Mf Newsletter 1207

Issue 1 December 2007

Welcome to the first issue of the Methuselah Foundation’s newsletter! We will be sending out this newsletter each quarter to keep you, our valued donors and supporters, up to date with solid progress in the Foundation's efforts to advance the engineered reversal of the molecular and cellular damage of aging - and also of the complementary efforts of scientists from all over the world.

Donations Tripled Until End of 2007! Thanks to the challenge pledges made by PayPal co-founder Peter Thiel and 300 Member Michael Cooper, donations to SENS research are currently tripled in value – but only until the end of 2007. If you’ve been considering making a donation, now is the time! All donors giving at least $100 are also eligible to receive a free, signed copy of Ending Aging, Dr. de Grey and Michael Rae’s recent book on rejuvenation science.

SENS3: A World-Class Success September 2007 saw the third SENS conference in Cambridge, England, which once again featured world-class research from an outstanding panel of scientists. The SENS conference series, although new, is now firmly established as the most exciting in the field, and the pace of discovery is already increasing. Videos of the sessions are now available at the Foundation’s website, alongside material from previous SENS conferences and the Edmonton Aging Symposium, organised by Foundation volunteer Kevin Perrott. Upgrading the Methuselah Foundation Website If you haven’t visited www.methuselahfoundation.org recently, you may be surprised to see how much has changed – we welcome feedback and suggestions on our new look and features. Among many other additions we now have an integrated Amazon store, all purchases from which help to support the Foundation. In This Issue We are currently sponsoring research in two of the seven strands of the SENS program; the preventing the harm caused by mitochondrial mutations (MitoSENS) and degrading damaging long-lived cellular debris (LysoSENS). Although this work began only recently, our teams have already seen interesting results and are moving forward rapidly. Their work is detailed in the Progress Report section of the newsletter, accompanied by highlights of related work from other groups. Next, Michael Rae presents a selection of key advances in areas important to SENS, accomplished by laboratories outside the Foundation. Dr. de Grey then describes a selection of prospective projects for which the Foundation is now actively marshalling funds.

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Finally, Elliot Bergman provides us with an update on the Foundation’s first funding program, the Mprize, whose total value is now well over $4 million.

Contents 3

Progress Report MitoSENS

3 4

Mark Hamalainen

MitoSENS: Several Promising Approaches

Michael Rae

LysoSENS

6 7

John Schloendorn

LysoSENS: Tackling Tau

Michael Rae

8

SENS Around The World

Michael Rae

9

Current Goals

Aubrey de Grey

10

Mprize Status

Elliot Bergman

We hope you enjoy the newsletter, and welcome any comments or suggestions for the March issue! Newsletter Editor: Ben Zealley ([email protected])

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Progress Report MitoSENS Mark Hamalainen Cambridge University / Quinze-Vingts National Center of Ophthalmology, Paris

The ultimate goal of MitoSENS is to transfer all 13 human mitochondrial proteinencoding genes from their present home in the mitochondria to the protected cell nucleus. This would represent a panacea for mitochondrial DNA related disease, including the gradual loss of mitochondrial function that occurs during aging, by allowing the genes to function in the nucleus even when damaged or destroyed in the mitochondria. Plenty of examples exist in nature: intracellular gene transfer from the mitochondrial DNA to the nucleus has occurred throughout the evolution of eukaryotes. Some mitochondrial genes, such as subunit a (A6) of ATP synthase are rarely found in the nucleus, however. One exception is found in the algae, Chlamydomonas reinhardtii, which in evolutionary terms has only recently transferred the A6 gene to its nucleus. In practice, the transfer of mitochondrial genes to the human nucleus (to produce what

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is called allotopic expression of the genes) has proved to be anything but straightforward. We’ve recently found that targeting messenger RNA (mRNA) to ribosomes proximal to mitochondria may be critical to achieving successful allotopic expression. Fibroblasts carrying the NARP or LHON mutations were rescued by this strategy, demonstrated by restoration of their ability to grow in galactose media. Experiments in 143B osteosarcoma cells have failed to replicate this result. However, it is possible that cancer derived cell lines are poor models for studying allotopic expression as mitochondrial dysfunction is thought to be important in carcinogenesis. Our current primary goal is to explore methods for reducing the toxic side effects of allotopic expression, by reducing protein hydrophobicity and reducing or regulating expression levels. Plasmids have been constructed containing a nuclear version of A6, each including up to seventeen naturally occurring polymorphisms that reduce hydrophobicity. A plasmid containing the human CF6 promoter, which drives expression of a mitochondrial protein associated with Complex V of the respiratory chain, has been constructed and is being tested. The ability of these methods to eliminate toxicity will be tested by expressing the constructs in cells that can be differentiated into a non-dividing state. The second objective of our work is to achieve co-expression of multiple mitochondrial genes with the eventual goal of complementing rho zero cells (which lack any mitochondrial DNA at all). Further Reading Holt IJ, Bokori-Brown M, Hamalainen M. “Allotopic expression: mitochondrial to nuclear gene transfer.” Rejuvenation Res. 2007 Sep;10(Suppl1):S32(Abs53).

MitoSENS – Several Promising Approaches Michael Rae Research Assistant to Dr. de Grey

Excitingly, work on allotopic expression and related "engineering" solutions to mitochondrial mutations is also taking place in other labs around the world, using a variety of different biotechnological approaches. Several groups reported their progress in these areas at the third SENS conference. Dr. Marisol Corral-Debrinski, of Paris' Quinze-Vingts National Center of Ophthalmology, presented her latest results in optimizing the import of allotopicallyexpressed mitochondrial proteins by shifting their production site closer to the mitochondria themselves. This technique is hoped to overcome a primary challenge in allotopic expression: how do the proteins now produced by nuclear DNA get back to the mitochondria where they are needed? She used this technique to insert an allotopic version of the defective human gene that causes the mitochondrial disease Leber’s Hereditary Optic Neuropathy (LHON) into mouse retinas, reproducing the same cell loss and abnormal lack of cell communications branching that appears in the disease. She next hopes to take this to the next level, and cure the disease in mice by introducing the healthy gene.

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Another way to ease the import of twisted-up (and thus hard to import back into mitochondria) allotopically-expressed proteins is through the introduction of special 'bracing bars' called inteins into them, to hold apart their snarling bends and kinks - an idea first proposed by Dr. de Grey in a 2000 paper in Trends in Biotechnology. After some preliminary work done by Japanese scientists, the University of Zaragoza's Dr. Antonio Enriquez has now picked up the ball, and described his early work with mitochondrial protein inteins during the meeting. In addition to the work on allotopic expression, two presentations at SENS3 covered progress on entirely novel ways of overcoming the problem of mitochondrial mutations. Dr. Volkmar Weissig of Northeastern University reported the import of whole new mitochondria into the cell, an effect that had actually previously been reported by Jerry Shay’s group in 1982 – and then forgotten! – but which Dr. Enriquez was able to confirm in his own lab. Finally, Dr. Samit Adhya of the Division of Molecular and Human Genetics at the Indian Institute of Chemical Biology is pursuing yet another innovative approach, in which he proposes to dispense with the need for mitochondrial DNA altogether, by instead providing the mitochondrial protein-making machinery directly with the "working instructions" (messenger RNA) that it normally receives in the form of a transcribed copy of the mitochondrial DNA. Dr. Adhya’s work borrows a trick used by a single-celled organism called Leishmania tropica to move messenger RNA into the mitochondria. He provided evidence that RNA imported into the mitochondria of human cells using this technique works as it should by introducing antisense RNA - RNA that is designed as a mirror-matched copy of the original, to which it binds and which it thereby inactivates. Introducing this antisense RNA exerted effects similar to those seen in people with defective copies of the genes that he had effectively kept from functioning as sources of mitochondrial proteins, supporting their functionality as silencing RNA. As with Dr. Corral-Debrinski's work, the next step will be to introduce functional RNA into animal models with dysfunctional mitochondrial genes, to see if it can restore normal function. Further Reading S. Ellouze, C. Bonnet, S. Augustin, V. Kaltimbacher, V. Forster, M. Simonutti, J-A. Sahel, M. CorralDebrinski. “Allotopic mRNA localization to the mitochondrial surface: a tool for rescuing respiration deficiencies.” Rejuvenation Res. 2007 Sep;10(Suppl1):S24(Abs 23). J.A. Enriquez. “Inteins and allotopic expression of mtDNA encoded proteins” Rejuvenation Res. 2007 Sep;10(Suppl1):S28(Abs 36). V. Weissig, E. Katrangi, S.V. Boddapati, G.G.M. D'Souza. “Manipulating (rejuvenating?) the mitochondrial genome” Rejuvenation Res. 2007 Sep;10(Suppl1):S50(Abs 124). S. Mukherjee, B. Mahata, B. Mahato, S. Adhya. “Use of a parasite-derived protein complex to modulate the function of mitochondria in human cells.” Rejuvenation Res. 2007 Sep;10(Suppl1):S19(Abs 2)

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LysoSENS John Schloendorn Biodesign Institute, Arizona State University

As we age, our bodies produce many types of junk molecules as a side-effect of normal functioning. For some of these molecules, no efficient removal system exists, and their accumulation gives rise to deposits of intracellular junk. This leads to age-related storage disease. The major diseases of this type are Alzheimer’s disease (beta-amyloid plaques in the brain) atherosclerosis (7-ketocholesterol/7KC, a cholesterol derivative in the artery wall), age-related macular degeneration (a compound called A2E in the eye), and diabetes (AGEs, sugar-derived protein-modifications, throughout the body). Medical Bioremediation is the field of research seeking environmental microorganisms that break these molecules down, whose gene products can then be harnessed for therapy in humans. For the past two and a half years, the Methuselah Foundation has been funding research into Medical Bioremediation at Arizona State’s Biodesign Institute in Tempe, Arizona and at Rice University, Austin, Texas. When these projects began in the summer of 2005, there was no strong evidence to suggest that any enzymes or organisms degrading intracellular junk existed in nature. But this did not intimidate Methuselah Foundation research volunteers Jacques Mathieu, Mark Hamalainen and John Schloendorn. With very limited funding and compensation, they visited leading environmental resarchers Pedro Alvarez, PhD and Bruce Rittmann, PhD at their labs and began culturing experiments. All three reseach volunteers had successfully cultured 7KC degraders before the end of 2005. This initial success laid the foundation for the scaling-up and professionalization of these projects. In 2006, Mathieu and Schloendorn enrolled as PhD candidates at their universities and won departmental support. Hamalainen has now enrolled at University in Paris, France to pioneer Mito-SENS as a second Methuselah-Foundation funded effort. 2006 saw further characterization of the 7KC degraders and corroboration of the results. In summer 2007, six undergraduate research assistants and one additional PhD candidate joined the effort at Biodesign Institute. They helped with synthesizing additional target compunds, such as A2E and CML (a major AGE). This veritable army of volunteers also was able to culture six independent degraders of CML, and identify two enzymes which break A2E in different ways. Today, researchers at the Biodesign lab are working on identifying the enzyme initiating the breakdown of 7KC and CML. They are also characterizing the A2E-degrading enzymes further, and are preparing to move them into a cell model of age-related macular degeneration for initial safety and efficacy testing. Various other projects at the Tempe lab are at earlier stages. Novel targets include artificial lipofuscin and the infamous glucosepane AGE-crosslink. Meanwhile, Mathieu at Rice University employs a modern microarray-based approach to identify genes that get expressed in the presence of 7KC, but not other nutrients. Such enzymes are likely involved in the breakdown of 7KC. This has the potential to characterize the complete genetics of 7KC degradation in one elegant experiment.

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Further Reading Rittmann BE, Schloendorn J. “Engineering away lysosomal junk: medical bioremediation.” Rejuvenation Res. 2007 Sep;10(3):359-65.

LysoSENS – Tackling Tau Michael Rae Research Assistant to Dr. de Grey

Neurofibrillary tangles (NFTs), aggregates of the protein tau, are a kind of molecular damage that accumulates inside our brain and other nerve cells with aging, and are associated with Alzheimer's and a variety of rare neurological diseases. There's good reason to think that removing these tangles would help to rejuvenate the aging brain. The cell already has an ‘incineration and recycling center’, the lysosome, which is responsible for the removal of damaged molecules within itself, so the age-related accumulation of NFTs in brain cells suggests some essential weakness in lysosomal functioning - either a failure to engulf NFTs, or a lack of the enzymes needed to shred them up into reusable parts once they have been taken in. This summer, scientists working in the New York University School of Medicine under Dr. Einar Sigurdsson reported that the burden of NFTs could be significantly reduced, and neurological function substantially preserved, in laboratory animals that normally suffer neurological damage because of a genetic predisposition to form NFTs, by immunizing them with a form of the tangles' precursors. This appeared to result from the antibodies' shepherding the NFTs into the lysosome for disposal. The benefits were mild, but could likely be enhanced by fortifying the lysosome with enzymes more suited to recycling the NFTs - a projected project for the LysoSENS strand of the SENS platform. Further Reading Asuni AA, Boutajangout A, Quartermain D, Sigurdsson EM. “Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements.” J Neurosci. 2007 Aug 22;27(34):9115-29.

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SENS Around The World Michael Rae Research Assistant to Dr. de Grey

Toxic Cells In January, Dr. Alan Saltiel and his colleagues at the Departments of Internal Medicine and Physiology at the University of Michigan Medical School reported results that will be central to the implementation of one of the SENS platform's planks: the removal of one of the several classes of toxic cells that accumulate in the body over time. We've known for some time that the systemic inflammation, insulin resistance, increased levels of blood fats, and other metabolic disorders that progress with aging is mostly related to the accumulation of body fat over the lifespan. Recently, however, we've come to understand that the metabolic effects of being overweight are not the result of having more fat generally, but are due to the attraction of inflammatory immune cells into the so-called "visceral" body fat (the depot around the internal organs of the gut). The removal of these cells would thus reverse the metabolic dysfunction that they induce, restoring a more youthful systemic metabolism. But to do that requires a way of selectively targeting such cells, while leaving healthy immune and fat cells alone. Dr. Saltiel's lab has now reported that these immune cells have distinctive binding sites and sugar-protein markers that distinguish them from their beneficial cousins elsewhere in the body. This will allow us to develop interventions similar to vaccines and some of today's targeted cancer therapies, removing the toxic cells with minimal collateral damage. Further Reading Lumeng CN, Bodzin JL, Saltiel AR. “Obesity induces a phenotypic switch in adipose tissue macrophage polarization.” J Clin Invest. 2007 Jan;117(1):175-84.

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Current Goals Aubrey de Grey Methuselah Foundation

A selection of projects within the SENS plan are ready to be launched as Foundationsponsored research programs, conditional only on the availability of sufficient funding. As for MitoSENS and LysoSENS, these projects will start small (likely with only a single researcher), with the aim of delivering high leverage in terms of the credibility of the approach.

Amyloid in tissues other than the brain Most people are aware of the amyloid deposits associated with Alzheimer's Disease: they are the main constituent of senile plaques, the aggregates that accumulate in the spaces between neurons as the disease progresses. Encouraging progress is being made in stimulating the body's immune system to eliminate these deposits. However, amyloid composed of different proteins also accumulates in other tissues during aging. Progress in removing these other amyloids has been much less intensive thus far, even though they are, if anything, more clearly linked to the progression of age-related illness than senile plaques are to Alzheimer's. I have been in preliminary discussion with one of the leaders in this area, with a view to initiating work as soon as possible.

Identifying genes essential for ALT WILT, the anti-cancer therapy incorporated into SENS, entails (among other things) the elimination of genes for the enzyme telomerase, which allows cancer cells to divide indefinitely without losing material from the ends of their chromosomes. Unfortunately, about 10% of cancers solve this problem in a different way, not using telomerase, via a process known as ALT, for alternative lengthening of telomeres. Even more unfortunately, ALT is still only very poorly understood. Recently, however, some intriguing observations in two different organs have given good reason to suspect a hitherto unsuspected gene. A relatively simple sequence of initial experiments could test this, and I am already in discussions with a leading ALT researcher concerning the possibility of launching this project.

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Mprize Status and Future Prospects Elliot Bergman Competitor Development Coordinator

The Mprize is designed to jumpstart scientific research into life-extending biomedicine with the twin incentives of (1) a large cash award, and (2) a prestigious public victory in a prominent research competition. The support of our generous donors is the key to this strategy: the magnetic draw of the Prize grows with every dollar pledged to this program. Therefore, we are pleased to report a substantial increase in the total amount of money in the Mprize fund, which now stands at $4.6 million. To broaden the field of competing research, a focused program was initiated this year to actively recruit new competitors, expanding the “mouse race” for breakthroughs in preventive and regenerative anti-aging biotechnology. Spearheading this campaign is Elliot Bergman, Ph.D., of biotech consulting firm ChemLifeSciences, as the Foundation’s Competitor Development Coordinator. Elliot’s efforts to date have brought in four new competitors for the Prize, for a total of eleven scientific teams independently racing to extend the lives of their furry subjects as of this writing. The four most recent additions are Professor Andrzej Bartke of Southern Illinois University; Professor Craig Cooney of the University of Arkansas for Medical Sciences; Alan Cash, founder of Terra Biological LLC; and Elise Sacane, co-founder of Neural Learning Systems. Each of these teams is testing a different anti-aging strategy, creating exactly the kind of wide-open, multi-strategy competition needed to weed out ineffective approaches and bring successful therapies into the spotlight. Several other potential competitors have been identified and are being qualified for participation. We hope to have a total of 13-15 competitors by the first quarter of 2008. We are particularly delighted that Professor Andrzej Bartke a renowned, world-class gerontologist has now announced that he will re-enter the fray in a second round of competition. In 2004, his Growth Hormone Receptor Gene Knockout (GHR-KO 11C) mice set the standard against which future competitors for the preventive (“Longevity”) Prize will be judged: a previously unheard-of lifespan of 1819 days, or nearly five years – a remarkable 50% extension of lifespan compared to normal, healthy mice. Dr. Bartke has not yet disclosed the new protocol that he will be testing in this second season of the anti-aging challenge, but we can be certain that he will be a major factor in the race. In addition to putting his own mice onto the gridiron, Professor Bartke is now also leading the Foundation’s efforts to promote the wider use of mice as an experimental model for anti-aging research. Today, the trend is increasingly toward lower cost studies using cheaper, shorter-lived organisms such as roundworms, yeast, and fruit flies – organisms that are progressively poorer proxies for human test subjects with every evolutionary step that they take away from us. Success in this initiative would not only attract many more new competitors to the Prize, but also broaden the range of interventions being tested within and without the Prize structure. We aim to enhance the impact of mouse research in general, and to the Methuselah Foundation in particular, while increasing the yield of results that are likely translatable into human interventions.

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We have also secured access, through cooperation with the National Institute on Aging (NIA) division of the National Institutes of Health (NIH), to a database of USgovernment-funded projects on mouse aging research, invaluable as a source of potential new competitors. It is notable that 3 of our 4 new competitors are supported, at least in part, by NIA/NIH grants, while two are scientific entrepreneurs who have raised private capital to fund part (Cash) or all (Sacane) of their Mprize research. We are scouring other external sources – such as reports in the scientific literature, and grant applications submitted to other non-governmental scientific organizations such as the Ellison Medical Foundation for additional potential competitors. In addition we follow up with researchers testing new aging treatments in rats, or in cultured cells or tissues, in an effort to identify new approaches which should result in lifespan studies in mice. Future prospects for the Mprize look bright, although there are some important challenges that continue to discourage use of mice as test subjects for anti-aging interventions. A major limiting issue is the relatively high cost of this research, which can range into the hundreds of thousands of dollars per year, as well as the length of time required to complete lifespan studies in an animal that normally lives for over three years. A hopeful sign is that despite these disincentives, several well-funded start-up companies in the new, important Nutritional Genomics field – such as Sirtris and IKARIA – are recognizing the importance of testing their interventions in mammals, and will likely be using mice extensively in their research. We are assessing the possibility of reducing research costs by outsourcing mouse trials to well-organized laboratories in Asia (mainly China, India, Japan, Taiwan, Thailand, and South Korea), where strict scientific standards can be assured. If such an international research facility becomes available, the scale of the investment required to perform mouse studies will be reduced considerably, broadening the scope of mouse treatments that can be tested and allowing a wider variety of new competitors to take up the gauntlet. Another way of limiting the cost and time required to perform anti-aging studies in mice is to encourage more researchers to compete in the Rejuvenation arm of the Mprize, which requires researchers to test their interventions in 16-month-old mice (instead of starting at weaning, as in the Longevity prize). Most of the Foundation’s donors already favor the Rejuvenation Prize over the Longevity Prize by a very large margin, as measured in completed pledges to the two Prize funds: $1.48 million vs. $0.16 million - a ratio of more than 9 to 1. This preference is based on the urgent need to develop interventions that can extend the healthy life spans of people who are already middle-aged, in hopes that people alive today can still be rescued from a death by biological decay brought on by the aging process.

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