells for research and drug testing purposes.  It appears that the engineering challenges of producing industrial-quantities of high grade cardiomyocytes suitable for drug-testing purposes have already been solved.  The article iPSC-Derived Human Cardiomyocytes appearing in the May 15 issue of Genetic Engineering & Biotechnology News describes the development, the work of a company Cellular Dynamics International.  Also, the development is covered in an article in PharmTech.  Cardiomyocytes are the cells that make up cardiac muscle.

The engineering challenges of obtaining large numbers of pure cardiomyocytes are not simple.  According to the Gen article “The pharmaceutical industry requires large numbers of purified cell types for screening candidate molecules for efficacy and unintentional toxicity, and the industrialized use of terminal cell types derived from iPSCs has been severely hampered, if not prohibited, by the difficulties of culturing stem cells. — iPSCs, while highly proliferative, are sensitive to manipulation; improper handling can severely restrict their pluripotency and drastically reduce the numbers of subsequently differentiated healthy cells. — Furthermore, while producing terminally differentiated cell types from stem cells using embryoid body (EB) and directed differentiation techniques are well known, the efficiency with which these methods produce terminally differentiated cells is highly variable; a common theme to both techniques is difficulty in producing highly pure (>90%) populations of terminally differentiated cells.”

“Therefore, the key to utilizing stem cell technology on an industrial scale is to develop processes that are both scalable and standardizable for both iPSC maintenance and differentiation. — Cellular Dynamics International’s (CDI) iCell™ Cardiomyocytes are human iPSC-derived cardiomyocytes that possess expected cardiac characteristics, form electrically connected syncytial layers, and exhibit expected electrophysiological and biochemical responses upon exposure to exogenous agents.  CDI’s new technology overcomes barriers in both iPSC maintenance, terminal cell type differentiation, and purification by generating standardized and scalable protocols. The primary production constraint of iPSC husbandry was eliminated by developing a culture system that uses standard single-cell splitting techniques and small molecules to minimize operator-specific effects(ref).”

Cardiomyocytes are highly specialized muscle cells(ref) which contract in a coordinated manner producing heart beats.  This video on the CDI web site shows a monolayer of iPSC-produced cardiomyocytes that is spontaneously beating.  The iPSC-produced cardiomyocytes’ biochemical and electrophysiological properties and gene expression profiles have been tested and appear to match those of human-derived cardiomyocytes(ref).  The cardiomyocytes can be customized.  “–because iPSCs can be derived from individuals with identifiable phenotypes and genotypes, targeted human subpopulation models can be employed early in the discovery and toxicity screening processes(ref).”

CDI has developed technology for directing differentiation of iPSCs, for their proliferation, and for purifying them.  Their basic breakthrough has been in engineering an accurate and efficient production process.  “CDI’s new technology overcomes barriers in both iPSC maintenance, terminal cell type differentiation, and purification by generating standardized and scalable protocols. The primary production constraint of iPSC husbandry was eliminated by developing a culture system that uses standard single-cell splitting techniques and small molecules to minimize operator-specific effects.– iPSC culture scalability was incorporated into the process by building the cell culture system in a parallel fashion to enable the production of billions of iPSCs through the use of CellSTACK® culture chambers (Corning).  — Differentiation of iPSCs into iCell Cardiomyocytes is built on CDI’s platform that utilizes recombinant genetic engineering and antibiotic selection. Prior to iPSC clonal expansion, genes encoding antibiotic resistance and an optional marker under control of a cell-type specific promoter (pan-cardiac for iCell Cardiomyocytes) are introduced into the iPSCs through homologous recombination. — After curation and quality control (QC), the iPSC clone carrying the selectable marker is expanded using iPSC maintenance procedures, harvested, and placed into the directed differentiation protocol of choice. Subsequent to differentiation initiation, the cultures are exposed to the selection agent to leave a pure, targeted cell population. — In the case of iCell Cardiomyocytes, the directed differentiation method produces cardiomyocyte purities greater than 50%, while antibiotic selection subsequently increases this purity to approximately 100%, a level that is necessary to ensure that the observed experimental outcome is due to an effect on cardiomyocytes rather than noncardiac “contaminating” cells. — This process, as currently practiced at CDI, is capable of meeting the foreseeable demand for purified iPSC-derived human cardiomyocytes and is scalable by more than two orders of magnitude, without difficulty, if necessary(ref).”

It is likely that variants of this technique can be used to produce a variety of other cell types “”We intend to launch liver, nerve, and blood vessel cell products over the next 18 months,” said Chris Parker, CDI’s Chief Commercial Officer(ref).”  CDI has recently raised $40.6 million in private equity funding with a total of $70 million since 2004(ref).

The iPSC-derived cells should be more reliable for drug toxicity testing than existing cell lines.  “Drug toxicity that emerges either in late-stage clinical trials or following market launch has been a long-term problem for the pharmaceutical industry. The fundamental challenge is that existing preclinical models do not adequately predict the toxicity of new chemical entities. Current cell models are either primary cell cultures derived from non-human animals or immortal cell lines derived from tumors. Because of their non-human nature or neoplastic life history, they are imperfect predictors of drug toxicity in humans(ref).” 

Developing a viable and robust cardiomyocyte product line based on iPSCs basically required overcoming a number of serious bioengineering challenges.  I think the same will be the case for closing the loop in the stem cell supply chain.

In my presentation Towards a Systems Theory of Aging I argue that the two theories Programmed epigenomic changes and Decline in functioning of the stem cell supply chain are complimentary and equivalent and have the potential for providing a framework for an overall systems view of aging that knits together a large collection of traditional special theories of aging.  In this post, I review some research that is relevant to this assertion, especially with respect to the relationships between the Programmed epigenomic changes theories and the aging theories 6. Chronic Inflammation, 7. Immune System Compromise, 8. Neurological Degeneration, 10. Susceptibility to Cancers, and 11. Susceptibility to Cardiovascular Disease.   

The 2010 publication Epigenetics in atherosclerosis and inflammation is a review study.  “Atherosclerosis is a multifactorial disease with a severe burden on western society. Recent insights into the pathogenesis of atherosclerosis underscore the importance of chronic inflammation in both the initiation and progression of vascular remodelling. — Besides genetic factors also epigenetic mechanisms play an essential and fundamental role in the transcriptional control of gene expression.  –. The concept of epigenetic regulation is gradually being recognized as an important factor in the pathogenesis of atherosclerosis. Recent research provides an essential link between inflammation and reprogramming of the epigenome.” The Programmed epigenomic changes theory of aging asserts that age-related reprogramming of the epigenome increases susceptibility to inflammation and inflammation-related diseases. 

The 2008 publication Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases highlights the same points, extending their scope to autoimmune diseases and cancers. “It is clear that the epigenetic state is a central regulator of cellular development and activation. Emerging evidence suggests a key role for epigenetics in human pathologies, including in inflammatory and neoplastic disorders. The epigenome is influenced by environmental factors throughout life. Nutritional factors can have profound effects on the expression of specific genes by epigenetic modification, and these may be passed on to subsequent generations with potentially detrimental effects. Many cancers are associated with altered epigenetic profiles, leading to altered expression of the genes involved in cell growth or differentiation. Autoimmune and neoplastic diseases increase in frequency with increasing age, with epigenetic dysregulation proposed as a potential explanation. In support of this hypothesis, studies in monozygotic twins revealed increasing epigenetic differences with age. Differences in methylation status of CpG sites, monoallelic silencing, and other epigenetic regulatory mechanisms have been observed in key inflammatory response genes. The importance of the epigenome in the pathogenesis of common human diseases is likely to be as significant as that of traditional genetic mutations.”A number of studies have been concerned with identifying epigenomic changes associated with particular cancers.  Although they are often highly technical, they show that complicated epigenomic/epigenetic changes are involved in cancer processes. 

 The 2007 publication Epigenetic profiling of multidrug-resistant human MCF-7 breast adenocarcinoma cells reveals novel hyper- and hypomethylated targets is an example.  “Presently, two hypotheses, genetic and epigenetic, have been proposed to explain mechanisms of acquired cancer drug resistance. In the present study, we examined the alterations in epigenetic mechanisms in the drug-resistant MCF-7 human breast cancer cells induced by doxorubicin (DOX) and cisplatin (cisDDP), two chemotherapeutic drugs with different modes of action. Despite this difference, both of the drug-resistant cell lines displayed similar pronounced changes in the global epigenetic landscape showing loss of global DNA methylation, loss of histone H4 lysine 20 trimethylation, increased phosporylation of histone H3 serine 10, and diminished expression of Suv4-20h2 histone methyltransferase compared with parental MCF-7 cells. In addition to global epigenetic changes, the MCF-7/DOX and MCF-7/cisDDP drug-resistant cells are characterized by extensive alterations in region-specific DNA methylation, as indicated by the appearance of the number of differentially methylated DNA genes. A detailed analysis of hypo- and hypermethylated DNA sequences revealed that the acquisition of drug-resistant phenotype of MCF-7 cells to DOX and cisDDP, in addition to specific alterations induced by a particular drug only, was characterized by three major common mechanisms: dysfunction of genes involved in estrogen metabolism (sulfatase 2 and estrogen receptor alpha), apoptosis (p73, alpha-tubulin, BCL2-antagonist of cell death, tissue transglutaminase 2 and forkhead box protein K1), and cell-cell contact (leptin, stromal cell-derived factor receptor 1, activin A receptor E-cadherin) and showed that two opposing hypo- and hypermethylation processes may enhance and complement each other in the disruption of these pathways. These results provided evidence that epigenetic changes are an important feature of cancer cells with acquired drug-resistant phenotype and may be a crucial contributing factor to its development. Finally, deregulation of similar pathways may explain the existence and provide mechanism of cross-resistance of cancer cells to different types of chemotherapeutic agents.”

The 2008 publication Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays states “Breast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. — . RESULTS: We integrated data from the tiling microarrays with targets identified by Ingenuity Pathways Analysis software and observed epigenetic variations in genes implicated in epithelial-mesenchymal transition and with tumor cell migration. We identified widespread genomic hypermethylation and hypomethylation events in these cells and we confirmed functional associations between methylation status and expression of the CDH1, CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our data also suggest that the complex genomic reorganization present in cancer cells may be superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes. CONCLUSION: This is the first whole-genome approach to identify genome-wide and gene-specific epigenetic alterations, and the functional consequences of these changes, in the context of breast cancer metastasis to lymph nodes. This approach allows the development of epigenetic signatures of metastasis to be used concurrently with genomic signatures to improve mapping of the evolving molecular landscape of metastasis and to permit translational approaches to target epigenetically regulated molecular pathways related to metastatic progression.”

The 2009 publication  Pituitary tumours: all silent on the epigenetics front states “Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to ‘uncover’ or ‘unmask’ silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.”Relating to almost all areas of medicine, starting in the early-2000s more and more research publications have been appearing pointing out the importance of epigenomic regulation in disease etiology and progression. 

 For example, the 2008 review publication Epigenetic Regulation of Vascular Endothelial Gene Expression “Epigenetics has emerged as an increasingly powerful paradigm to understand complex non-Mendelian diseases. For example, epigenetics provides a newer perspective for understanding how gene expression is perturbed in prevalent diseases of the human vascular system characterized by a dysfunctional endothelium.⁴” 

The 2009 publication Epigenetics and periodontal disease: future perspectives states “Periodontitis is a multifactorial infection characterized by inflammation and destruction of tooth supporting tissues, as a result of the response of a susceptible host to bacterial challenge. Studies have demonstrated that epigenetic events are able to influence the production of cytokines, contributing to the development of inflammatory diseases. Epigenetic events act through the remodeling of chromatin and can selectively activate or inactivate genes, determining their expression. The epigenetic process, by inducing a change in cytokine profile, may subsequently influence the pathogenesis and determine the outcome of many infectious diseases. These findings may have relevance for inflammatory diseases in which the expression of cytokines is unregulated. The purpose of this review is to show evidence that supports the hypothesis that epigenetic alterations, such as hyper and hypomethylation, of cytokine genes, could help to understand the mechanisms related to periodontal disease activity. Therefore, epigenetics may have future impact on diagnosis and/or therapeutics of periodontal disease.“

The 2009 publication Epigenetic mechanisms that underpin metabolic and cardiovascular diseases relates to other critical disease fronts and focuses on lifelong changes in the epigenome and their affect on disease susceptibilities:

·     “Developmental plasticity enables an organism to respond to environmental cues and adjust its phenotypic development to match its environment.

·     Developmental plasticity is effected, at least in part, by epigenetic changes that are established in early life and modulate gene expression during development and maturity.

·     In mammals, the window during which the epigenome is susceptible to nutritional cues extends from conception to at least weaning.

·     Mismatch between the early and mature environments may result in inappropriate patterns of epigenetic changes and gene expression that increase subsequent susceptibility to metabolic and cardiovascular diseases.

·     The available evidence suggests that interventions to prevent metabolic and cardiovascular diseases should focus on the prenatal and early postnatal periods.”   

The 2008 publication Epigenetic principles and mechanisms underlying nervous system functions in health and disease states “Epigenetics and epigenomic medicine encompass a new science of brain and behavior that are already providing unique insights into the mechanisms underlying brain development, evolution, neuronal and network plasticity and homeostasis, senescence, the etiology of diverse neurological diseases and neural regenerative processes. Epigenetic mechanisms include DNA methylation, histone modifications, nucleosome repositioning, higher order chromatin remodeling, non-coding RNAs, and RNA and DNA editing. RNA is centrally involved in directing these processes, implying that the transcriptional state of the cell is the primary determinant of epigenetic memory. This transcriptional state can be modified not only by internal and external cues affecting gene expression and post-transcriptional processing, but also by RNA and DNA editing through activity-dependent intracellular transport and modulation of RNAs and RNA regulatory supercomplexes, and through trans-neuronal and systemic trafficking of functional RNA subclasses. These integrated processes promote dynamic reorganization of nuclear architecture and the genomic landscape to modulate functional gene and neural networks with complex temporal and spatial trajectories. Epigenetics represents the long sought after molecular interface mediating gene-environmental interactions during critical periods throughout the lifecycle. The discipline of environmental epigenomics has begun to identify combinatorial profiles of environmental stressors modulating the latency, initiation and progression of specific neurological disorders, and more selective disease biomarkers and graded molecular responses to emerging therapeutic interventions. Pharmacoepigenomic therapies will promote accelerated recovery of impaired and seemingly irrevocably lost cognitive, behavioral, sensorimotor functions through epigenetic reprogramming of endogenous regional neural stem cell fate decisions, targeted tissue remodeling and restoration of neural network integrity, plasticity and connectivity.”

The above is just a small sample of the river of applicable literature but enough to make the point: The Programmed epigenomic changes theory of aging is critically implicated in a large number of disease and disease progression processes. It will clearly take a lot more research to establish that this theory is capable of explaining all the phenomena described by all the other theories of aging but I strongly suspect that this will in time happen.

Epigenomic interventions to deal with various diseases are now in the clinical trials phase.  Specifically, a number of histone deacetylase inhibitors are now in clinical trials, basically substances that keep apoptosis and other critical genes active.  Describing these will be the subject of a subsequent post.  I also hope to characterize some interesting research relating lifelong nutrition patterns to epigenomic changes.

The p21 gene has long been known for its role in cell cycle arrest and apoptosis.  In case of DNA damage it signals to the p53 gene to initiate apoptosis of the cell, averting the possibility of tumorgenesis.  Very-recent research indicates something else – expression of P21 is part of what keeps us from growing new limbs or other body parts like salamanders or newts.  This blog post reviews the new research in the context of what has long been known about P21.

Some background on P21

P21 is a cell cycle regulator, specifically a CDK (cyclin-dependent kinase) inhibitor(ref).  It has long been known to impede stem cell differentiation and proper embryonic development.  For example, the 1996 publication Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration states “The CDK inhibitor p21 (WAF-1/CIP-1/SDI-1) has been implicated in DNA damage-induced p53-mediated G1 arrest, as well as in physiological processes, such as cell differentiation and senescence, that do not involve p53 function. — These results provide the first in vivo evidence that appropriate p21 levels are critical in normal development and further implicate p21 in the control of multiple cell-cycle phases.”

P21 has been studied for its role in fibrosis and other lung diseases.  A 2004 publication is entitled Induction of CDK inhibitor p21 gene as a new therapeutic strategy against pulmonary fibrosis.  The 2008 publication P21 regulates TGF-beta1-induced pulmonary responses via a TNF-alpha-signaling pathway relates “Transforming growth factor (TGF)-beta(1) is an essential regulatory cytokine that has been implicated in the pathogenesis of diverse facets of the injury and repair responses in the lung. The types of responses that it elicits can be appreciated in studies from our laboratory that demonstrated that the transgenic (Tg) overexpression of TGF-beta(1) in the murine lung causes epithelial apoptosis followed by fibrosis, inflammation, and parenchymal destruction. Because a cyclin-dependent kinase inhibitor, p21, is a key regulator of apoptosis, we hypothesized that p21 plays an important role in the pathogenesis of TGF-beta(1)-induced tissue responses. — Collectively, our studies demonstrate that p21 regulates TGF-beta(1)-induced apoptosis, inflammation, fibrosis, and alveolar remodeling by interacting with TNF-alpha-signaling pathways.”

Expression of P21 is a barrier to stem cell differentiation.   The 2000 publication Hematopoietic Stem Cell Quiescence Maintained by p21^cip1/waf1 states “Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.”  In the absence of P21, hematopoietic stem cells would not remain quiescent in their niches but would instead prematurely differentiate when stress occurs exhausting the pools of those cells and interrupting the normal functioning of the stem cell supply chain leading to premature death.  The 2009 paper Accelerating stem cell proliferation by down-regulation of cell cycle regulator p21 offers a consistent message.  “Inhibition of the cell cycle regulator p21 results in significant acceleration of mesenchymal stem cell proliferation without promoting spontaneous cellular differentiation.”

P21 is also implicated in active disease processes especially in certain cancers.  For example, the 2009 paper Cell-cycle restriction limits DNA damage and maintains self-renewal of leukemia stem cells argues that p21 gives cancer cells the chance to repair their DNA and keep living.  “Here we demonstrate that expression of the cell-cycle inhibitor p21 is indispensable for maintaining self-renewal of leukemia stem cells. Expression of leukemia-associated oncogenes in mouse haematopoietic stem cells (HSCs) induces DNA damage and activates a p21-dependent cellular response, which leads to reversible cell-cycle arrest and DNA repair.  Activated p21 is critical in preventing excess DNA-damage accumulation and functional exhaustion of leukemic stem cells. These data unravel the oncogenic potential of p21 and suggest that inhibition of DNA repair mechanisms might function as potent strategy for the eradication of the slowly proliferating leukemia stem cells.”

There is a lot more that can be said about P21 but the above is sufficient for this post.

P21 and organ renewal

Interesting news relating P21 to limb and appendage regeneration is reported in a March 2010 report Lack of p21 expression links cell cycle control and appendage regeneration in mice.  “Animals capable of regenerating multiple tissue types, organs, and appendages after injury are common yet sporadic and include some sponge, hydra, planarian, and salamander (i.e., newt and axolotl) species, but notably such regenerative capacity is rare in mammals. The adult MRL mouse strain is a rare exception to the rule that mammals do not regenerate appendage tissue. Certain commonalities, such as blastema formation and basement membrane breakdown at the wound site, suggest that MRL mice may share other features with classical regenerators.  As reported here, MRL fibroblast-like cells have a distinct cell-cycle (G2/M accumulation) phenotype and a heightened basal and wound site DNA damage/repair response that is also common to classical regenerators and mammalian embryonic stem cells.”  MRL mice do not express P21 and are like salamanders in an important respect.  When wounded “The super-healing mice form a “blastema”, a clump of immature cells that behave like stem cells, at the injury site. The blastema cells differentiate into the proper cells, leaving virtually no scar tissue or other trace of the injury. Such regenerative power is almost unknown in mammals. But it is common in amphibians such as the newt and axolotl, which can regrow entire limbs(ref).”  MRL mice are known to be able to at least partially regenerate digits and “have a far superior ability to regenerate cardiac tissue than do regular mice, and humans(ref).”  

The researchers discovered that the wound-injury response of P21-knockout mice was like that of MRL mice.  In response to a hole punched in a mouse’s ear, the MRL and P21- mice formed healing blastemas which closed up the holes like they were never there while ordinary P21+ mice formed scars and the holes remained open. 

What about the absence of the protective role of P21 in P21- and MRL mice?  Are they more cancer prone? “DNA damage is a hallmark of cancer, and regeneration in the healer mice and p21 knockout mice features an increase in DNA damage in the dividing cells at the blastema. — The link between regeneration and cancer notwithstanding, the researchers who originally created the p21 knockout mice haven’t found any evidence that the lack of a p21 gene leads to increased rates of cancer. — Along with the cell proliferation and DNA damage in the heightened regeneration, there’s an increased rate of apoptosis, which kills off cells too severely damaged to be repaired, —  “The combined effects of an increase in highly regenerative cells and apoptosis may allow the cells of these organisms to divide rapidly without going out of control and becoming cancerous,” Heber-Katz (an author of the study) said. “In fact, it is similar to what is seen in mammalian embryos, where p21 also happens to be inactive after DNA damage. The down regulation of p21 promotes the induced pluripotent state in mammalian cells, highlighting a correlation between stem cells, tissue regeneration, and the cell cycle(ref).”

The line of research was conducted in the Wistar Institute in the laboratory of Dr. Ellen Heber-Katz.  Dr. Heber Katz has been involved in highly related research since the 1990s.  In fact they accidentally discovered the regenerative capability of MRL mice back in the mid 90s when they punched holes in the ears of some of these animals to identify them, a standard laboratory procedure.  In normal mice the holes stay permanently open.  In the MRL mice the holes healed closed without a trace.  This set Dr. Katz and her group off on a chain of discoveries based on working with normal and super-healer mice.  In 1998 the group reported on multiple regions in 5 chromosomes connected with wound healing.  The latest discovery is that reported here(ref).The hope is that it may ultimately be possible to regenerate damaged or missing organs, possibly even fingers or limbs in human beings through temporary inactivation of P21. “If humans can be induced to heal like these healer mice, it would be possible to repair skin wounds without scarring, and to induce regrowth of cartilage. Internal healing could be improved, including that of damage from heart attacks, as a 2001 study in PNAS said(ref).”  Spinal cord tissue injury is another potential area of application.

On a personal note, I have been concerned about loss of nerve connections in two fingers and spinal cord tissue damage.  See my posts Nerve regeneration and Spinal cord injury pain – a personal story and a new paradigm.  And I have been looking forward to the still far-off days when these parts of me can be regenerated.  I believe the current research about P21 moves us a step further up the long ladder leading to practical regenerative medicine.

I have finished drafting the Power Point presentation for the Society’s 39^th annual meeting in Portland Oregon early next month.   I am still polishing up the presentation, fixing mysteriously broken links, adding citations, etc., but I have put it online where it can be previewed by my readers at Towards a Systems Theory of Aging.  The conclusion of the presentation is “The two theories Programmed epigenomic changes and Decline in functioning of the stem cell supply chain are complimentary and equivalent and reflect cutting-edge developments in molecular and cell biology and genomics.  They have the potential for providing a framework for an overall systems view of aging that knits together a large collection of traditional special theories of aging.”  Expect minor changes and improvements between now and the time of the conference. 

With this done, I am happily getting back to my regular blogging activities.  As always, any and all comments are most welcome.

Melanoma is the most serious form of skin cancer. It is a cancer “–  of melanocytes which are found predominantly in skin but also in the bowel and the eye (see uveal melanoma).  — Melanocytes are normally present in skin, being responsible for the production of the dark pigment melanin.^[2] Despite many years of intensive laboratory and clinical research, the greatest chance of cure is in the early surgical resection of thin tumors(ref).”  In 2009, 68,720 new cases of melanoma were reported in the US and the disease led to 8,650 deaths(ref). “According to a WHO report about 48,000 melanoma related deaths occur worldwide per year(ref).”^[6] 

Melanoma is also among the most-studied cancers.  The US government clinical trials database shows 910 clinical trials for melanoma in various stages.  This blog entry reports a selection of recent (2010) published research findings.

An April 2010 e-publication Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial provides a testimony to the relative inefficacy of a high-technology vaccine in the face of progressive malignant melanoma.  See the earlier blog post Dendritic cell cancer immunotherapy.  According to the new publication “Dendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2(+) patients) or with autologous/allogeneic tumor lysate (HLA-A2(-) patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-alpha2b. Results: Of 46 patients who initiated treatment, 10 stopped treatment within 1-4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2(+) patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25(high) CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD. Conclusions: Vaccination was feasible and safe.  Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells”  I decode this to mean that about a quarter of the treated patients extended their mean survival time from 9 months to 18.4 months.  While this is only a Phase I/II study, it casts doubt on whether dendritic cell cancer immunotherapy, a major focus of current cancer research, will provide a magic bullet against aggressive cancers.

A study published this week in the journal Cell A Temporarily Distinct Subpopulation of Slow-Cycling Melanoma Cells Is Required for Continuous Tumor Growth may help explain several key aspects of melanoma, such as its aggressiveness.  The study suggests that there are two important temporary subpopulations of melanoma cells, both tumorigenic but different in their differentiation and replication potentials.  There is at any time a relatively small slowly-dividing population of cells characterized by the JARID1B demethylase biomarker.  This population is stem-cell-like and necessary for continuous tumor growth.  The JARID1B- cells, on the other hand, divide very rapidly but are insufficient for continuous tumor maintenance.  Moreover, JARID1B- cells can transform into being JARID1B+ cells and the other way around.  “Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.  º The H3K4 demethylase JARID1B marks a subpopulation of slow-cycling melanoma cells.  º The JARID1B+ subpopulation is required for continuous tumor maintenance .  º Cells can lose or gain JARID1B expression and do not follow a stem cell hierarchy.  º Tumor initiation is not necessarily linked with tumor maintenance.”  A possible implication if the study is that an effective therapy regimen for melanoma will have to target both the fast (JARID1B-) and slow-growing (JARID1B+) cell populations if it is to be effective.  The study is also important for pointing out how the JARID1B- cells can convert themselves into the stem-cell-like JARID1B+ cells, an effect not observed in other cancers.   For background relating to cancer stem cells you can check out the earlier blog entries On cancer stem cells, Big pharma is targeting cancer stem cells, Update on cancer stem cells,  and News on disabling cancer stem cells.

The April 2010 publication Melanoma: Stem cells, sun exposure and hallmarks for carcinogenesis, molecular concepts and future clinical implications reports on a review study, the purpose of which was to identify molecular biomarkers for melanoma.  A vivid view is provided of how melanoma originates: “RESULTS: Melanocyte precursors undergo several genome changes -UV-induced or not- which could be either mutations or epigenetic. These changes provide stem cells with abilities to self-invoke growth signals, to suppress antigrowth signals, to avoid apoptosis, to replicate without limit, to invade, proliferate and sustain angiogenesis. Melanocyte stem cells are able to progressively collect these changes in their genome. These new potential functions, drive melanocyte precursors to the epidermis were they proliferate and might cause benign nevi. In the epidermis, they are still capable of acquiring new traits via changes to their genome. With time, such changes could add up to transform a melanocyte precursor to a malignant melanoma stem cell. CONCLUSIONS: Melanoma cannot be considered a “black box” for researchers anymore. Current trends in the diagnosis and prognosis of melanoma are to individualize treatment based on molecular biomarkers. Pharmacogenomics constitute a promising field with regard to melanoma patients’ treatment. Finally, development of novel monoclonal antibodies is expected to complement melanoma patient care while a number of investigational vaccines could find their way into everyday oncology practice.”  I find it interesting here that the genome changes leading up to a cancer could be epigenetic as well as mutations in the genome.  Until recently the conventional wisdom has been that cancers are caused only by gene mutations. 

The January 2010 review publication Molecular cytogenetics of cutaneous melanocytic lesions – diagnostic, prognostic and therapeutic aspects is concerned with cytogenetic alterations in melanocytic tumours.  The focus in this paper is on molecular biology and genetic changes rather than on cancer stem cells.  “This review demonstrates that at present cytogenetics has mainly increased our understanding of the pathogenesis of melanocytic tumours, with an important role for activation of the mitogen-activated protein kinase (MAPK) signaling pathway in the initiation of melanocytic tumours. Mutations in BRAF (in common naevocellular naevi), NRAS (congenital naevi), HRAS (Spitz naevi) and GNAQ (blue naevi) can all cause MAPK activation. All these mutations seem early events in the development of melanocytic tumours, but by themselves are insufficient to cause progression towards melanoma. Additional molecular alterations are implicated in progression towards melanoma, with different genetic alterations in melanomas at different sites and with varying levels of sun exposure. This genetic heterogeneity in distinct types of naevi and melanomas can be used for the development of molecular tests for diagnostic purposes. However, at the moment only few molecular tests have become of diagnostic value and are performed in daily routine practice. This is caused by lack of large prospective studies on the diagnostic value of molecular tests including follow-up, and by the low prevalence of certain molecular alterations. For the future we foresee an increasing role for cytogenetics in the treatment of melanoma patients with the increasing availability of targeted therapy. Potential targets for metastatic melanoma include genes involved in the MAPK pathway, such as BRAF and RAS. More recently, KIT has emerged as a potential target in melanoma patients. These targeted treatments all need careful evaluation, but might be a promising adjunct for treatment of metastatic melanoma patients, in which other therapies have not brought important survival advantages yet.”  As pointed out in the previously-cited paper, there is a need for identifying biomarkers and developing better molecular tests related to them.

One potentially useful biomarker for melanoma is Nestin, as pointed out in the January 2010 publication Stem cell marker nestin expression in peripheral blood of patients with melanoma.  “There is continued interest in markers indicative of circulating melanoma cells. Nestin is a neuroepithelial intermediate filament protein that was found to be expressed in melanoma and in various cancer stem cells. Objective: We investigated expression of nestin in peripheral blood of melanoma patients. Patients/Methods: We analyzed nestin expression by flow cytometry and by quantitative reverse transcription polymerase chain reaction (qRT-PCR) both in tissues (n=23) and blood samples (n=102) from patients with AJCC stage III-IV melanoma. Forty-six negative controls were also added. Results: Flow cytometry did not reveal nestin expressing cells in peripheral blood of healthy volunteers. In melanoma patients, however, nestin protein was expressed in a proportion of melanoma cells enriched from peripheral blood by immunomagnetic sorting. In melanoma tissue samples a significant correlation was found between mRNAs encoding for nestin and tyrosinase (p=0.001) and Mart-1 (p=0.002), whereas in blood a significant correlation was only observed for tyrosinase (p=0.015), but not for Mart-1 (p=0.53). Nestin expression was higher in stage IV patients compared to stage III/IV with no evidence of disease (NED), positively correlated to tumour burden, and positively correlated to expression of tyrosinase and Mart-1. Conclusions: Nestin showed to be an additional marker of interest for circulating melanoma cells.”

The February 2010 publication Prognostic significance of the hair follicle stem cell marker nestin in patients with malignant melanoma also looked at the use of Nestin as a predictive biomarker, this time related to prognosis of outcome in melanoma patients. “Nestin is an intermediate filament protein, and serves as a hair follicle stem cell and neural stem cell marker. Recent studies have suggested that nestin expression is also important for tumorigenesis. Previous reports from our laboratory have revealed that nestin is a marker of HMB-45-negative melanoma cells in dermal invasive lesions of nodular malignant melanoma. The present study examines nestin expression in malignant melanoma and investigates the relationship between nestin expression and prognosis in patients.  We immunohistochemically stained 78 formalin-fixed and paraffin-embedded malignant melanomas for nestin, HMB-45 and S100 reactivity. We found that nestin, HMB-45 and S100 protein were detected in 56.5%, 88.4% and 100% of malignant melanomas, respectively. The 5-year survival rate of stage I and II nestin-positive cases was significantly decreased compared to the nestin-negative cases (p < 0.05). In addition, the 5-year survival rate exceeded 80% in nestin-negative malignant melanomas at all stages of tumor development. We conclude that nestin expression may be a predictor of poor prognosis in patients with malignant melanoma.”

Similar points are made in the January 2010 paper The stem cell marker nestin predicts poor prognosis in human melanoma.  “These results suggest that nestin expression in both tumoral and endothelial cells may be considered an important early prognostic marker in melanoma.”

Collectively, these papers indicate the importance of developing biomarker tests for melanoma, and the emergence of Nestin as such a biomarker.  I note that the emphasis on discovering reliable predictive biomarkers applies to diseases across the spectrum.  See the recent blog entry Harnessing the engines of finance and commerce for life-extension .  The discovery of disease biomarkers is identified there as central to a new paradigm in medicine, Personalized Predictive Preventative Participatory Medicine.

There is much more going on in the world of Melanoma research and I will pick this thread up again in a later blog entry.  I will also discuss research relating various supplements to melanomas. 

As a final note, by far the most effective protection against melanomas and other skin cancers is UV avoidance.  Wear sunhats, clothes, sunglasses and sunscreen protective against both UVa and UVb when outdoors in the summer and at high altitudes, and stay away from tanning beds.

This blog entry is about three recent research results where the outcomes were the opposite of what might have been expected. 

1.     Chocolate consumption and depression are correlated 

The April 2919 publication Chocolate and Depressive Symptoms in a Cross-sectional Analysis indicates a surprising relationship between chocolate consumption and mood, namely that consumption and depression seem to be correlated.  “A sample of 1018 adults (694 men and 324 women) from San Diego, California, without diabetes or known coronary artery disease was studied in a cross-sectional analysis. The 931 subjects who were not using antidepressant medications and provided chocolate consumption information were the focus of the analysis. Mood was assessed using the Center for Epidemiologic Studies Depression Scale (CES-D). Cut points signaling a positive depression screen result (CES-D score, 16) and probable major depression (CES-D score, 22) were used. Chocolate servings per week were provided by 1009 subjects. — Results  Those screening positive for possible depression (CES-D score 16) had higher chocolate consumption (8.4   servings per month) than those not screening positive (5.4 servings per month) (P = .004); those with still higher CES-D scores ( 22) had still higher chocolate consumption (11.8 servings per month) (P value for trend, <.01). These associations extended to both men and women. These findings did not appear to be explained by a general increase in fat, carbohydrate, or energy intake.” 

The study does not indicate a causal connection or, if one exists, direction of the cause.  Does eating chocolate induce depression or do depressed people eat more chocolate?  If it is the first case, the result is the opposite of what I would have thought.  In the second case, eating chocolate while feeling depressed seems completely understandable to me since I am a chocolate-eater.  I have mentioned health benefits of consuming chocolate in several blog entries.  For example, see Health and longevity benefits of dark chocolate. 

2.     Cognition and HIV HAART therapy

A rather interesting recent result is that cognition improves in AIDS patients when HAART therapy is discontinued.  HAART stands for highly active antiretroviral therapy and is normally administered as a cocktail of multiple antiretroviral drugs combined into a single pill.  For a long time it has been thought that anti-retroviral therapy in AIDS patients improves cognitive functioning.  For example. the 1999 publication Positive and sustained effects of highly active antiretroviral therapy on HIV-1-associated neurocognitive impairment reports for a sample of 26 patients” “Conclusion: HAART produces a positive and sustained effect on neurocognitive impairment in HIV-infected patients. A reduction of plasma viral load was associated with the regression of neuropsychological test abnormalities.” The recent result associated with discontinuation of HAART therapy again is the opposite of what was expected. 

The April 2010 publication Neurocognitive effects of treatment interruption in stable HIV-positive patients in an observational cohort reports “Methods: Neurocognitive function was assessed as part of ACTG 5170, a multicenter, prospective observational study of HIV-infected subjects who elected to discontinue ART. Eligible subjects had CD4 count >350 cells/mm3, had HIV RNA viral load <55,000 cp/mL, and were on ART ( 2 drugs) for 6 months. Subjects stopped ART at study entry and were followed for 96 weeks with a neurocognitive examination. — Results: A total of 167 subjects enrolled with a median nadir CD4 of 436 cells/mm3 and 4.5 median years on ART. Significant improvements in mean neuropsychological scores of 0.22, 0.39, 0.53, and 0.74 were found at weeks 24, 48, 72, and 96 (all p < 0.001). In the 46 subjects who restarted ART prior to week 96, no significant changes in neurocognitive function were observed. — Conclusion: Subjects with preserved immune function found that neurocognition improved significantly following antiretroviral treatment (ART) discontinuation. The balance between the neurocognitive cost of untreated HIV viremia and the possible toxicities of ART require consideration.”   

It is interesting that the scores continued to improve during the 96 week period following discontinuation of the HAART therapy.  An important point is that the typical 2010 HAART therapy is different than the 1999 therapy and more effective in its anti-viral effects. So, it might be that one or more of the drugs in the 2010 HAART therapy that were not in the 1999 therapy are responsible for cognitive impairment.

3.     Vitamin-B therapy does not help diabetic nephropathy

We supplement-oriented types often have a default assumption that vitamin therapies are likely to have positive effects.     Specifically, B vitamins have been thought to be useful for treating neurological pathologies.  Not necessarily so, according to the April 2010 publication Effect of B-Vitamin Therapy on Progression of Diabetic Nephropathy.  “Context  Hyperhomocysteinemia is frequently observed in patients with diabetic nephropathy. B-vitamin therapy (folic acid, vitamin B₆, and vitamin B₁₂) has been shown to lower the plasma concentration of homocysteine.  Objective  To determine whether B-vitamin therapy can slow progression of diabetic nephropathy and prevent vascular complications.  Design, Setting, and Participants  A multicenter, randomized, double-blind, placebo-controlled trial (Diabetic Intervention with Vitamins to Improve Nephropathy [DIVINe]) at 5 university medical centers in Canada conducted between May 2001 and July 2007 of 238 participants who had type 1 or 2 diabetes and a clinical diagnosis of diabetic nephropathy.  Intervention  Single tablet of B vitamins containing folic acid (2.5 mg/d), vitamin B₆ (25 mg/d), and vitamin B₁₂ (1 mg/d), or matching placebo.  Main Outcome Measures  Change in radionuclide glomerular filtration rate (GFR) between baseline and 36 months. Secondary outcomes were dialysis and a composite of myocardial infarction, stroke, revascularization, and all-cause mortality. Plasma total homocysteine was also measured.  Results  The mean (SD) follow-up during the trial was 31.9 (14.4) months. At 36 months, radionuclide GFR decreased by a mean (SE) of 16.5 (1.7) mL/min/1.73 m² in the B-vitamin group compared with 10.7 (1.7) mL/min/1.73 m² in the placebo group (mean difference, –5.8; 95% confidence interval [CI], –10.6 to –1.1; P = .02). There was no difference in requirement of dialysis (hazard ratio [HR], 1.1; 95% CI, 0.4-2.6; P = .88). The composite outcome occurred more often in the B-vitamin group (HR, 2.0; 95% CI, 1.0-4.0; P = .04). Plasma total homocysteine decreased by a mean (SE) of 2.2 (0.4) µmol/L at 36 months in the B-vitamin group compared with a mean (SE) increase of 2.6 (0.4) µmol/L in the placebo group (mean difference, –4.8; 95% CI, –6.1 to –3.7; P < .001, in favor of B vitamins).  Conclusion  Among patients with diabetic nephropathy, high doses of B vitamins compared with placebo resulted in a greater decrease in GFR and an increase in vascular events.” GFR stands for  glomerular filtration rate, the best test to measure level of kidney function and determine the stage of  a kidney disease.  Lower is worse and scores below 15 indicate a pathology.  The B-vitamins did lower homocysteine but they also significantly lowered GFR indicating an overall negative effect in the population studied.

A few observations with respect to the life sciences and longevity:

·        Reasonable conjectures and conclusions might not be valid.

·        What we know we don’t know expands faster than what we know.  Uncertainty expands faster than the certainty. 

·         We have to live with that growing uncertainty.  For example, does eating chocolate cause depression or is that a good thing to do if you have depression?  In the past this was not something to be concerned about.. 

I have posted several blog entries related to Alzheimer’s Disease (AD), including  New views of Alzheimer’s disease and new approaches to treating it,  The social cost of Alzheimer’s disease and late-life dementia, Diet and cognition, Warding off Alzheimer’s Disease and things in my diet,  and a short post Deconstructing Alzheimer’s Disease – role of mitochondria.  The purpose of this blog entry is to report on some additional mostly-recent research findings, focusing particularly on dietary substances and supplements and their impacts on AD.  Topics covered are research related to 1.  Relationship of SIRT1 and taking resveratrol to AD, 2. Dietary substances and supplements and AD, 3. Clinical trials of dietary substances for AD, and 4. Curcumin and AD. 

1.      SIRT1 and Resveratrol and Alzheimer’s Disease 

I have discussed the SIRT1 gene and protein in numerous contexts in this blog.  In the blog entry SIRT1, mTOR, NF-kappaB and resveratrol I mentioned links between SIRT1 activation, mTOR signaling suppression, and inhibition of NF-kappaB.  And I discussed how some researchers think activation of SIRT1 might confer a strong a therapeutic effect for control of Alzheimer’s disease.  Several review articles published in the last couple of years articulate that hypothesis and suggest a potential role for resveratrol in controlling AD.  These articles include the March 2010 e-publication Resveratrol as a Therapeutic Agent for Neurodegenerative Diseases, the 2009 publication Resveratrol and neurodegenerative diseases: activation of SIRT1 as the potential pathway towards neuroprotection and the 2008 publication Modulation of sirtuins: new targets for antiageing. “ — increasing SIRT1 has been found to protect cells against amyloid-beta-induced ROS production and DNA damage, thereby reducing apoptotic death in vitro. Moreover, it has been demonstrated that Alzheimer’s and Huntington’s disease neurons are rescued by the over-expression of SIRT1, induced by either caloric restriction or administration of resveratrol, a potential activator of this enzyme. The therapeutic use of resveratrol (a polyphenol present in red wines) and other related compounds, which utilize SIRT1 pathway modulators, in treating aging-related brain disorders will be discussed in this review(ref).”   

While these and other review articulate this hypothesis (that resveratrol and SIRT1 activation could be useful in controlling AD), current experimental evidence to this effect seems to be scarce.  There were earlier experiments indicating that resveratrol is neuroprotective against beta-amyloid-induced neurotoxicity in rat neurons(ref), but I am unaware of any tests of the effects of taking resveratrol on warding off or treating human Alzheimer’s disease.  Personally, I think resveratrol is a great supplement but the hypothesis that resveratrol and SIRT1 activation could be useful in controlling AD remains an untested conjecture as far as I know.  Theoretical reasons for taking resveratrol may be strong but clinical data on effectiveness in AD is nonexistent. 

2.     Alzheimer’s Disease, dietary substances and supplements

A large number publications based on in-vitro and mouse studies suggest that polyphenols occurring in common dietary substances and supplements may be useful for treating or preventing the onset of Alzheimer’s or other neurodegenerative diseases.  Examples are:

·          (2010) l-theanine protects the APP (Swedish mutation) transgenic SH-SY5Y cell against glutamate-induced excitotoxicity via inhibition of the NMDA receptor pathway. 

·         (2010) [Review on the neuroprotective effects of green tea polyphenols for the treatment of neurodegenerative diseases] 

·         (2010) Fish oil enhances anti-amyloidogenic properties of green tea EGCG in Tg2576 mice.

·         (2010) Nanolipidic particles improve the bioavailability and alpha-secretase inducing ability of epigallocatechin-3-gallate (EGCG) for the treatment of Alzheimer’s disease.

·          (2010)  Naturally occurring phytochemicals for the prevention of Alzheimer’s disease.

·         The 2009 study Cinnamon extract inhibits tau aggregation associated with Alzheimer’s disease in vitro reports “An aqueous extract of Ceylon cinnamon (C. zeylanicum) is found to inhibit tau aggregation and filament formation, hallmarks of Alzheimer’s disease (AD). The extract can also promote complete disassembly of recombinant tau filaments and cause substantial alteration of the morphology of paired-helical filaments isolated from AD brain.”

·         (2009) Natural Compound In Extra-Virgin Olive Oil — Oleocanthal — May Help Prevent, Treat Alzheimer’s 

·         (2009)  l-Theanine, an amino acid in green tea, attenuates beta-amyloid-induced cognitive dysfunction and neurotoxicity: reduction in oxidative damage and inactivation of ERK/p38 kinase and NF-kappaB pathways.

·         (2009) Neuroprotective molecular mechanisms of (-)-epigallocatechin-3-gallate: a reflective outcome of its antioxidant, iron chelating and neuritogenic properties. 

·         (2009) Green tea (-)-epigallocatechin-3-gallate inhibits beta-amyloid-induced cognitive dysfunction through modification of secretase activity via inhibition of ERK and NF-kappaB pathways in mice.

·         (2009) Green tea polyphenol (-)-epigallocatechin-3-gallate enhances the inhibitory effect of huperzine A on acetylcholinesterase by increasing the affinity with serum albumin.

·         (2009) Neuroprotective effect of epigallocatechin-3-gallate against beta-amyloid-induced oxidative and nitrosative cell death via augmentation of antioxidant defense capacity.

·         (2009) Botanical phenolics and brain health.

·         (2009) Natural antioxidants protect neurons in Alzheimer’s disease and Parkinson’s disease.

·         (2008) Simultaneous manipulation of multiple brain targets by green tea catechins: a potential neuroprotective strategy for Alzheimer and Parkinson diseases.

·          (2008) Cell signaling pathways and iron chelation in the neurorestorative activity of green tea polyphenols: special reference to epigallocatechin gallate (EGCG).

·          (2008) Lipoic acid as an anti-inflammatory and neuroprotective treatment for Alzheimer’s disease. 

·         (2008) Targeting multiple neurodegenerative diseases etiologies with multimodal-acting green tea catechins.

·         (2008) Green tea epigallocatechin-3-gallate (EGCG) reduces beta-amyloid mediated cognitive impairment and modulates tau pathology in Alzheimer transgenic mice.

·         (2008) Green tea catechins prevent cognitive deficits caused by Abeta1-40 in rats. 

·         (2008) Epicatechin gallate increases glutamate uptake and S100B secretion in C6 cell lineage. 

·         (2008) Neuroprotective effects of (-)-epigallocatechin-3-gallate (EGCG) on paraquat-induced apoptosis in PC12 cells. 

Again, the clinical evidence for the effectiveness of most of these substances against Alzheimer’s disease seems to range from being very limited to nonexistent.  Pharmaceutical companies are understandably only willing to spend millions or hundreds of millions of dollars on clinical trials of substances that they own the rights to – and that excludes foods and plant-based supplements.  However, some public agencies have been stepping in to fill the gaps. 

3.     Clinical Trials of dietary substances for AD

The clinicaltrials.gov database shows some 781 clinical trials for AD of which approximately 100 are ongoing.  I went only through the first 300 trials listed to come up with the following related to dietary substances or supplements.  

The Department of Veterans Affairs is currently sponsoring two such trials now in the recruiting phase, A Single Center, Multi-site, Randomized, Double-blind, Placebo-controlled Trial of Resveratrol With Glucose and Malate (RGM) to Slow the Progression of Alzheimer’s Disease.  And A Randomized, Clinical Trial of Vitamin E and Memantine in Alzheimer’s Disease (TEAM-AD)   Another clinical trial Lipoic Acid and Omega-3 Fatty Acids for Alzheimer’s Disease sponsored by the Oregon Health and Science University is not yet recruiting.  The clinical trial Lutein and Alzheimer’s Disease Study (LAD) sponsored by the Oregon Health and Science University is listed as still in the recruiting phase.

A clinical study Prevention of Alzheimer’s Disease by Vitamin E and Selenium (PREADVISE) sponsored by the National Institute on Aging (NIA) is ongoing.

4.     Completed clinical trials of dietary substances for AD

A clinical trial VITAL – VITamins to Slow ALzheimer’s Disease (Homocysteine Study) sponsored by the National Institute on Aging has been completed.  The substances tested were Folate, Vitamin B6 and Vitamin B12.  The 18-month double blind study involved in 409 participants in 38 study locations and was designed to measure differences in decline of cognitive functioning.  The study results, reported in the publication High-dose B vitamin supplementation and cognitive decline in Alzheimer disease: a randomized controlled trial, were negative. “A higher quantity of adverse events involving depression was observed in the group treated with vitamin supplements. CONCLUSION: This regimen of high-dose B vitamin supplements does not slow cognitive decline in individuals with mild to moderate AD.” 

Another completed clinical trial is OmegAD (Omega-3 and Alzheimer’s Disease), sponsored by Karolinska University Hospital.  The randomized double-blind trial involved omega-3 fatty acid treatment of 174 patients with mild to moderate Alzheimer’s Disease .  The results were underwhelming as reported in the 2006 paper Omega-3 fatty acid treatment in 174 patients with mild to moderate Alzheimer disease: OmegAD study: a randomized double-blind trial.  “CONCLUSIONS: Administration of omega-3 fatty acid in patients with mild to moderate AD did not delay the rate of cognitive decline according to the MMSE or the cognitive portion of the Alzheimer Disease Assessment Scale. However, positive effects were observed in a small group of patients with very mild AD.” 

A clinical trial Long-term Use of Galantamine Versus Nootropics (Memory Enhancing Drugs) in Patients With Alzheimer’s Dementia Under Conditions of Daily Routine sponsored by Janssen-Cilag G.m.b.H has been completed but no results of it have yet been published.  The nootropics were evaluated secondarily, galantamine being the main focus of the study.  The nootropics included ginkgo biloba, nicergoline, and piracetam. 

A clinical trial Effect of Panax Ginseng on the Cognitive Performance in Alzheimer’s Disease sponsored by the Seoul National University Hospital has been completed.  The results are published in the 2008 publication Panax ginseng enhances cognitive performance in Alzheimer disease.  “Consecutive AD patients were randomly assigned to the ginseng (n=58) or the control group (n=39), and the ginseng group was treated with Panax ginseng powder (4.5 g/d) for 12 weeks. Cognitive performances were monitored using the mini-mental state examination (MMSE) and Alzheimer disease assessment scale (ADAS) during 12 weeks of the ginseng treatment and at 12 weeks after the ginseng discontinuation. MMSE and ADAS scales showed no baseline difference between the groups. After ginseng treatment, the cognitive subscale of ADAS and the MMSE score began to show improvements and continued up to 12 weeks (P=0.029 and P=0.009 vs. baseline, respectively). After discontinuing ginseng, the improved ADAS and MMSE scores declined to the levels of the control group. These results suggest that Panax ginseng is clinically effective in the cognitive performance of AD patients.” 

A number of clinical trials are shown as completed but no publications report on their results.A competed pilot clinical trial Alzheimer’s Disease: Potential Benefit of Isoflavones shows no publication history up to this point. A clinical trial Fish Oil and Alpha Lipoic Acid in Treating Alzheimer’s Disease Phases I and II sponsored by the Oregon Health and Science University appears to be completed in 2007 but no associated publication is listed.  The study Curcumin in Patients With Mild to Moderate Alzheimer’s Disease sponsored by the John Douglas French Foundation is shown as completed but I could find no publication reporting on it. 

Please note that the above is only a sample of the available clinical trial information.  I reviewed only 300 out of 781 studies listed.  Assuming the above-mentioned trials are representative, I am struck by how thin our clinical trial information is with respect to the effectiveness of supplements against Alzheimer’s disease 

4.     Curcumin and Alzheimer’s Disease

There seems to be a considerable literature with respect to curcumin and AD(ref).  While I have not independently verified the statements and citations therein, the October 2, 2007  item Turmeric and Alzheimer ‘s disease by Jacob Schor, ND appears to me to be particularly informative.  He makes a compelling case for why curcumin may be helpful in prevention of AD. He points out that the incidence of AD in India where curcumin is commonly found in foods is a quarter that in the US. (Note that some of this might be explainable by differing demographic factors like life expectancy.)  As I have been pointing out, we lack the information that could be provided by a large and well-designed clinical trial of curcumin in AD.  According to the Pauling Foundation web site: “In Alzheimer’s disease, a peptide called amyloid beta forms aggregates (oligomers), which accumulate in the brain and form deposits known as amyloid plaques (72). Inflammation and oxidative damage are also associated with the progression of Alzheimer’s disease (73). Curcumin has been found to inhibit amyloid beta oligomer formation in vitro (74). When injected peripherally, curcumin was found to cross the blood brain barrier in an animal model of Alzheimer’s disease (74). In animal models of Alzheimer’s disease, dietary curcumin has decreased biomarkers of inflammation and oxidative damage, amyloid plaque burden in the brain, and amyloid beta-induced memory deficits (74-77). It is not known whether curcumin taken orally can cross the blood brain barrier or inhibit the progression of Alzheimer’s disease in humans. As a result of the promising findings in animal models, clinical trials of oral curcumin supplementation in patients with early Alzheimer’s disease are under way (59, 78). The results of a 6-month trial in 27 patients with Alzheimer’s disease found that oral supplementation with up to 4 g/day of curcumin was safe (4). Larger controlled trials are needed to determine whether or not oral curcumin supplementation is efficacious in Alzheimer’s disease.” 

Please see the medical disclaimer for this blog.