The home run reported this week was in a practice game in a minor league.  But nonetheless the results are impressive and lead to hope for those suffering from certain forms of congenital blindness and vision impairment.  The report leading to this optimism, Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial, appeared in the November 7 edition of The Lancet.

The trial involved 12 patients (aged 8—44 years), all having  Leber’s congenital amaurosis (LCA), “a rare inherited eye disease that appears at birth or in the first few months of life, and affects around 1 in 80,000 of the population.”

People with this disease are born with impaired vision and their  vision deteriorates until they are totally blind, usually within 10-15 years. “Typically a baby with LCA will have very reduced vision at birth although the retina may appear normal when first examined. Within months, however, parents will usually notice nystagmus – an involuntary, rhythmical, repeated movement of the eyes. Children with LCA account for 10-18% of all cases of congenital blindness. Vision in individuals with LCA varies greatly from relatively mild acuity problems (20/70) to no light perception(ref).”  Up to this point, the disease has been incurable.

LCA is an autosomal recessive disorder, which means that if both parents are carriers of a defective gene as well as a good gene, there is a 1 in 4 chance the child will end up with two defective genes and manifest the disease.  The 12 patients in the recent trial had a variant of LCA caused a defect in the RPE65 gene(ref)(ref), a gene responsible for encoding the retinal pigment epithelium-specific 65 kDa protein. This protein “is located in the retinal pigment epithelium and is involved in the conversion of all-trans retinol to 11-cis retinal during phototransduction, which is then used in visual pigment regeneration in photoreceptor cells(ref).” 

The  therapy used in “home run” trial  is a simple one.   Each of the 12 patients were given one given one subretinal injection using a thin needle in the worst eye of a “defanged” adeno-associated virus (AAV) into which the RPE65 gene had been inserted.  “The engineered virus then invaded retinal cells and inserted the gene into the cells’ DNA. — Within two weeks, the treated eyes began to become more sensitive to light, and within a few more weeks, vision began to improve. The younger the patients were, the better they responded.  — By both objective and subjective measures, vision improved for all the patients. They were able to navigate obstacle courses, read eye charts and perform most of the tasks of daily living. The children who were treated “are now able to walk and play just like any normally sighted child —  The improvement has now persisted for as long as two years(ref).”  There appeared to be no safety issues or adverse effects involved.   

“The study “holds great promise for the future” and “is appealing because of its simplicity,” wrote researchers from the Nijmegen Medical Center in the Netherlands in an editorial accompanying the report –(ref).” The study had its limits of course.  The patients would like to have their other eye treated and optimum dosage of the adenovirus/RPE65 is not yet determined.  Yet the results lend hope to the other some 130,000 victims of LCA worldwide as well as to victims of other more common hereditary eye diseases such as Retinitis pigmentosa.

After many years of false starts and problematical trials, it appears that gene therapies may finally be getting off the ground.  See also the recent post ALD and lentivirus vectors for gene therapy.

Senesco is a small research-based company specializing in “genetic technologies designed to regulate cell death.”  A company that started out with focus on commercial agriculture, it has been exploring a proprietary gene, Factor 5A1, that might well also have a role in treating human diseases, particularly cancers.  The story of Senesco is interesting because it illustrates the serendipity that can lead to drug discovery, because it describes a research company that has no research labs, because it points to another gene that could be a key to curing certain cancers, and because after five years the possible cancer therapy is still in a preclinical stage tested only on small animals.

The trade publication Gen featured a story this week, Senesco Attempts to Exploit Cell Death, which tells the early history of the company.  “At the University of Waterloo, Ontario, John Thompson, Ph.D., now Senesco’s CSO, discovered that lipase is regulated by two genes that work together to control senescence in plants. Because the finding held promise for extending the life of plants, Sascha Fedyszyn co-founded Senesco in 1998 to explore agricultural applications. A few years later, company researchers learned that the same senescence cascade in plants operates in humans, too. “That was the start of our life science work,” says Fedyszyn, vp, corporate development. — Senesco scientists found that two genes—eukaryotic translation initiation Factor 5A and deoxyhypusine synthase (DHS)—are powerful regulators of programmed cell death, known as apoptosis in human cells and senescence in plant cells. Although cell death is a normal function, premature apoptosis leads to inflammatory conditions, cancer, and tumor growth.”

The company outsources its research.  “To take greatest advantage of our early stage discoveries, we have formed a highly focused and efficient R&D department headed by John Thompson, Ph.D., the inventor of our technology. This R&D takes place at several highly regarded academic institutions around North America. While most early-stage biotechnology companies spend millions on R&D, we are conducting top-notch research with relatively minimal cash burn by utilizing these institutions(ref).” 

The company maintains relationships with a number of agricultural product firms.  “Agricultural research and development programs continue through licensing and joint ventures with agricultural biotechnology companies such as Monsanto, Bayer Crop Science, and Scotts. Efforts are under way to improve corn, soybeans, rice, cotton, bananas, canola, turf grass, and ornamental plants(ref).”

Relating to the life sciences area, “Senesco’s technology is based on the discovery that Factor 5A1, one of two human genes encoding eucaryotic translation initiation Factor 5A, regulates apoptosis as well as certain execution genes, pro-inflammatory cytokines, receptors and transcription factors and therefore plays a key role in cell death and inflammation. We believe that Factor 5A1 is a novel and potentially powerful therapeutic target for a broad range of apoptotic diseases, including inflammatory/ischemic diseases and cancers(ref).” 

In plain language, the following from the company’s web site says that Factor 5A1 kills cancer cells without affecting normal cells.  “Factor 5A1 functions as a shuttle protein, selectively translocating mRNAs required for apoptosis and cytokine function from the nucleus to cytosolic ribosomes for translation. Our preclinical studies have shown that Factor 5A1 kills cancer cells through both the intrinsic (p53) and the extrinsic (cell death receptor) pathways. We have specifically shown that Factor 5A1 regulates the expression of p53, caspases, TNFR1 (TNFa receptor) and the IFN-gamma receptor, and that it also negatively regulates bcl-2 and telomerase. Our studies to date have shown that Factor 5A1 may be non-toxic to normal cells, presumably because of its function as a shuttle protein. Normal cells are not actively expressing mRNAs of cell death genes, and therefore the Factor 5A1 protein, even if present in normal cells, is non-functional(ref).”

According to the Gen article “The company’s first drug candidate based on this combination therapy, SNS-01, targets multiple myeloma. The cytokines IL-1, IL-6, and NF-kB are known to contribute to the proliferation of this disease. The design of SNS-01 “provides a perfect combination for killing multiple myeloma cells,” says Richard Dondero, Senesco’s vp of R&D. Multiple myeloma is not only a cancer, but also a disease of the immune system with a proinflammatory side. “Our technology lends itself to that,” Dondero says. — Collaborators at the Mayo Clinic tested SNS-01 in in-vitro experiments and on mice. Within two weeks of intravenous delivery of SNS-01, “tumor reduction was visible, and some multiple myeloma tumors were even completely eradicated within six weeks,” says Dondero. Senesco plans to file an IND application for SNS-01 in the treatment of multiple myeloma early in 2010.”

Promotion or inhibition of Factor 5A1 may also serve other therapeutic purposes and Senesco’s experiments with therapies  based on Factor 5A1  go back at least five years.   For example, according to a 2004 report Factor 5A1 may be useful for controlling glaucoma. “By inhibiting Factor 5A1, Senesco blocked TNF-alpha-induced apoptosis by 80% in lamina cribrosa cells of the human optic nerve head. — TNF-alpha is strongly upregulated in the optic nerve head of the glaucomatous eye, and TNF-alpha-induced apoptosis appears to be an important determinant of the progressive neurodegeneration characteristic of glaucoma. Thus, inhibition of TNF-alpha-induced apoptosis may reduce damage to the optic nerve during glaucoma.”  Another 2004 report indicated Proprietary gene Factor 5A1 induces atopsis in lung cancer tumors of mice. 

It will be interesting to see whether Senesco will be successful in moving treatments involving the targeting of Factor 5A1 into clinical trials and, if so, how long it will take for this to happen.

If you want to live a very long life and are tired of the highly technical stuff in this blog, you might want to appeal to one of the traditional longevity gods or goddesses.  Some of these gods and goddesses, courtesy of the Encyclopedia Mythica website are:

1.   Fukurokuju 

2.   Gama 

3.   Djambu Baros 

4.   Shou-xing 

5.   Atira 

6.   Peng-zi 

7.   San-xing 

8.   Shou-lao 

9.   Heammawihio 

10.       Pancaraksa 

Gene therapy – the substituting of inherited defective genes with good ones – does not have an outstanding success record but now is getting off the ground.  As pointed out in an editorial in the Nov 6 2009 issue of Science magazine, “Gene therapy has recently had some important successes in treating severe inherited diseases after years of skepticism from the scientific community and neglect by the pharmaceutical industry.  On page 818 in this issue, Cartier et al. (4) report another major advance—the successful first clinical testing of an HIV-derived vector in hematopoietic stem cell (HSC)–based gene therapy. The procedure was used to treat a severe neurodegenerative disease, X-linked adrenoleukodystrophy (ALD), and the results indicate stable expression of a therapeutic gene in a substantial fraction of patients’ hematopoietic cells, as well as clinical benefits.”  

ALD is a rare disease that typically affects boys between the ages of six and 10. Featured in the Hollywood film Lorenzo’s Oil, “ALD is a severe hereditary condition caused by a deficiency of a protein called ALD that is involved in fatty acid degradation. Sufferers steadily lose their myelin sheath, the protective layer that coats nerve fibers in the brain. Without myelin the nerves lose function, leading to increasing physical and mental disability in patients. X-linked ALD, the most common form of the disease, affects boys starting at age 6-8 years of age and death usually occurs before the patients reach adolescence(ref).”

The November 6 2009 research article Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-Linked Adrenoleukodystrophy was reported widely in the press and describes successful treatment by a French team of two patients with ALD.  “X-linked adrenoleukodystrophy (ALD) is a severe brain demyelinating disease in boys that is caused by a deficiency in ALD protein, an adenosine triphosphate–binding cassette transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). We initiated a gene therapy trial in two ALD patients for whom there were no matched donors. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1, and then re-infused into the patients after they had received myeloablative treatment. Over a span of 24 to 30 months of follow-up, we detected polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. These results strongly suggest that hematopoietic stem cells were transduced in the patients. Beginning 14 to 16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped, a clinical outcome comparable to that achieved by allogeneic HCT. Thus, lentiviral-mediated gene therapy of hematopoietic stem cells can provide clinical benefits in ALD.”

“The healthy ALD protein was expressed in about 15 percent of blood cells, yet surprisingly this low level was sufficient to slow brain disease in ALD. This percentage of correction will not be sufficient for all diseases,” warns Aubourg (Patrick Aubourg, co-author of the study). “There is a lot of work to be done to make this gene therapy vector more powerful, less complicated, and less expensive. This is only the beginning,” he said(ref).”

What  is particularly interesting in the study is use of a defanged version of the HIV virus as a delivery vector for getting the corrected genes into the hematopoietic stem cells extracted from the patients.  “In most gene therapy studies, a working gene is inserted into the genome to replace a dysfunctional, disease-causing gene. A carrier molecule called a vector is used to deliver the therapeutic gene into the patient’s cells. Vectors are typically the backbones of viruses that have been genetically altered to carry normal human DNA. Scientists have recently turned to vectors based on the lentivirus genus of retroviruses, which includes HIV. Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells, and are thought to provide long-term and stable gene expression, unlike other retroviruses(ref).” 

“Although studies with larger cohorts of patients are needed, these results suggest that gene therapy with lentiviral vectors, which are derived from disabled versions of human immunodeficiency virus (HIV), could potentially become instrumental in treating a broad range of human disorders(ref).”  A concern has been that the lentiviral DNA could integrate itself into the stem cells and their descendents and in time create mischief.  “Gene therapy is not without serious risks. Like other retrovirus vectors, the HIV-derived lentivirus vector is tasked with inserting the therapeutic gene in the chromosomes of the patients’ cells. In a worst case scenario, this action could disturb the biology of the cells and patients could end up with leukemia; this outcome has occurred in past gene therapy trials.  “The HIV-derived lentivirus vector basically has this same risk, although the design of the vector makes patients less prone to this side effect,” said Aubourg(ref).” It seems like the two boys are doing fine so far.

Falling is one of the major causes of debilitation and accelerated death among the elderly.  Older as well as recent research indicates that old folks in and out of nursing homes who take regular Vitamin D supplements fall a lot less than those who don’t.

The research literature on injuries and deaths in the elderly due to falling goes way back.  In 1985 it was written “Injuries are the sixth leading cause of death in the 75-and-over population, with falls the leading cause of injury-related deaths. Hospitals and residential centers for the elderly have high rates of falls and injuries. With increasing age, patients in nursing homes have a corresponding increase in the proportion of fatal falls.” My own mother fell and very seriously injured herself in her late 80s, contributing to her decline and death at age 93.  A 1996 report Epidemiology of hip fractures indicated “There were an estimated 1.66 million hip fractures world-wide in 1990. According to the epidemiologic projections, this worldwide annual number will rise to 6.26 million by the year 2050. This rise will be in great part due to the huge increase in the elderly population of the world.”  Over 90% of hip fractures result from falling. 

According to the CDC website on hip fractures,

• “In 2004, there were more than 320,000 hospital admissions for hip fractures, a 3% increase from the previous year.
• About one out of five hip fracture patients dies within a year of their injury.
• Most patients with hip fractures are hospitalized for about one week.
• Up to one in four adults who lived independently before their hip fracture has to stay in a nursing home for at least a year after their injury.
• In 1991, Medicare costs for hip fractures were estimated to be $2.9 billion.” (Imagine what the cost must be now!)

There is also a long history relating vitamin D to falling and hip fractures.  Citing data based on women living in nursing homes in France, in the 1996 paper Prevention of hip fractures by correcting calcium and vitamin D insufficiencies in elderly people the author state “We have shown in a 3-year controlled prospective study that the daily use of these supplements (1.2 g of calcium and 800 IU of vitamin D3) given in a large population of 3270 elderly ambulatory women living in nursing homes reduced of 23% (intention-to-treat analysis) the number of hip fractures and other non vertebral fractures.”

Another study report, published in 2008 was entitled A Higher Dose of Vitamin D Reduces the Risk of Falls in Nursing Home Residents: A Randomized, Multiple-Dose Study.  “PARTICIPANTS: One hundred twenty-four nursing home residents (average age 89). INTERVENTION: Participants were randomly assigned to receive one of four vitamin D supplement doses (200 IU, 400 IU, 600 IU, or 800 IU) or placebo daily for 5 months. MEASUREMENTS: Number of fallers and number of falls assessed using facility incident tracking database. RESULTS: —  Participants in the 800 IU group had a 72% lower adjusted-incidence rate ratio of falls than those taking placebo over the 5 months (rate ratio=0.28; 95% confidence interval=0.11-0.75). — CONCLUSION: Nursing home residents in the highest vitamin D group (800 IU) had a lower number of fallers and a lower incidence rate of falls over 5 months than those taking lower doses. Adequate vitamin D supplementation in elderly nursing home residents could reduce the number of falls experienced by this high falls risk group.”  A discussion of this study treating its limitations and comparing the results with those of other studies can be found here.

An October 2009 report Extended Physiotherapy and Vitamin D Protect Against Second Hip Fractures appears to describe the latest work on this topic. “Extended physiotherapy significantly reduced the rate of falls among patients with a prior hip fracture, and high-dose vitamin D significantly reduced the rate of hospital readmissions in a study of 173 patients. — A program of extended physiotherapy reduced the fall rate by 25%, compared with standard postfracture physiotherapy; high-dose vitamin D therapy reduced the hospital readmission rate by 39%, compared with a lower dose, the researchers found.”  Half of the patients had a severe vitamin D deficiency when they started the study and the mean age of the patients was 84 years.  Some patients were given 800 IU of vitamin D, others 2000 IU. “There was no difference in the fall rate for the two vitamin D groups, but high-dose vitamin D did reduce the rate of hospital readmission by 39%, which was significant. There was also a significant 60% reduction in fall-related injuries. “This was mainly driven by a nonsignificant reduction in repeat nonvertebral fractures by 52%,” said Dr. Bischoff-Ferrari of the Centre on Aging and Mobility at the University Hospital Zurich.”

Interestingly, I found one 2007 dissenting study Does Vitamin D Stop Inpatients Falling? A Randomised Controlled Trial.  “Objective: to determine whether routine supplementation with vitamin D plus calcium reduces numbers of fallers and falls in a cohort of hospital admissions while they are inpatients.  Design: randomised, double-blind, controlled study. Participants: two hundred and five acute admissions > 65 years to a geriatric medical unit. Methods: patients were randomised to intervention of daily vitamin D 800 IU plus calcium 1,200 mg or control group of daily calcium 1,200 mg, until discharge or death.  — Results: median age 84 years and a median length of stay = 30 days.   —  Although there were fewer fallers in the vitamin D cohort, this did not reach statistical significance –Conclusions: in a population of geriatric hospital inpatients, vitamin D did not reduce the number of fallers. Routine supplementation cannot be recommended to reduce falls in this group.”  I think the explanation is that the lengths of the hospital stays involved (30 days mean) were too short for the effects of vitamin D to be manifest and too short for many falls to happen (36 of those on vitamin D, 45 for those doing only calcium).  The other studies were longer and involved a lot more falls providing statistical significance.

So, it appears that Vitamin D works two ways to prevent fall-related injuries: 1. it reduces the number of falls, probably by a combination of muscle strengthening and improving coordination, and 2. it reduces the number of fractures in those who do fall, via bone strengthening.  Of course, preventing falls is only one of an impressive array of benefits due to vitamin D supplementation.

Chronic nagging back pain can result from spinal cord injury (SCI) and can lead to pain in other parts of the body that are in fact not injured.  It can be very difficult to diagnose and a person suffering from it can find their quality of life seriously compromised.  I know, for I have been suffering from such pain for three months now and only this week have I been able to put together a diagnosis and identify an effective treatment plan for myself.  There are two stories to be told here.  One is a science story about a new paradigm for viewing and treating chronic neuropathic pain and the other is a personal story about how I managed to connect the dots about my personal problem and arrive at insight as to what is going on in me.   I relate the science story here.

Neuropathic pain is pain “gone wild,” normally pain that was initiated by some injury to the nervous system but that assumes a life of its own and is out of proportion to the original injury and can show up in other locations. It creates “allodynia, meaning “other pain”, a pain due to a stimulus which does not normally provoke pain(ref).”   “As much as 7% to 8% of the population is affected by neuropathic pain and in 5% it may be severe(ref)(ref)(ref).”  Neuropathic pain may result from a variety of conditions including injuries to or disorders of the peripheral nervous system or the central nervous system (brain and spinal cord).

The traditional view of pain is that it is caused by firing of neurons which transmit signals up to the brain.  The conventional way to handle even persistent neuropathic pain has therefore been to block the neural signals.  This approach has a basic limitation.  Opiates like morphine are the most effective drugs for blocking acute pain.  But in the case of chronic pain opiates seem to become less and less effective in time requiring the use of higher doses leading to addiction. “Previous studies have shown opiates to be relatively ineffective for neuropathic pain in animals, and the animals typically develop tolerance over a short period of time, in that the drugs lose their ability to relieve pain(ref).”

In the mid-90s a new view of neuropathic pain started to emerge, which is to pay attention to the glial cells, the microglia and astrocyte cells which accompany and support  the neurons in nervous tissues in the spine.   Before pain is perceived in the brain, pain-producing signals must pass through stages of neural circuitry including ones that pass through the spine. There are more glia than neurons in the spinal cord and brain and they play essential functions like providing nourishment  to neurons, maintaining the chemical environment surrounding neurons and absorbing neurotransmitter molecules given off by them.  Glia listen carefully to signaling molecules from the neurons they accompany and respond with signaling molecules of their own.  This mutual molecular signaling process serves many important functions, including facilitating repair of damaged nerves.  However, it can get out of control and produce neuropathic pain.   

Quoting from a 2004 paper Glial activation: a driving force for pathological pain “Pain is classically viewed as being mediated solely by neurons, as are other sensory phenomena.   The discovery that spinal cord glia (microglia and astrocytes) amplify pain requires a change in this view. These glia express characteristics in common with immune cells in that they respond to viruses and bacteria, releasing proinflammatory cytokines, which create pathological pain. These spinal cord glia also become activated by certain sensory signals arriving from the periphery. Similar to spinal infection, these signals cause release of proinflammatory cytokines, thus creating pathological pain.  Taken together, these findings suggest a new, dramatically different approach to pain control, as all clinical therapies are focused exclusively on altering neuronal, rather than glial, function.”  An excellent and detailed explanation of this new view is in the article New Culprits in Chronic Pain in the November 2009 issue of Scientific American.  This 2009 paper further explains the molecular biology of how activated microglia creates pain without apparent cause (allodynia).

A simplified explanation is that neurons respond to an injury by firing rapidly to signal pain and releasing stress-related messenger molecules.  These changes are picked up by the microglia which begin emitting molecules that ease stress on the neurons and increase their sensitivity.  Among these molecules are proinflammatory cytokines like Interleukin-1, TNFα and IL-6, and TNFr, urgent messengers requesting mobilization of the body’s repair mechanisms.  The cytokines can greatly increase the sensitivity of the injured neurons as well as other neighboring neurons causing them to fire all the more.  This in turn leads to  additional production of cytokines by neighboring microglia, etc.   A vicious pro-inflammatory cycle ensues.  As this happens the pain is amplified.   Further, the cytokines fan out and affect other tissue systems leading them to give out their own pain signals.  The result is neuropathic pain – pain amplified in the nervous system. 

“Pathological pain has long been described as the result of dysfunctional neuronal activity.  While neuronal functioning is indeed altered, there is significant evidence showing that exaggerated pain is regulated by the activation of astrocytes and microglia. In exaggerated pain, astrocytes, and microglia are activated by neuronal signals including substance P, glutamate, and fractalkine. Activation of glia by these substances leads to the release of mediators that then act on other glia and neurons. These include a family of proteins called “proinflammatory cytokines” released from microglia and astrocytes. These cytokines have been shown to be critical mediators of exaggerated pain. Some patients with pathological pain also report “extra-territorial” and/or “mirror” image pain (Allodynia). That is, exaggerated pain is experienced not only in the area of trauma.  In extra-territorial pain, pain is also perceived as arising from neighboring healthy tissues outside of the site of trauma. In the rare cases of mirror-image pain, such pain is perceived as arising from the healthy, corresponding body part on the opposite side of the body. New data suggest that activation of astrocyte communication via gap junctions may mediate such spread of pain(ref).”

The paper Activated Microglia Contribute to the Maintenance of Chronic Pain after Spinal Cord Injury probably tells the central story of my own pain experience “Given that there are data supporting the involvement of microglia in pain after peripheral injury and work showing chronic post-SCI activation of microglia, we hypothesized that activated spinal microglia play a role in chronic central pain after SCI. Here, we report that thoracic SCI causes chronic activation of microglia in the lumbar spinal cord and that these activated microglia contribute to the maintenance of neuronal hyperresponsiveness and pain-related behaviors.”

The new view of pain also explains why opiates like morphine lose their pain-killing effectiveness in time(ref).  It turns out that since morphine or other opiates lowers the sensitivity of neurons to pain, the accompanying glia whose job is to maintain balance detect that fact and send out proinflammatory molecules that ratchet the neuron’s sensitivity back up again.  So, the blunting effect of the narcotic on pain is unblunted.  The agony of withdrawal experienced by a heroin addict who suddenly stops taking the drug is also explained.  While on the drug, the addicted person’s microglia have ratcheted up the pain sensitivity of the person’s neurons so that the person’s pain sensitivity is restored to near normal.  Suddenly withdrawing the drug leaves the person’s pain neurons super-sensitized without the blunting effect of the narcotic and it takes several days for them to calm back down.

The way to treat chronic pain according to this new perspective is to inhibit microglia from expressing cytokines, a story told by the title of the 2009 paper Early microglial inhibition preemptively mitigates chronic pain development after experimental spinal cord injury.  “These results suggest that inhibition of early neuroimmune events can have a powerful impact on the development of long-term pain phenomena following SCI and support the conclusion that modulation of microglial signaling may provide a new therapeutic strategy for patients suffering from post-SCI pain.”  One of the first substances identified that calm down microglia is the drug minocycline. “The current study examined the hypothesis that early administration of the microglial-inhibiting drug minocycline could ameliorate the development of pain after SCI. — Adult male Sprague-Dawley rats underwent SCI at the ninth thoracic spinal segment and received either vehicle or minocycline treatment for 5 days postinjury. Time course studies revealed that over 4 weeks post-SCI, microglial activation in vehicle-treated animals was progressively increased. Minocycline treatment resulted in reduction, but not prevention, of microglial activation over time. — These results suggest that inhibition of early neuroimmune events can have a powerful impact on the development of long-term pain phenomena following SCI and support the conclusion that modulation of microglial signaling may provide a new therapeutic strategy for patients suffering from post-SCI pain.”

The aforementioned Scientific American article mentions nine different of drugs being tested for their capability to quiet overactive glia, either in cell or animal tests and in some cases in clinical trials.  But that article says nothing about any drug that is available right now that quiets glia and is generally safe to use on a protracted basis for neuropathic pain.  Through serendipity I believe I have possibly identified such a substance, a familiar drug Gabapentin (Neurontin).  Indeed, I have been using this drug and in the course of four days it has brought my pain level down from 7-8 to 1-3 and greatly enhanced my comfort and functionality.

Gabapentin, is widely prescribed for chronic spinal pain and other pain conditions though its means for controlling pain have not been understood.   Gabapentin is approved by the FDA as an anti-convulsive drug and only for the Post-Therapeutic Neuralgia pain indication. Neurontin has an interesting history.  While several scientific studies and clinical trials support its use for pain management and a large percentage of the prescriptions for Neurontin are off-label, the company owning the rights to it, Pfizer, has gotten into trouble for promoting such uses.  “A division of Pfizer Inc., the world’s largest drug maker, has agreed to plead guilty to two felonies and pay $430 million in penalties to settle charges that it fraudulently promoted the drug Neurontin for a string of unapproved uses(ref).”  And there are still many unsettled lawsuits connected with such uses.  The key point here is that, strictly speaking, Gabapentin is an off-label treatment for neuropathic pain.  Moreover, the drug is now a low-cost generic so it is unlikely that Pfizer would ever want to incur the high costs of clinical trials for other indications.The 2003 review paper Gabapentin dosing for neuropathic pain: Evidence from randomized, placebo-controlled clinical trials concludes “At doses of 1800 to 3600 mg/d, Gabapentin was effective and well tolerated in the treatment of adults with neuropathic pain.”  The 2005 review paper Gabapentin in the treatment of neuropathic pain states:  “Clinically, several large randomized controlled trials have demonstrated its effectiveness in the treatment of a variety of neuropathic pain syndromes. Patients with neuropathic pain can expect a mean reduction in pain score of 2.05 points on an 11 point numerical rating scale compared with a reduction of 0.94 points if they had taken the placebo. Around 30% of patients can expect to achieve more than 50% pain relief and a similar number will also experience minor adverse events –.“  The 2004 paper Gabapentin is a first line drug for the treatment of neuropathic pain in spinal cord injury and the paper Gabapentin effect on neuropathic pain compared among patients with spinal cord injury and different durations of symptoms report on studies suggesting  that Gabapentin may be particularly effective for treating neuropathic pain associated with spinal cord injuries.

The paper I have found that suggests that Gabapentin moderates pain via the microglia is Gabapentin reverses microglial activation in the spinal cord of streptozotocin-induced diabetic rats.  “In addition, an attenuation of microglial activation correlated with reduced Allodynia following Gabapentin treatment, while Gabapentin had no effect on the number of astrocytes. Here we show a role of microglia in STZ-induced mechanical Allodynia and furthermore, that the anti-Allodynia effect of Gabapentin may be linked to a reduction of spinal microglial activation. Astrocytic activation in this model appears to be limited and is unaffected by Gabapentin treatment. Consequently, spinal microglial activation is a key mechanism underlying diabetic neuropathy. Furthermore, we suggest that Gabapentin may exert its anti-allodynic actions partially through alterations of microglial cell function.”

While this last conclusion is based on experiments with pain due to induced spinal injury in rats, I suspect strongly it also applies to spinal neuropathic pain in humans.  I have found a number of other papers suggesting possible molecular channels through which Gabapentin may inhibit pain, related to Gabapentin and cytokines and how Gabapentin inhibits the expression of NF-kappaB in certain cells, but none others that directly deal with the effects of Gabapentin on microglia.

I am on day 4 of Gabapentin treatment and the pain/discomfort level due to my spinal injury is running 1-3 in a scale of 1 to 10, down from a month or more of 6-8.  Time will tell whether I can maintain this comfort level and whether the original injury will heal.  So, I now have: 1.  a good working diagnosis of my condition; it is neuropathic pain created by a probably-minor spinal injury incurred while swimming, 2.  what so far is an effective therapeutic regimen, 3. a significantly enhanced personal state of comfort and functionality associated with pain reduction, and 4. an understanding of the basic mechanisms of the pain that was torturing me, as explained in this blog entry.

I got into this topic indirectly, starting out by researching what is known about DNA demethylation in response to a comment by Res on my blog post Homicide by DNA methylation.  I found a great deal of interesting material related to epigenomics, cancers, DNA demethylation, histone acetylation and other topics  –  too much to cover in one blog post.  I am going to concentrate here mostly on cystine DNA demethylation as a new approach to preventing and treating cancers, leaving a number of other interesting epigenomic topics for other posts.  The path of the discussion leads around to an old longevity topic – drinking green tea.

1.      Cancer is an epigenetic disease

The point is made in the 2006 publication New therapeutic targets in cancer: the epigenetic connection “Cancer is an epigenetic disease, a combination of DNA modifications, chromatin organization and variations in its associated proteins configure a new entity that regulates gene function throughout methylation, acetylation and chromatin remodeling. Irregular de novo DNA methylation, mainly promoter hypermethylation, histone deacetylation or methylation are important means for the transcriptional repression of cancer-associated genes. Reverse these epigenetic processes restoring normal expression of malignancy- preventing-genes has consequently become a new therapeutic target in cancer treatment. Aberrant patterns of epigenetic modifications will be, in a near future, crucial parameters in cancer diagnosis, prognosis and therapy.”

2.     One of the key things that happen in cancers is aberrant methylation of the promoter region of tumor suppressor genes, rendering them inexpressive, that is, silencing them.

This kind of DNA methylation takes place at CpG dinucleotides.   “CpG islands, which are regions of more than 500 base pairs in size and with a GC content greater than 55% (ref. 3), have been conserved during evolution because they are normally kept free of methylation. These stretches of DNA are located within the promoter regions of about 40% of mammalian genes and, when methylated, cause stable heritable transcriptional silencing. Aberrant de novo methylation of CpG islands is a hallmark of human cancers and is found early during carcinogenesis(ref).”

I am talking about methylation-induced silencing of familiar anti-tumor genes like p53 and p16(INK4a), plus a number of others like BRCA1, BRCA2, APC, RB1. WIF1, MLH1, TIMP3, PTEN, APC, CD95, RASSF1A, E cadherin, RECK and GSTP1.  The role of methylation of tumor suppressor genes in cancers is pretty established knowledge by now. For example, this 2005 report looked at “DNA methylation patterns of the tumour suppressor gene p16INK4A promoter in colon carcinoma cell lines.” Around that time, microarray technology started to become available for efficient detection of DNA methylation(ref)(ref) and the microarray detection methods have continued to improve since allowing much research progress.

3.     There has been a significant amount of research aimed at developing cancer therapies that work by inhibiting methylation of tumor suppressor genes or by demethylating them and restoring their expression.

A number of substances have been effective in demethylating tumor suppressor cells in cancers and restoring their functionality in in-vitro and small animal experiments.  For example, one substance that does the trick is described in the 2006 paper Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells.  “In the present study, we investigated methylation status of the CpG islands of some major tumor suppressor genes both in human hepatocellular carcinoma and liver cancer cell lines and examined whether demethylation by arsenic trioxide (As2O3) could restore their expression in the cell lines. HepG2 and Huh-7 cells were treated with 2 to 10 micromol/L of AS2O3 and/or 1 micromol/L of 5-aza-2′-deoxycytidine for 24, 48, and 72 hours. The methylation status of the CpG island around the promoter regions of p161NK4a, RASSF1A, E cadherin, and GSTP1 was detected by a methylation-specific polymerase chain reaction (MSP). — In conclusion, a low concentration of As2O3 induces CpG island demethylation of tumor suppressor genes by inhibition of DNMT and reactivates the partially/fully silenced genes in liver cancer cells.”  Of course, arsenic trioxide may not be a very friendly substance to use in humans.

A 2009 paper A New Class of Quinoline-Based DNA Hypomethylating Agents Reactivates Tumor Suppressor Genes by Blocking DNA Methyltransferase 1 Activity and Inducing Its Degradation mentions two demethylating drugs that have made it through the clinical trials process but that have toxic properties, Vidaza,and Decitabine.  “Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and its congener 5-aza-2′-deoxycytidine (decitabine) has provided an alternate approach to cancer therapy. We have shown previously that these drugs selectively and rapidly induce degradation of the maintenance DNA methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of these compounds is largely due to their incorporation into DNA, it is critical to explore novel, nonnucleoside compounds that can effectively reactivate the silenced genes.”

So, there has been a continuing search for effective agents that can demethylate tumor suppressor genes and that are free of toxic side effects.  One class of potentially useful substances is described in the previously mentioned paper, substances based on Quinoline, a familiar organic substance. “Here, we report that a quinoline-based compound, designated SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable IC50 (6-13 µmol/L) by competing with S-adenosylmethionine in the methylation reaction. — Prolonged treatment of RKO cells with SGI-1027 led to demethylation and reexpression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This study provides a novel class of DNA hypomethylating agents that have the potential for use in epigenetic cancer therapy.”

4.     Some familiar substances can demethylate DNA in tumor suppressor genes.

One of the directions of search is plant-derived substances, our old friends, phytochemicals.  A 2009 publication Modulation of DNA Methylation by a Sesquiterpene Lactone Parthenolide states “Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. — Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.”  

Parthenolide occurs naturally in the plant feverfew (Tanacetum parthenium), after which it is named.  “The plant is well known in natural medicine. Tablets and tinctures are used for the relief of migraine, to help prevent blood clots,^[  as an anti-inflammatory providing relief in cases of arthritis, to relieve some types of menstrual problems, and as a digestive aid. Parthenolide, the main active ingredient, is a potential anticancer drug. It destroys acute myelogenous leukemia (AML) cells by inducing apoptosis, leaving normal bone marrow cells relatively unscathed. Moreover, the compound may get at the root of the disease because it also kills stem cells that give rise to AML(ref).” Parthenolide is a powerful inhibitor of the expression of NF-kappaB(ref).

A couple of demethylating substances showing potential promise are surprisingly familiar: the anesthetic procaine, and the green tea polyphenol epigallocatechin-3-gallate.  

The 2003 paper Procaine Is a DNA-demethylating Agent with Growth-inhibitory Effects in Human Cancer Cells was ahead of its time, before the wave of strong interest in demethylation-based cancer therapeutics. “Using the MCF-7 breast cancer cell line, we have found that procaine is a DNA-demethylating agent that produces a 40% reduction in 5-methylcytosine DNA content as determined by high-performance capillary electrophoresis or total DNA enzyme digestion. Procaine can also demethylate densely hypermethylated CpG islands, such as those located in the promoter region of the RARß2 gene, restoring gene expression of epigenetically silenced genes. This property may be explained by our finding that procaine binds to CpG-enriched DNA. Finally, procaine also has growth-inhibitory effects in these cancer cells, causing mitotic arrest. Thus, procaine is a promising candidate agent for future cancer therapies based on epigenetics.”  This was followed by a 2007 study Procaine inhibits the proliferation and DNA methylation in human hepatoma cells. “All the genes transcriptionally suppressed by DNA hypermethylation were demethylated and reactivated with PCA treatment. PCA treatment led to partial demethylation and significant reduction in tumor volume in vivo.”

This leads me to wonder if something as familiar as procaine could become an important anti-cancer tool.  Another relevant paper in this regard are Procaine and procainamide inhibit the Wnt canonical pathway by promoter demethylation of WIF-1 in lung cancer cells.  “Our results provide the first evidence that procaine and procainamide reactivate WIF-1 in these cancer cells and downregulate the Wnt canonical pathway. These results further suggest that procaine and procainamide may have a potential use for preventing the development of lung cancer.”

Finally, if you read this blog, you are likely to have a powerful tumor suppressor gene demethylating agent already sitting on your kitchen shelf – green tea.  The 2003 publication Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines tells the basic story.  “Hypermethylation of CpG islands in the promoter regions is an important mechanism to silence the expression of many important genes in cancer. The hypermethylation status is passed to the daughter cells through the methylation of the newly synthesized DNA strand by 5-cytosine DNA methyltransferase (DNMT). We report herein that (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol from green tea, can inhibit DNMT activity and reactivate methylation-silenced genes in cancer cells. — EGCG dose-dependently inhibited DNMT activity, showing competitive inhibition with a K(i) of 6.89 microM. — Treatment of human esophageal cancer KYSE 510 cells with 5-50 microM of EGCG for 12-144 h caused a concentration- and time-dependent reversal of hypermethylation of p16(INK4a), retinoic acid receptor beta (RARbeta), O(6)-methylguanine methyltransferase (MGMT), and human mutL homologue 1 (hMLH1) genes as determined by the appearance of the unmethylation-specific bands in PCR. This was accompanied by the expression of mRNA of these genes as determined by reverse transcription-PCR. The re-expression of RARbeta and hMLH1 proteins by EGCG was demonstrated by Western blot. Reactivation of some methylation-silenced genes by EGCG was also demonstrated in human colon cancer HT-29 cells, esophageal cancer KYSE 150 cells, and prostate cancer PC3 cells. The results demonstrate for the first time the inhibition of DNA methylation by a commonly consumed dietary constituent and suggest the potential use of EGCG for the prevention or reversal of related gene-silencing in the prevention of carcinogenesis.” 

A 2008 paper Effects of green tea polyphenol on methylation status of RECK gene and cancer cell invasion in oral squamous cell carcinoma cells provides an additional take.  “RECK is a novel tumour suppressor gene that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumour invasion, angiogenesis and metastasis. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, on the methylation status of the RECK gene and cancer invasion in oral squamous cell carcinoma cell lines. Our results showed that treatment of oral cancer cells with EGCG partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression level of RECK mRNA.”

So, some of the substances that can demethylate and reactivate tumor suppressor genes appear to be procaine, polyphenols in green tea and the herb feverfew.  Research in this area is quite new and I would bet that a number of other very familiar phyto substances will turn out to also have this property.  Hey, I have been systematically demethylating my tumor suppressor genes every day (by consuming lots of green tea) without even knowing that I was doing that!

The lowly naked mole rat is in the news again.  I talked about the little critter in my in my February 2009 post Animal models of aging – the African naked mole rat.  I said “This little critter is the size of a tiny mouse but lives about eight times longer.  Living up to 28 years, it is the longest-living rodent.  Its secret to longevity is not known but there are clues.  For example they are very cool, they can all but shut down their metabolism, and they spend a great deal of their life sleeping.  Surprisingly, the markers of oxidative damage in these tiny rats exceed those of mice when they are relatively young.   However the rate of accrual of oxidative damage in these rats does not appear to markedly ramp up with age as it does with mice.  They change very little as they age and females more than 20 years old can give birth.  It seems that the mole rat has a powerful long-lived antioxidant defense system which mice do not have.”

The news items that surfaced yesterday reported on work by Vera Gorbunova and her team at the University of Rochester and included a nice story in the New York  Times entitled The Life Span of a Rodent May Aid Human Health.  Even apart from their longevity these little naked mole rat critters are fascinating.  They cannot see and spend their entire lives underground.  They “live in large colonies, presided over by a queen, in which only the queen and a few privileged males breed while the rest of the colony—all members of the same family—work together to raise young and maintain the colony. Wild colonies range in size from 20 to 300 individuals, with an average colony consisting of 75 individuals(ref).”  They have a very hierarchical social system with the queen at the top, then her favorite mates, ranging down to the workers.  She is extremely bossy.  They dig extensive tunnels looking for tubers which they share for food with other members of their colony.   Besides dead reckoning, they use the earth’s magnetic field to help them navigate underground, at least for longer distances(ref).  A colony of naked mole rats can build a system of tunnels stretching up to two or three miles in cumulative length. ^“Ensconced in the arid soils of Africa, these three-inch-long creatures must continually dig tunnels in search of sporadic food supplies and evade the deadly jaws of snakes(ref).”  If you are interested in learning more about their lifestyle, you can check the article The Naked Truth about Mole-Rats.

Physically, the naked mole rats are quite unique.  Apparently they have no pain sensors on their skins and use their fang-like teeth to dig their tunnels. Their large, protruding teeth are used to dig, and their lips are sealed just behind the teeth to prevent soil from filling their mouths while digging(ref).” They look a little like tiny walruses.  “They have little hair (hence the common name) and wrinkled pink or yellowish skin. — The naked mole rat is well adapted for the limited availability of oxygen within the tunnels that are its habitat: its lungs are very small and its blood has a very strong affinity for oxygen, increasing the efficiency of oxygen uptake. It has a very low respiration and metabolic rate for an animal of its size, about 2/3 that of a mouse of the same size, thus using oxygen minimally. In long periods of hunger, such as a drought, its metabolic rate can be reduced by up to 25 percent(ref).”

“Some of the “hottest” research on naked mole rats today concerns senescence, or aging. Naked mole rats in the lab have reached up to 28 years of age. And it’s not just the controlled environments of their captivity that are doing this. –Braude has observed mole rats in the wild that are 17 years and older. But these are the breeders. Lab researchers didn’t realize that in the wild workers only live two or three years(ref).” 

Though there are many interesting things about these little beasties, let me get down to the questions of why they live so long and what might be the lessons for us.  To start, there are two clues from my previous post on them:

1.      Naked mole rats go inactive for periods and turn their metabolism way down.

2.     Naked mole rats have a powerful long-lived antioxidant defense system which mice do not have. 

The next point is based on the research reported yesterday:

3.     Naked mole rats never get cancers.

Despite their very long lives which provide plenty of time for their cells to grow cancerous, naked mole rat have never been found with tumors of any kind. A report just published in The Proceedings of the National Academy of Sciences, Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat, suggests why.   “Here we show that naked mole–rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed “early contact inhibition.” Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole–rat fibroblasts arrest at a much lower density than those from a mouse. — We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27/Kip1. In contrast, early contact inhibition in naked mole–rat is associated with the induction of p16(INK4a).” 

In essence, the observed results were that expression of p16(INK4a) “makes the cells “claustrophobic,” stopping the cells’ proliferation when too many of them crowd together, cutting off runaway growth before it can start. The effect of p16 is so pronounced that when researchers mutated the cells to induce a tumor, the cells’ growth barely changed, whereas regular mouse cells became fully cancerous(ref).”  [I have discussed  p16(INK4a) at some length in my treatise and in this blog(ref), a tumor suppressor protein also very active in humans.  But nowhere previously have I seen any discussion of p16(INK4a) making cells claustrophobic.  Apparently this does not happen in humans where weaker contact inhibition is instead triggered by p27/Kip1.]  In any event, whether or not contact resistance is the central element, naked mole rats have an incredibly effective defense against cancers.

Finally, to top it off, it is believed that

4.     Naked mole rats body cells express telomerase.

In 2006 the same lead researcher, Vera Gorbunova, studied small rodents to see how their telomerase expression varied.  She “investigated 15 rodents from across the globe to determine what level of telomerase activity each species expressed, to see if there were some correlation she could find. — The species ranged from tiny field mice to the 100-pound capybara from Brazil. Lifespans ranged from three years for the mice, to 23 or more for common backyard squirrels(ref).”  She found the smaller the critters the higher degree of expression of telomerase – presumably also in the naked mole rat.  Her results were published in the paper Telomerase activity coevolves with body mass, not lifespan where she concludes “Here we show that telomerase activity does not coevolve with lifespan but instead coevolves with body mass: larger rodents repress telomerase activity in somatic cells. These results suggest that large body mass presents a greater risk of cancer than long lifespan, and large animals evolve repression of telomerase activity to mitigate that risk.”  Of course, we are like very large rodents in the respect that telomerase activity in our somatic cells is very repressed.

These studies provide interesting insights and food for speculation.  First of all, for my readers who see telomerase activation as a one-track approach to life extension, telomerase expression by itself does not correlate with lifespan.  Second, it could well be that the message of the naked mole rat is that a combination of a very powerful anti-cancer defense with activated telomerase might lead to significantly greater longevity.  That, by the way, has been my personal view for some time now.  In my treatise I have written “I speculate that protection against carcinogenesis in the course of such telomerase stimulation can probably be achieved through strengthening of apoptotic mechanisms such as P53, P16 and P21. Credence is given to this view by a very recent finding that mice which possess extra copies of both telomerase-creating and antitumor genes live 26% to 40% longer than their normal cohorts(ref).”  Both the Lifestyle Regimen and the Supplement Regimen in my anti-aging firewall program suggest numerous provisions for the avoidance of cancers.  The treatise contains an extensive discussion of telomerase activation and the supplement regimen suggests use of a telomerase activator.

MicroRNAs (miRNAs) are short (22 nucleotides,more or less) single-stranded RNA molecules which do not encode proteins. Discovered in 1993 they are recently coming under intense research scrutiny because of the important roles they play in post-transcriptional regulation of gene expression. According to a feature article published last year in Gen Tapping miRNA-Regulated Pathways, “miRNAs are master regulators of gene expression, according to William S. Marshall, Ph.D., president and CEO of miRagen Therapeutics. “You can have one microRNA that controls multiple genes and one gene that is controlled by multiple microRNAs.” They exert negative regulation and have been shown to control expression of entire signaling pathways(ref).” “About 3% of human genes encode for miRNAs, and up to 30% of human protein coding genes may be regulated by miRNAs. MicroRNAs play a key role in diverse biological processes, including development, cell proliferation, ifferentiation, and apoptosis(ref).”

The number of discovered human miRNAs continues to grow. “ – It can be argued that, based on computational analysis “there may be as many as 50,000 miRNAs in the human genome and each may have as many as a few thousand potential targets(ref).” “Approximately 400 to 500 miRNAs have been characterized in humans to date, according to Dr. Marshall, and 80–150 are typically expressed in any particular cell type(ref).” That was written some 18 months ago. I suspect that by now the number of characterized miRNAs is climbing up over a thousand.

MicroRNAs work by turning gene expression off. “MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation(ref).” “–more short regulatory RNAs were identified in almost all multicellular organisms, including flowering plants, worms, flies, fish, frogs, mammals [38, 40, 41, 48, 71], and in single cellular algae and DNA viruses [66, 75]. — Computational predictions of miRNA targets suggest that up to 30% of human protein coding genes may be regulated by miRNAs [46, 68]. This makes miRNAs one of the most abundant classes of regulatory genes in humans. MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation(ref).”

MicroRNAs play many roles in organisms “The literature includes examples of miRNAs that function as oncogenes, with their overexpression contributing to tumorigenesis, and of others that act as tumor suppressors, which when downregulated contribute to cancer(ref).” A single miRNA may simultaneously impact in complicated ways on several different genes and gene-activation pathways. “There is accumulating evidence that short RNAs can not only affect the levels of proteins, but that proteins may also affect the production of microRNAs(ref).”

Large complements of miRNAs are expressed in embryonic stem cells as pointed out in the publication MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries. “A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs, and 56 candidate miRNAs.” Further, interestingly, some of the miRNAs found in embryonic stem cells seem not to be found in other cells and seem to be regulated by many of the same transcription factors used to revert somatic cells back to induced pluripotent stem status “Finally, integration of our data with genome-wide chromatin immunoprecipitation data on OCT4, SOX2, and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here(ref).” miRNAs in embryonic stem cells seem to play important roles in cell differentiation.

Another study MicroRNA expression patterns and function in endodermal differentiation of human embryonic stem cells concludes “Our results demonstrate that expression of specific miRNAs correlates with that of specific genes upon differentiation, and highlight the potential role of miRNAs in endodermal differentiation of hESC.”

Dozens of new papers related to miRNAs have been published in the last few months, and the perceived regulatory importance of these little critters continues to grow.  “Thousands of miRNA genes have been found in diverse species, and many of them are highly conserved. With the miRNA roles identified in nearly all aspects of biological processes, evidence is mounting that miRNAs could represent a new layer of regulatory network, and their regulatory effect might be much more pervasive than previously suspected(ref).”A number of the more-recent publications have focused on the roles of miRNAs in cancers(ref)(ref)(ref), in Alzheimer’s disease(ref)(ref), in herpes infections(ref), in coronary artery cell senescence(ref) and restenosis(ref) and in many other disease processes. Examples of efforts by university research scientists and biotech companies to develop therapeutic products based on miRNA approaches are outlined in the Gen article.

Of special interest to me is yet-another view of aging in which miRNAs play the lead roles. Quoting again from the Gen article, “Eugenia Wang, Ph.D., professor at the University of Louisville, has proposed that miRNAs have a critical role in “a universal or system-specific programmatic shift of signaling control” that occurs at mid-life and brings about a decline in cellular health status associated with aging, which may precipitate increased risk of late-life diseases. In her presentation, she will review the hypothesis that the changes in expression of most if not all aging-related genes are controlled by underlying hubs and the belief that miRNAs, acting as molecular master switches, are candidate hubs.”

In fact the May 2009 issue of Current Genomics focuses on the roles of miRNAs in the aging process and Dr. Wang wrote the editorial Hot Topic: MicroRNA Regulation and its Biological Significance in Personalized Medicine and Aging. “This issue focuses on a discussion of microRNA’s post-transcriptional control of the aging process.” The ground covered in that issue is large and interesting enough for me to make it the subject of a subsequent post. For the moment I will comment that Dr. Wang’s view of aging appears to suggest specific mechanisms for the operation of the 13^th theory of aging covered in my treatise, Programmed Epigenomic Changes. As I understand it, this view says that miRNAs are the signaling messengers and “hit men” for programmed aging, progressively and systematically switching off disease-protecting genes as an organism ages.

It is interesting that in the previous post Homicide by DNA methylation I outlined a view of a Russian scientist suggesting a different mechanism for operation of the Programmed Epigenomic Changes theory. This view suggests that mutated genes due to progressive DNA methylation might be the cause for genomic and therefore somatic deterioration and loss of disease protection with age. Of course, both the miRNA explanation and the DNA methylation explanations for aging could be compatible, and molecular links between the two sets of process could be discovered. I plan to come back to this topic.

A recent publication suggests that DNA methylation may be the cause of aging and death in higher organisms.  The May 2009 publication by Alexander L. Mazin from Lomonosov Moscow State University is entitled Suicidal function of DNA methylation in age-related genome disintegration and presents a very dark view of DNA methylation as possibly being at the heart of the aging process.

The 13^th theory of aging covered in my treatise is called Programmed Epigenomic Changes and envisages aging as a systematically articulated set of epigenomic changes, involving changes in DNA methylation in cells accumulated with aging.  The new Mazin review publication proposes a mechanism for such programmed aging, seeing DNA methylation as a wrecking-ball process that progressively introduces mutations in key genes, eventually wrecking the genome causing aging and death.

I introduced the topic of DNA methylation in this blog in the post Epigenetics, epigenomics and aging and have mentioned it in a number of subsequent posts.  Its role in aging is outlined in the post DNA methylation, personalized medicine and longevity. As I said there, DNA methylation is a process by means of which sites adjacent to genes on chromosomes (promoter regions) are chemically methylated after a cycle of DNA replication(ref).   The methylation is passed on in the course of cell divisions and through generations of people. The methylation pattern captures the ancestral history of the cell that is not in the genes themselves and is unique to every cell. DNA methylation is thought to be one of the main ways epigenetic information is captured and passed on.

The Mazin paper states “This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome.” He is saying that cell differentiation and specialization requires silencing of certain genes, which ones depending on the destination cell type, and that task – necessary for the development of complex organisms – is accomplished by DNA methylation. Further, he is saying that DNA methylation does something else too, and that is eventually wrecking the genome by creating mutations in certain genes. The paper goes on to say “The proposed model considers DNA methylation as the generator of 5mC > T transitions that induce 40–70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes.” “The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.”

The theory is plausible. DNA methylation is known to be capable of exercising mutagenic and epigenetic effects. Multiple publications discuss mutations in relationship to methylation in CpG sites within genes(ref)(ref)(ref). In a previous paper DNA Cytosine Methylation Produces CpG and CpNpG Hotspots for Various Types of Mutations in Human Genes Mazin stated “The evidence is presented that both CpG and CpNpG sites of DNA methylation and their 5`-, 3`-neighboring nucleotides are hotspots not only for 5mC>T transitions, but also for most types of mutations. 40-70% of all spontaneous mutations are found at these sites, and mutation frequencies at the hotspots are 10-40 times higher than the average for the genes studied. 52-77% of CpG sites could be lost because of relict germ-line 5mC>T substitutions, and 10-20% of somatic mutations result in the emergence of new sites of methylation in these genes. Various mutagenes induce significant changes in mutation spectra at sites of methylation. Thus, one of the basic functions of DNA methylation is mutation destruction of most host genes that responsible for human genetic diseases, aging, and cancer.”

The most recent post in this blog, The NRG1 Gene – an important new tumor suppressor gene, points to a specific mechanism of how DNA methylation can lead to cancers – through silencing the NRG1 tumor suppressor gene. According to the Mazin hypothesis more than silencing of genes is involved through methylation – they are mutated, irreparably damaged. Numerous other “housekeeping” genes are likewise affected by the methylation. Such gene damage, according to my reading of the hypothesis, leads to multiple disease susceptibilities and degenerative processes associated with aging.

If the Mazin hypothesis is correct, the mutational damage due to DNA methylation is in the genes themselves and therefore cannot be corrected through demethylation or any form of epigenomic reversal. It says you start out in life with mostly good genes and end up with enough bad ones to kill you. Further old cells with such damage reverted to iPSC pluripotent status would continue to contain age-related mutational damage and, in that way, would be genetically different than embryonic stem cells were for the same individual. Because the reverted cells would continue to contain the genetic seeds for age-related self-destruction, they could not be used to close the loop in the stem cycle supply chain as suggested in the blog post The stem cell supply chain – closing the loop for very long lives. This is a rather gloomy outlook on life extension for it says that without constant gene-correction therapy to reverse the results of constant mutations due to DNA methylation, aging is indeed irreversible.

I would strongly prefer a scenario where our genes are more or less constant through life and aging mainly goes on in the epigenome where it might, in principle at least, be reversible. We will have to see what happens if and when other researchers pay attention to Mazin’s hypothesis. Meanwhile there is more that can be said about DNA methylation and you can probably expect another post on that topic soon.