For a decade we have known that Alzheimer's (AD) amyloid-beta protein (Abeta) exists normally as a soluble, molecule and is associated in blood and CSF with high density lipoproteins (HDL) [detailed in 2].

Lipoproteins provide the proper thermodynamic environment for Abeta, which shares with other apolipoproteins the unique structural property of amphipathicity [2]. In the absence of lipids, such molecules easily cross- and self- aggregate and undergo oligomerization. Some also form amyloid fibrils, structures that are present in separate types of human amyloidosis (for ex., apoA-I, serum amyloid A). Previous reports have indicated that natural Abeta does not cross link with other apoliporoteins in normal cerebrospinal fluid (CSF) high density lipoproteins (HDL), but does crosslink with HDL apolipoproteins in CSF samples from Alzheimer's patients [2]. The AD condition thus may represent a pathological alteration of the lipoprotein structural integrity in a way that breaks monomeric Abeta-to-lipid interaction and favors the interaction of Abeta-to-apolipoprotein (shown for ApoE and ApoJ) [2] or Abeta-to-Abeta, which creates the oligomers that are the subject of the Science study (18 April, p. 486).

Of major importance for this report, in our view, is the potent inhibition by lipoproteins of the neural toxicity of Abeta [3, 4, 5]. Such inhibition of Abeta toxicity by lipoproteins was shown in SH-SY5Y cells [3]. These same cells were used in the Science study under different experimental conditions to prove Abeta oligomer toxicity; the effect of lipoproteins on oligomer toxicity was not tested in this report.

We also think that introducing a control of specificity (perhaps by pre-adsorbing anti-oligomer antibodies with Abeta oligomers) in an immunofluorescent staining of Alzheimer's brain tissue would add confidence that the structures detected are indeed Abeta oligomers. This is especially important, because the micellar Abeta used for production of the anti-oligomer antibodies may mimic the conformation of lipid-bound Abeta (and of other membrane-bound proteins [6]), which was similarly shown to have distinct immunoreactivity [7].

Sincerely,

Alexei Koudinov, MD, PhD
neuroscientist and editor
http://anzwers.org/free/neurology
http://neurobiologyoflipids.org

Footnote: This 300 word letter was first submitted to Science magazine (online submission no. 30545, April 21, 2003; rejected on May 1, 2003).

Competing financial interests: I do not have any competing financial interest. I aim free information dissemination and an unbiased development of Alzheimer's neuroscience. I observe the Society for Neuroscience Guidelines for Responsible Conduct Regarding Scientific Communication. Discussed herein Science report (18 April, p. 486) and earlier Science viewpoint article on oligomeric Ab by Dennis J Selkoe (25 Oct 2002, p. 789) lack appropriate competing interest declaration. Such interests are disclosed in Forbes magazine and in Science (Corrections and clarifications. Science 27 Sept 2002, 297: 2209), respectively. For further details please see my Open letter to Public Citizen's Health Research Group on Alzheimer's disease research (SAGE KE, 21 Feb 2003).

References:

1. Shuster AM, Gololobov GV, Kvashuk OA, Bogomolova AE, Smirnov IV, Gabibov AG. DNA hydrolyzing autoantibodies. Science 256: 665-7 (1992) [ PubMed ].

2. Koudinov AR, Berezov TT, Koudinova NV. The levels of soluble Ab in different HDL subfractions distinguish Alzheimer's and normal aging CSF: implication for brain cholesterol pathology? Neurosci Lett. 314: 115-118 (2001) [ PubMed ][ Author Related Articles and Scientific Correspondence ].

3. Cedazo-Minguez A, Huttinger M, Cowburn RF. Beta-VLDL protects against A beta(1-42) and apoE toxicity in human SH-SY5Y neuroblastoma cells. NeuroReport 12: 201-206 (2001) [ PubMed ].

4. Farhangrazi ZS, Ying H, Bu G, Dugan LL, Fagan AM, Choi DW, Holtzman DM. High density lipoprotein decreases beta-amyloid toxicity in cortical cell culture. NeuroReport 8: 1127-1130 (1997) [ PubMed ].

5. Koldamova RP, Lefterov IM, Lefterova MI, Lazo JS. Apolipoprotein A-I directly interacts with amyloid precursor protein and inhibits Abeta aggregation and toxicity. Biochemistry. 40: 3553-3560 (2001) [ PubMed ].

6. Soreghan B, Pike C, Kayed R, Tian W, Milton S, Cotman C, Glabe CG. The influence of the carboxyl terminus of the Alzheimer Abeta peptide on its conformation, aggregation, and neurotoxic properties. Neuromolecular Med. 1: 81-94 (2002) [ PubMed ].

Friday, April 21, 2006

Dear Dr. Gray and Dr. Bürkle:

I enjoyed your article in SAGE-KE about the recent SENS-II conference, which gave a nice overview of the meeting and the areas of controversy around Aubrey’s ideas and activities.

As one of the authors of the EMBO Reports article, I thought I ought to respond to your speculation about an area of apparent ambiguity. You write:

[quotation] Positing that the SENS agenda is 'so far from plausible that it commands no respect at all within the informed scientific community,' these authors wish to 'dissociate themselves from the cadre of those impressed by de Grey's ideas in their present state.' This statement is both forceful and ambiguous. It can be read as the relatively benign wish to be placed in a nonoverlapping circle on the Venn diagram of who believes what in aging-related research, or it can be read as a more sinister threat of shunning the apostates. If the authors intended the latter, what are the requirements for admission into the shunned cadre? It could not be attendance at one SENS conference, for some of the signatories have attended. Would attendance at both suffice? Further, is it not possible to express interest in or contribute to some of the scientific objectives of SENS without being judged a SENS acolyte? [end quotation]

I am very sorry that the article may have left the misimpression of its cosignators as sinister and threatening bullies, the kind of people who take attendance at meetings and make lists of evil-doers destined to be confined to some scientific Guantanamo, where they are denied access to their copies of Rejuvenation Research, and from which much-needed shipments of liquid nitrogen and telomerase are diverted to other purposes. It was intended, as you speculated, to express a fully benign wish, inviting comrades to see the light and step into the part of the Venn diagram where people support aging research, do some of it themselves, go to any conferences they want to attend, and talk to and be friendly with anyone they wish, but are careful to try to distinguish fact from speculation, and speculation from fantasy.

I don’t see any need to define a shunned cadre, with or without admission requirements. Nearly all of the scientific objectives on the SENS menu are interesting, and people working on them are likely to make discoveries of great importance. But when colleagues, like Aubrey, feel a need to exaggerate the speed with which biogerontology will produce practical advances, or exaggerate the likelihood that old people can be converted into spanking new ones, I think this is in general bad for society and harmful to the scientific enterprise. I think it’s important for scientists to do their part to help administrators, journalists, and interested laypersons distinguish strategy from wish-fulfillment, and critical thought from advertising. Your SAGE-KE article is, in my view anyway, a fine example of how to approach this kind of knotty issue.

First, I have never remotely suggested that participation in my SENS conferences constitutes tacit agreement with me as to SENS's feasibility. (It is not clear from Gray and Buerkle's text whether they think I have been doing this, but a reader might easily form such an impression.) That would be a ludicrous inference, given that I am on abundant record as knowing full well how radical SENS is, and also that I have repeatedly taken a decidedly more established approach when I seek to determine whether other scientists support a component of SENS, namely, first to expose them to it for a whole day and then to ask them to co-author a paper on it [2-5]. (It is notable that only one of my co-authors on any of these papers signed Warner et al.'s recent denunciation of SENS [6]; one can draw one's own conclusions from the fact that her name was mis-spelled in the list of authors.) There is no reason whatever for a SENS conference participant to be concerned that his or her opinions regarding SENS will be misrepresented either by me or by any other SENS advocate. If some of my more energetic detractors seek to make such a link, it is those detractors who should be condemned for impugning the reputations of eminent scientists in an attempt to marginalise me, and if anything, scientists should resist that intimidation by attending SENS conferences in greater numbers than ever. Perhaps this was Gray and Buerkle's point, but if so, I fear that some readers might have missed it.

The converse point also merits a slight correction. While attendance at the SENS conferences means nothing about support for SENS, it does confer at least circumstantial authority to opine on the matters that were presented there. Gray and Buerkle point out that some of the 28 signatories to the recent denunciation of SENS [6] attended, but this may give an exaggerated impression; in fact, and as I pointed out in my response to Warner et al. [7], not one of them attended SENS 2 and only five attended its altogether less SENS-centric predecessor, IABG10, in 2003 (when SENS had less notoriety).

Gray and Buerkle quote the "Position Statement on Human Aging" [8] out of context: the comment there about humans living forever referred to literally that, i.e. no death from age-independent causes either. My agreement with that paper's main thrust, the present non-existence of any true life- extending products, is the reason I endorsed it. I and some other endorsers lobbied the authors vigorously to remove assertions about the probability of such products arriving within our lifetime, but without success; but that was not the paper's main theme, so I judged that an endorsement was still appropriate.

I am perplexed at Gray and Buerkle's implication that I have in some way been tarred with the Hwang brush as a result of his participation in a conference that I organised. If I have, all I can say is that I am in very good company.

Finally, I must take sharp exception to Gray and Buerkle's assessment of the SENS Challenge. When scientists disagree as to the plausibility of a hypothesis or the feasibility of an experiment, they generally do so in cautious terms, and in such circumstances it is entirely proper to be able to express one's view without being required to justify it rigorously. When they express their opinion of a colleague's work using words like "fantasy" and "pretence," however, the object of their derision is entitled to ask for an explanation - and, if years pass with no explanation being forthcoming, to bring his critics' reticence to public notice. Gray and Buerkle say that the terms of the Challenge mean that it allows me to taunt my detractors "baselessly," but that is not true at all: rather, the conclusion that a submission must persuade a panel of independent experts of in order to win (namely, that SENS is so absurd that it should not be dignified with learned debate) is precisely the view that my critics have been expressing off the record for some years and more recently (though only after my strong criticism of their public silence [9]) in print. It is public knowledge [10] that Technology Review began the search for a mainstream gerontologist's demolition of SENS entirely on its own initiative, as a result of having been given a strongly negative but non- specific evaluation of it by experts whom they consulted during 2004, and that the SENS Challenge was begun (with input from the Methuselah Foundation, yes) only after a number of experts declined to write such an article and Technology Review began to wonder whether they had been misled. Thus, Gray and Buerkle are absolutely wrong to suggest that "[t]he TR Challenge serves no purpose but to attract attention to Aubrey de Grey and the increasingly bitter dispute with his detractors" -- rather, it serves the wholly legitimate purpose of testing the hypothesis that my detractors have formed their negative opinions of SENS without the attention to its details that their audience will tend to presume that they have paid. If Gray and Buerkle know of a better way to distinguish that hypothesis from the competing one, advanced by my detractors, that no such commentary has been written simply because experts are too busy to ridicule something so ridiculous, they should suggest it.

In conclusion, my high media profile results from the coherence of the SENS agenda and the incoherence of the criticism that SENS has so far received [7]. This is demonstrated by the fact that SENS has been accorded an almost uniformly positive media treatment (Technology Review's articles in February 2005 being almost the only exception). I am not a soundbite expert, nor am I fond of them. The SENS conferences will thus continue to bring together the world's best science in areas that I consider relevant to extreme life extension, and participants will continue to be able to make up their own minds as to whether that science adds up to a viable approach to greatly postponing aging.

1. D. A. Gray, A. Buerkle, SENS and the Polarization of Aging-Related Research. Sci. Aging Knowl. Environ. 2006 (7), pe8 (2006).

2. de Grey ADNJ, Ames BN, Andersen JK, Bartke A, Campisi J, Heward CB, McCarter RJM, Stock G. Time to talk SENS: critiquing the immutability of human aging. Annals NY Acad Sci 2002; 959:452-462.

3. de Grey ADNJ, Baynes JW, Berd D, Heward CB, Pawelec G, Stock G. Is human aging still mysterious enough to be left only to scientists? BioEssays 2002; 24(7):667-676.

4. de Grey ADNJ, Campbell FC, Dokal I, Fairbairn LJ, Graham GJ, Jahoda CAB, Porter ACG. Total deletion of in vivo telomere elongation capacity: an ambitious but possibly ultimate cure for all age-related human cancers. Annals NY Acad Sci 2004; 1019:147-170.

5. de Grey ADNJ, Alvarez PJJ, Brady RO, Cuervo AM, Jerome WG, McCarty PL, Nixon RA, Rittmann BE, Sparrow JR. Medical bioremediation: prospects for the application of microbial catabolic diversity to aging and several major age- related diseases. Ageing Res Rev 2005; 4(3):315-338.

6. H. Warner, J. Anderson, S. Austad, E. Bergamini, D. Bredesen, R. Butler, B. A. Carnes, B. F. Clark, V. Cristofalo, J. Faulkner et al., Science fact and the SENS agenda. What can we reasonably expect from ageing research? EMBO Rep. 6, 1006-1008 (2005).

7. de Grey ADNJ. Like it or not, life extension research extends beyond biogerontology. EMBO Reports 2005; 6(11):1000.

8. S. J. Olshansky, L. Hayflick, B. A. Carnes, Position statement on human aging. J. Gerontol. A Biol. Sci. Med. Sci. 57, B292-B297 (2002).

9. de Grey ADNJ. Resistance to debate on how to postpone ageing is delaying progress and costing lives. EMBO Rep 2005; 6(S1):S49-S53.

I cannot comment, the SAGE KE 'pay per article' does not display a 'padlock' symbol alongside the 'internet zone' symbol, indicating that my Visa debit card details are not secure in transit.

I would be happy to pay the fee, and very much wish to study this work, I understand it's importance, however there must be some way of obtaining this article without the possibility of breaching my Visa security.

It's puzzling, as the other 'Science' subsciption sites do display the padlock symbol. It may just be an oversight.

In the 309 volume of Science (26th August 2005) the SAGE KE section published a paper by James McC. Noble et al. entitled “Brain Tumor- Associated Dementia”. For general readers this paper needs some comments regarding the case, its clinical interpretation as well as some statements made by the authors.

First of all, the title of the paper is misleading. The authors describe the impairment of cognitive functions, however the neuropsychological examination performed was incomplete and not consistent with the diagnosis of dementia. Mini-Mental State Examination showed decreased score (27/30), nevertheless it is not sufficient to diagnose dementia (MMSE score should be in such cases below 24). MMSE is the screening test and for the diagnosis of dementia, particularly if the authors like to differentiate it with Alzheimer disease there are more precise neuropsychological tests, to mention ADAS-cog (Alzheimer’s Disease Assessment Scale) as the most adequate one. DSM IV criteria for the diagnosis of Alzheimer disease exclude brain tumor as pathology involved in etiology for dementia of such a kind (1).

The patient clinically manifests frontal lobe syndrome (change in behavior and personality, social withdrawal). Among the symptoms indicating the lesion of frontal lobe, depression is very frequent, so there exists a strong clinical need for psychological examination of the mood of the patient. The differentiation of depression – associated cognitive impairment and dementia requires further testing (e.g. Cornell scale).

Cognitive function impairment in the course of malignancy, which is an important clinical question, needs to be recognized at two levels. One is dementia associated with brain tumor (besides neoplastic brain tumor, the abscess, subdural hematoma, arterio-venous malformation should be also taken into consideration), and the other is cognitive dysfunction as paraneoplastic syndrome (remote effect of cancer). Paraneoplastic limbic encephalitis (2,3,4) may manifest with dementia as it is also the case for subacute cerebellar degeneration (5,6). The last two syndromes of high clinical importance, however not common, should be taken into consideration during differential diagnosis of dementia.

The authors have undertaken an important question of cognitive impairment associated with malignancy, however the case presented is not consistent with contemporary diagnostic standards and clinical knowledge and needs elucidation as a local effect of tumor, manifesting more likely as frontal lobe syndrome rather than dementia. 1. American Psychiatric Association, Desk Reference to

Diagnostic Criteria from DSM-IV. DC:APA, Washington

(1994). 2. S. Alamowitch, F. Graus, M.Uchuya, R.René,

E. Bescansa, J. Y. Delattre, Limbic encephalitis and

small cell lung cancer. Clinical and immunological

features. Brain 120, 923–928 (1997). 3.R.Voltz, S. H. Gultekin, M.R. Rosenfeld, E.Gerstner,

J.Eichen, J.B. Posner, J. Dalmau, A serologic marker of paraneoplastic limbic and brain-stem encephalitis in patients with testicular cancer. N Engl J Med 340,1788-95 (1999). 4. J. Kassubek, E.U. Nitzsche, F.D. Juengling,

C.H.Lücking, Limbic Encephalitis Investigated by 18FDG-

PET and 3D MRI. J Neuroimaging 11, 55–59 (2001). 5. N.E.Anderson, J.B.Posner, J.J.Sidtis, J.R.Moeller,

S.C.Strother, V.Dhawan, A.Rottenberg, The metabolic

anatomy of paraneoplastic cerebellar degeneration. Ann

Neurol 23(6), 533-40 (1988).

6. J.B.Posner, Paraneoplastic syndromes in Neurology in

Clinical Practice, Bradley W.G. et al. (eds),

Butterworth-Heinemann, Boston, 1299-1307 (2000).

Slawomir Michalak

Department of Neurochemistry and Neuropathology

Chair of Neurology, University of Medical Sciences,

SantaCruz et al. describe a transgenic mouse model that expresses a repressible human tau variant that is linked to hereditary tauopathies. Repression of tau is in these mice regulated by a tretracycline-operon responsive element. These mice develop neuronal tau changes in a similar sequence as seen in Alzheimer’s disease (AD) starting with “Pre-tangles” and finally resulting into severe neuronal loss. When repressing tau by application of doxycyclin tau levels decreased and progression of neuronal loss stopped. However, the progression of tangle formation continued after repression of tau. The authors conclude that tau may be capable of inducing neuronal death in a neurofibrillary tangle independent pathway and that neurofibrillary tangles not necessarily induce neurodegeneration.

This study points out that the generation of neurofibrillary tangles once initiated does not require overexpression of abnormal tau variants. Moreover, neurofibrillary changes can be present without altering neuronal function for a long time. This result is in line with neuropathological studies that have shown that neuronal function of tangle-bearing neurons prevails for years before tangle formation leads to neuronal cell death (1 -3). Neurofibrillary independent mechanisms of cell death in tau transgenic mice can be discussed as a similar mechanism as in AD. However, several arguments may favor alternative explanations for this type of neurodegeneration in this mouse model. 1) In the tau transgenic mice a mutant form of tau is overexpressed that leads to fronto-temporal dementia but not to AD. 2) Tau overexpression in neuronal cells blocks the intracellular kinesin-dependent transport (4) and may, in so doing, lead to neurodegeneration. 3) In AD the amyloid beta-protein is present and it has an additional neurotoxic capacity that can alternatively explain neurodegeneration of non-tangle-bearing neurons.

The article on twins chromosomes reminded me of the fact that paternal age correlates with achondroplasia in the offspring. It would seem logical that is a similar reflection of a change in the DNA of the father over time.

Stindl R. Tying it all together: telomeres, sexual size dimorphism and the gender gap in life expectancy. Med Hypotheses 2004;62(1):151-154.

www.stindl.info

To Whom It May Concern: I just wanted to say taht the article that you wrote is extremely inspiring and I think it is one of the best articles I have ever read. Thank you for your time.

Dear Dr. Davenport,

In your article you state that "Daughters have a normal life span even when they come from older moms..." However, Kennedy et al. (1994) states the opposite. And, the current paper which you are discussing, "Asymmetric Inheritance of Oxidatively Damaged Proteins During Cytokinesis," comes to the conclusion that asymmetry of oxidized proteins does break down with increasing mother cell age (which would support Kennedy's findings, if you believe that oxidized proteins are causative to the aging process and not just correlated).

Perhaps I have missed something in the literature refuting Kennedy. Could you explain the basis for the statement that daughters of old mothers have a normal lifespan?

Aging and life extension research has been stuck in the CR mode for quite some time without delving into an real understanding of the biochemical, hormonal, and physiological bases for the effects of CR. In addition, human beings are biased with notions of aging that are being overturned almost daily by an advancing baby boom generation that is providing fresh data to shed some light on antiquated concepts. For example, how long ago was tooth loss considered an inevitable consequence of aging, but now is regarded as a chronic disease process (periodonditis) with an infectious component?

One does not need to look too hard to identify other 'dogmas' of aging that are being revised in light of new data. Most textbooks cite reductions in growth hormone (GH) as a consequence of aging, yet recent evidence suggest that increases in visceral adipose tissue and fasting insulin levels are better predictors, independent of age. In addition, glucose homeostasis has long been thought to deteriorate with aging, yet again, visceral adipose tissue mass appears more signficant. The basis for the former mistake is simple, adipose tissue mass and visceral adipose tissue mass typically accumulates with aging. Unless one can indentify all the relevant variables and statistically control for them, age may appear predictive, but only because it is highly correlated with increasing overall adiposity.

Adipose tissue is gradually coming into its own as an important tissue. Again, older textbooks simply relegated adipose tissue to an energy storage site. Today, adipose tissue is regarded as a true endocrine organ involved with many systems that clearly can derange and contribute to chronic diseases that shorten lifespan. In addition, current imaging techniques are now able to quantitate different adipose tissue stores. Discrete anatomical sites of adipose tissue accumulation manifest different effects, such as leptin secretion by subcutaneous adipose tissue and insulin resistance with increasing visceral adipose tissue.

Thus, we can view CR as a means to restrict adipose tissue and promote leaness, rather than limit energy expenditure. In this light, perhaps rather than an overall caloric reduction, a carbohydrate limitation would demonstrate better glucose homeostasis and prevent the host of chronic disease where hyperinsulinemia is believed to play a role (hypertension, dyslipidemias, diabetes, etc.). In fact, the recent work by Donald Layman on altered protein to carbohydrate ratios during dietary restriction provides for a sound physiological basis with regards to carbohydrate restriction effects on glucose homeostasis. Exercise in this light can also be seen as contributory since exercise would serve to create another source for carbohydrate utilization.

“Extended Longevity in Mice Lacking the Insulin Receptor in Adipose Tissue” (Jan.24, pp. 572-574) by Bluher et al. is an exciting report. It is the first demonstration that maximal length of life of a mammalian population can be significantly increased by a manipulation that does not decrease caloric intake on a per animal basis. Moreover, the fact that the life extension is due to the knockout of the insulin receptor specifically in the adipose tissue (FIRKO mouse) provides a potentially valuable new approach to the study of mammalian aging.

However, the authors also infer that their findings indicate that the increased life span in both the FIRKO mouse and in calorie-restricted rats and mice is due to the decrease in body fat. This inference dismisses the findings in the literature that strongly indicate that extended longevity induced by caloric restriction is not due to a reduction in fat mass. In calorie-restricted rats, for example, fat mass and life span are positively correlated; in ad libitum-fed rats, there is no correlation (1). Also, calorie-restricted ob/ob mice (48% body fat) have a life span that is longer than the ad libitum-fed, highly congenic C57BL/6J mice (22% body fat), and as long a life span as the very lean (13% body fat) calorie-restricted C57BL/6J mice (2). Even in the case of the FIRKO mouse, the reduction in fat mass may well be adventitious in regard to the increased longevity. Indeed, Bluher et al. point out that the insulin receptor loss-of-function and the metabolic sequelae thereof may be responsible for the increase in life span. In summary, the author’s implication that reduction of fat mass plays a causal role in life extension in calorie-restricted rodents is not in accord with currently available facts.

EDWARD J. MASORO
Department of Physiology, University of Texas Health Science Center, San Antonio, TX 78229, USA

We are pleased that SAGE decided to highlight our paper with a commentary. M. Leslie accurately describes the first part of the paper in which we discuss evidence for JNK signals protecting fruitflies against oxidative stress. We feel, however, that the second main conclusion of the paper (i.e. that JNK signaling can extend lifespan) is dismissed based on a misrepresentation or misunderstanding of the experimental evidence. Citing John Tower from USC, Leslie implies that the conclusion that JNK signaling extends lifespan is wrong and derived by flawed experimentation. The text indicates that in the critical experiments we compared an inbred strain to a cross between two genetically diverse strains and that therefore the results that we see are due to alleviation of inbreeding depression (i.e. heterosis; hybrid strains being fitter than inbred strains) and not to changes in JNK signaling. This is not how the experiment was done and thus the implication is quite erroneous. Specifically,

- by backcrossing the puc strain 10 times into its parental background (ry506), we have generated isogenic controls where no heterosis effects can be expected.

- the rescue of the observed longevity of puc heterozygotes by crossing in the hypomorphic JNKK mutation hep1 demonstrates that JNK signaling is required for increased lifespan in puc heterozygotes

- the lifespan extension observed in flies that over-express Hep in neurons (compared to the same strain without Hep) independently demonstrates that higher JNK signaling levels extend lifespan.

These experiments are clearly described in the publication (see Fig. 3 in Wang et al., 2003), and, in our opinion (and that of the reviewers and editors of Developmental Cell), support the drawn conclusions.

I am always pleased to find that the scientific community are prepared to adopt new thinking and to judge such hypotheses on their scientific credentials (merit). This has been done for the research of Ashley Bush and colleagues who have, rightly, pioneered the idea of a role for copper and zinc in the aetiology of Alzheimer's disease.

However, such hypotheses should not be built through the discrediting of other equally viable hypotheses. For some reason, Bush and colleagues seem to like nothing more than to pooh-pooh the possible role of aluminium in AD as if their own hypothesis involving zinc and copper actually requires the Al hypothesis to be discredited ? This has all the makings of great politics but very poor science. It is equally disappointing that Science has taken up Bush's cudgel in ridiculing the Al hypothesis. Flippant remarks in this weeks hard copy of Science advertising the Sage article refer to aluminium pots and pans being 'exonerated' of causing AD !

I am not aware of pots and pans being aetiological factors in AD though I am aware of a burgeoning amount of high quality scientific research which does, if nothing else, describe the link between Al and AD (see; Exley C (Editor) (2001) Aluminium and Alzheimer’s Disease: The Science that Describes the Link. Elsevier Science, Amsterdam, The Netherlands. 441p.)

Whilst Bush and colleagues actively ignore the question of Al this ignorance should not be a substitute for science and it certainly should not be perpetuated in the pages of Science.

Unlike Bush and colleagues I do not know whether Al is a contributory factor in AD. However, if needs be I can tell a story with at least the scientific content of that told by Bush and colleagues for copper and zinc which does implicate Al in AD and does not require me to ridicule any possible role of any other aetiological factor such as copper and zinc.

This is the difference between doing science and pandering to the whims and fashions of science. Bush elucidates upon the opposition that he initially faced in implicating copper and zinc in AD. He even goes so far as to blame the Al hypothesis for this opposition ! and yet he is quite prepared to make life equally difficult for researchers who would like to test the Al hypothesis scientifically.

In the UK, the comments by individuals such as Bush have now made it impossible to do research on Al and AD. There is not a single grant of any substance from any funding body to any individual anywhere to do research in this area. This is in spite of the fact that the scientific case for an involvement of Al in AD is stronger today than at any other previous time.

Spurious comments made by irresponsible people cannot be avoided. What can be avoided is that such comments are not carried and thus perpetuated (without any form of peer review) by the leading scientific journals of the day.

Dear DR. Martin, Kudos on content and policy implications of your submission. The case is building for a systems approach to aging and frailty. A helpful corollary constuct is " Symmorphosis" PNAS 1991, 88:10357-10361.

Dear Dr. Davenport,

Thank you very much for your nice comment at SAGE-KE. Since our paper may be complicated for the people who are not familiar with the cellular senescence, I appreciate the way you introduced our paper.

Sincerely yours,

After reading the paper by Lai et al., I can't help but wonder whether ATP2 is really relevant to the normal aging process in wild type yeast. The authors have most likely already thought about these issues and I'd be interested to hear their reasoning.

My first reason for questioning the validity of this mutant as a model for aging is the change in shape of the viability curves for atp2 mother and daughter cells grown at the restrictive temperature and transferred to the permissive temperature after 6 generations (Fig 2B). Unlike wild-type (Fig 2C), the mutants no longer show Gompertz-Makeham kinetics. The change in the shape of the curve can't simply be explained by a 6 generation shifting of the curves (the length of incubation at the restrictive temperature), as the slope is quite different from wild type. In fact, the mutant viability curves strongly suggest a large stochastic component to mortality that is not present in wild type cells. I would interpret this to suggest that some sort of unrepaired damage persists in atp2 cells after the switch back to 30C, which results in a fixed probability of death with each generation. When a high rate of stochastic death is combined with Gompertz-Makeham kinetics, the result is a curve very similar to those present in Fig 2B (McVey et al., 2001).

I am also forced to question the selection scheme for differentiating between a mutant that makes a "vital substance" and a true segregation mutant. First of all, I don’t think these are the only two possibilities. What about a mutant that results in some sort of persistent, unrepaired damage at the restrictive temperature? If the damage results in an increased probability of terminal arrest or cell lysis, you might expect to get a viability curve with a large stochastic component – which is exactly what the data show. I would predict that many clonal senescence or weak ts mutants would behave exactly the same way as this allele of ATP2. Was a control experiment performed on other ts clonal senescence mutants (hdf1 for example)?

I don't think it's necessarily true that a segregation-defective mutant would result in clonal senescence. In the case of rDNA circles, a mutation that results in no segregation bias should cause an increase in the steady-state level of ERCs (assuming negligible reintegration of ERCs into the rDNA array) in the population, but wouldn’t necessarily cause clonal senescence, due to dilution of the ERCs among mothers and daughters.

I also have a few questions that perhaps the authors would be willing to answer:

I may have missed it, but what is the life span of the atp2 mutant strain (CS16) when grown at 30C for the entire experiment? What does the viability curve look like? This seems like an important piece of information.

What is the explanation for the increase in ERCs in the ATP2 deletion mutant at 37C (Fig5)?

Why was the YPK9 strain background chosen for these experiments? If I remember correctly, this was the only strain out of 4 where petite mutants had an extended life span (Kirchman et al., 1999). Were these results verified in any other strain background? Given that YPK9 behaves differently from every other yeast strain tested for petite life span extension, this may be important information. It is stated in the paper that deletion of ATP2 results in clonal senescence in W303. Since it's pretty well established that ERCs are limiting for life span in W303 (Defossez et al., 1999, Kaeberlein et al., 1999, Sinclair et al., 1997), this also suggests to me that the clonal senescence phenotype of ATP2 mutants is unrelated to normal (wild-type) aging.

I read with great interest two recent News focus stories by Laura Helmuth on a developing spectrum of Alzheimer's disease therapeutic approaches (Science 23 Aug. 2002, p. 1260, p. 1262 ; SAGE KE 28 Aug. 2002, or9, or10 ). One report by L. Helmuth is entitled "Protecting the brain while killing the pain?" and discusses possible prevention of Alzheimer's disease by NSAIDS, a nonsteroidal anti-inflammatory drugs.

"An NIA-funded prevention trial that aims to determine whether NSAIDs do indeed protect against Alzheimer's disease" was the subject of the recent letter of the Public Citizen's Health Research Group addressed to Health and Human Services Secretary Tommy Thompson [1].

The group letter urged "the NIA to immediately stop this unethical trial" and particularly stated [1]:

Public Citizen claimed that "there is no longer any biological basis for this [ADAPT] trial". Their preference was the amyloid hypothesis. The major support for the amyloid hypothesis in the group letter come from the Science magazine review article by Hardy and Selkoe [2].

What the letter missed is the fact that the key reference for the article by Hardy and Selkoe [2] is red flaged by Science magazine erratum [3]. This is because of the competing financial interest by Dennis J. Selkoe, MD. Dr. Selkoe is not only a Harvard Professor and a recent member of the governmental NIH National Advisory Council on Aging [4]. He also serves as a director for Elan, a company, involved in developing amyloid hypothesis based Alzheimer's therapy. Moreover, Howard L. Weiner, M.D., a co-inventor and co-author of Dr. Selkoe nasal amyloid vaccine [5] serves as a member of the Federal Drug Administration (FDA) Peripheral and Central Nervous System Drugs Advisory Committee [6].

The most eye-catching is the following journalist message [7, also see Ref. 8]:

Moreover, shortly after D. Selkoe sale of his Elan shares in February 2001 [7] American Academy of Neurology (AAN) Potamkin Prize Selection committee (chaired since 1999 by Dennis Selkoe) awarded the Prize 2001 to Elan scientist Dr. Dale Schenk [9, 10]. As CNN stated, "The Potamkin -- seen by some as a potential precursor to Nobel consideration -- is a peer award, and the AAN has some 17,500 neurology professionals in its membership" [10].

In my previous letter to BMJ [8] I alarmed the public about Dr. Selkoe conflict and expressed the necessity for the attention to D.Selkoe case and disciplinary actions by the societies' ethics committees and editorial boards of journals where he published or served as board member without acknowledging his competing financial interest.

The letter by Public Citizen (signed by Elizabeth Barbehenn, PhD, Peter Lurie, MD, MPH, and Sidney M. Wolfe, MD) states that the authors "have done a search of the medical literature on AD and NSAID use and have discovered that the drugs being used in this study are not only unlikely to be effective but have the potential to inflict harm on these otherwise healthy individuals with little if any possibility of benefit." The letter claims that this is due to "no lowering of amyloid levels" [1]. I have two concerns in this regard.

First, I am puzzled why the group did not discover several contributions that question amyloid hypothesis [see Ref. 11 for bibliography details, also see Ref. 12].

Second, although I do not agree with the focus of the letter discussion, I believe that the group statement is not fully supported by the currently available literature. To support the claim Public Citizen cites journal article by Koo, Golde and colleagues [the group letter citation #1], and two meeting reports by the research teams of Paul Aisen and Monique Breteler presented as Alzheimer Forum (AlzForum) news reports, not the primary literature [Public Citizen letter citations #21 and #22, respectively]. AlzForum is an important subject information source [13] that, however, commits no public responsibility to disclose competing financial interests of its' commentators and authors [14].

The reaction of Dr. Aisen on the Public Citizen letter is the following: "I would describe the letter as an inflammatory tirade rather than a scientific argument. While it does review some relevant scientific observations, the discussion is one-sided. I do not agree with the conclusions and recommendations of the letter" [15]. Monique Breteler adds: "I profoundly disagree with the interpretation of the existing evidence and the scientific argument in mentioned letter" [15].

The data of the other citation for a journal article [Public Citizen citation #1] were corrected in Science magazine two weeks after the publication of the letter [16], thus further invalidating the group argument. The later Science correction note stated that "the results of research by Edward Koo, Todd Golde, and colleagues were misrepresented... As the article noted, all experiments showing an effect of nonsteroid anti-inflammatory drugs on beta amyloid production used very high doses" [16]. The above is also present in Dr. Koo testimony to AlzForum [15]. Dr. Koo states that "many of Sidney Wolfe's accusations of ADAPT are unfounded". Dr. Golde further charges Public Citizen of being "irresponsible". He adds: "I am disturbed by the actions of Public Citizen in that they use our data to support their argument, but never once spoke to either Eddie [Koo] or myself for our take on it" [15].

Three weeks after the publication of the group letter [1] the story was communicated to Alzheimer's research community and openly commented on by twelve scientists at the AlzForum web site [17]. Public Citizen's Health Research Group letter also raised significant media interest [18] but did not become the voice of "many researchers in the field of AD research", as no one shared the letter enthusiasm [17].

Oppositely, many scientists now disagree with the group favorite amyloid hypothesis [11, 12], the fact that is erased in their letter to HHS Secretary Tommy Thompson. Below is the citation of a director of a respected institute [19]:

Another world class neuroscientist added with regard to amyloid-based Alzheimer's immuno-therapeutics [20]:

The above statements call for a task force of safeguarding equal opportunities for different hypotheses of Alzheimer's research [21] and investigating the factual view that amyloid hypothesis is about business [7, 8, also see Ref. 22].

In light of the above I would like to know who stands behind ill-advised confidence in amyloid hypothesis of Sidney M. Wolfe and the Public Citizen's Health Research Group.

Alzheimer's research field reserves to know this. I and my colleagues look forward hearing from Dr. Wolfe.

Sincerely,

Alexei Koudinov, MD, PhD
neuroscientist and editor
http://anzwers.org/free/neurology
http://neurobiologyoflipids.org

Competing financial interests: I do not have any competing financial interest. I aim free information dissemination and an unbiased development of Alzheimer's neuroscience. I observe the Society for Neuroscience Guidelines for Responsible Conduct Regarding Scientific Communication.

References:

1. Barbehenn E, Lurie P, Wolfe SM. Letter to HHS Secretary Tommy Thompson that raises ethical concerns about the “Alzheimer’s Disease Anti-Inflammatory Prevention Trial” (ADAPT) (HRG Publication #1637) Public Citizen publications. Public Citizen web site. (4 Sept 2002) [ FullText ].

2. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 297, 353-6 (19 July 2002) [ PubMed ] Erratum in: Science. 297, 2209 (27 Sep 2002) [ FullText ].

3. Corrections and clarifications. Science. 297, 2209 (27 Sept 2002) [ FullText ].

4. Attachment A: Membership Roster. National Advisory Council on Aging. National Institute on Aging. National Institute of Health web site. Summary Minutes for [ The Eighty-Sixth Meeting May 21-22, 2002 ] [ The Eighty-Fifth Meeting Jan 29-30, 2002 ] [ The Eighty-Fourth Meeting Sept 24-25, 2001 ]. Make a note of Dr. Selkoe citation at the Comments from Retiring Members.

5. Cromie WJ. Alzheimer's vaccine looks promising. Harvard Gazette. (18 Jan 2001) [ FullText ]; Weiner HL, Selkoe DJ. Inflammation and therapeutic vaccination in CNS diseases. Nature. 420, 879-84 (19-26 Dec 2002) [ PubMed ].

6. Members: Howard L. Weiner, M.D. FDA Roster of the Peripheral and Central Nervous System Drugs Advisory Committee. FDA web site. (last viewed 16 Feb 2003) [ FullText ].

7. Crowley D. Elan Alzheimer's expert in pre-slump share sale. The Sunday Business Post. (18 Aug 2002) [ FullText ]; Crowley D. Elan directors sold $43.5 million of shares before disastrous downturn. The Sunday Business Post. (11 Aug 2002) [ FullText ]; Elan Corporation PLC. Schedule 11. Notification of interests of directors and related persons. Dennis Selkoe [ 13 Feb 2001 ][ 18 Dec 2001 ].

8. Ready T. Science for Sale: A Harvard researcher stands to profit from a product he "independently" reviewed for the National Institutes of Health. The Boston Phoenix (29 April 1999) [ FullText ]; Waldholz M, King RT, Jr. Did ties to Alzheimer's test maker sway NIH report? The Wall Street Journal (30 Nov 1998) [ FullText ]. Also see: Koudinov AR. Ethical conundrums: an Alzheimer's case. British Medical Journal (12 Sept 2002) [ FullText ].

9. Rosenberg RN. The Potamkin Prize for Pick's, Alzheimer's Disease and Related Disorders. AAN web site. (last viewed 16 Feb 2003) [ Original .DOC file ] [ HTML cached version ].

10. Dale Schenk: Alzheimer's researcher. 'The synthesis of a hundred little ideas'. CNN web site. (23 July 2001) [ FullText ].

11. Koudinov AR. Amyloid was never clearly implicated in Alzheimer's disease, so look at Abeta from a different angle. BMJ online. (1 Dec 2002) [ FullText ]; Koudinov AR, Smith MA, Perry G, Koudinova NV. Alzheimer's disease and amyloid beta protein. Science online. (14 June 2002) [ FullText ].

12. McGeer and McGeer. Is there a future for vaccination as a treatment for Alzheimer's disease? Neurobiol Aging. In Press article, available online 3 December 2002 [ Abstract ]; Obrenovich ME, et al. Amyloid-beta: a (life) preserver for the brain. Neurobiol Aging. 23, 1097-9 (Nov-Dec 2002) [ PubMed ]; Smith MA, et al. Ill-fated amyloid-beta vaccine. J Neurosci Res. 69, 285 (1 Aug 2002) [ PubMed ]; Atwood CS, et al. Cerebral Hemorrhage and Amyloid-beta. Science. 299, 1014 (14 Feb 2003) [ FullText ]; Koudinov AR, Koudinova NV. Alzheimer’s anti-amyloid vaccination and statins: two approaches, one dogma. The time for change. British Med J. (20 March 2002) [ FullText ][ Related Correspondence ]; Koudinov AR, et al. Alzheimer's disease and amyloid beta protein: dogma is bad for science. 32nd Soc Neurosci Meeting, Program #21.11 (2002) [ Abstract and further reading ].

13. Site visit: Alzheimer's Roundtable. Science. 286, 1643 (1999) [ FullText ].

14. E.mail response by June Kinoshita, AlzForum Executive Editor, (23 Sept 2002) on A. Koudinov e.mail letter of 20 Sept 2002. Also see: Guidelines: Responsible Conduct Regarding Scientific Communication. Society for Neuroscience web site. (2002) [ Preface ] [ Acrobat .PDF FullText ]; Smith R. Beyond conflict of interest. British Medical Journal. 317, 291-2 (1998) [ FullText ].

15. Comments by Scientists. Trials and Tribulations: Does ADAPT Have to Adapt? AlzForum web site. (4 Oct 2002) [ Comment by Paul Aisen ] [ Letter by Monique Breteler ] [ Comment by Eddie Koo ] [ Q&A with Todd Golde ].

16. Corrections and clarifications. Science. 297, 1996 (20 Sept 2002) [ FullText ].

17. Trials and Tribulations: Does ADAPT Have to Adapt? AlzForum web site. (Updated 4 Oct 2002) [ FullText ].

18. Mitchell S. Govt urged to halt Alzheimer's study. The Washington Times. (4 Sept 2002) [ FullText ].

19. E.mail response to A.Koudinov by a senior scientist (1 Jul 2002) regarding Koudinov et al. Science dEbate letter of 14 June 2002.

20. E.mail response to A.Koudinov by a senior scientist (19 Aug 2002) regarding Sunday Business Post article of 18 Aug 2002.

21. Current hypotheses. AlzForum web site. (last updated 5 Dec 2002) [ FullText ].

Dear Sir, This is a very good review article, covering mainly the potential role of complement system in AD. Quite candidly, however, the authors did not discuss anything about other important immune factors that may also be involved in the immunopathogenesis of this disease. Specifically, I am referring to the autoimmune hypothesis that involves brain-specific autoantibodies, circulating cytokines, and activated T cells. Abnormalities of all of these autoimmune factors in AD has been very well documented in the literature (see not eblow) but the authors of this review article did not describe and/or discuss their involvement in AD.

Note: Reports by Singh, VK and co-workers will give ample literature pertinent to this topic.

Once again, its a very good review article but deficient in some important aspects of AD immunopathology.

First let me state that I have an inherent conflict of interest by the fact that the company that I am employed by is focused on developing treatments targeting a bio-molecular pathway we believe is responsible for aging. I also want to state that I fully support and agree with the author’s effort to discourage the use of ineffective and potentially dangerous anti-aging therapies. However, I disagree with several assertions made in the paper. Foremost is your definition of aging as being impaired cellular function which is attributed to the accumulation of random damage”. If the damage is random then why do certain tissues exhibit senescence while other tissues are yet to complete development? Hearing acuity begins to deteriorate at about age 10 in the human. In addition, the free radical theory of aging is far from universally accepted and fails to explain numerous observations such as the extremely rapid aging seen in many semelparous species.

I would also suggest statements such as, “it impossible for us to possess genes that are specifically designed to cause physiological decline with age or to control how long we live” are not prudent. This is especially true since the authors go on to state, “Genes or genetic variants that prove detrimental in the postreproductive part of the life span can become commonplace, but only if they participate in important processes early on.” Indeed, this represents a separate theory of aging known as the antagonistic pleiotrophic theory.

If it turns out that the antagonistic pleiotrophic theory is correct then the author’s statement that, “The lack of a specific genetic program for aging and death means that there are no quick fixes that will permit us to treat aging as if it were a disease,” may be false. Just because there is no consensus as to which specific genetic program might be responsible for aging does not mean that such a program does not exist. This is similar to statements such as “we now know that cancer is a multifactorial disease and there will never be a single cure for cancer.” While it may be true, it could also be true that there is a single cure for cancer which is yet to be identified. Current research in C. elegans and drosophila appear to be close to identifying such a genetic program for aging.

Two comments and a minor correction to offer:

1. I think my friend Dave Harrison may have his causal arrow on backwards. Just because the wild mice are light does not mean that they have low appetite and are for that reason similar to food restricted rodents. Short people, Shetland ponies, and miniature dogs, for example, aren't small because they hate to eat; on the contrary, they don't eat much because their small size means they don't need lots of calories to maintain body temperature. Wild-trapped mice placed into laboratory housing do indeed gain weight, though they do not reach the same weight as lab-derived stocks. They are shorter than lab-type mice, too, and do not show the exceptional leanness characteristic of food-restricted mice.

2. The article suggests that these mice are too tough to handle, but I think the author is being too pessimistic about this. It does take an experienced animal technician a week or so to learn to out-wit these mice, and an ability to tape over escape routes and use live traps to recapture the occasional escapee are requirements, but the mice are less scary than Stitch. We are hoping that other labs will want to use these mice for aging research, and would be glad to provide breeding stock to anyone who wants to use them for his/her research work. Free to good homes!

3. Minor point: the wild-derived mice are, at 4 months of age, about 55% of the weight of lab-type mice; the statement in the article that they are 75% smaller than ordinary mice isn't quite accurate.

Studies performed by our group and by Flurkey and his colleagues resulted in demonstration that two spontaneous mutations in the mouse increase average and maximal life expectancy by 40 - 60 %. These mutations cause multiple endocrine defects including growth hormone/insulin-like growth factor 1 (GH/IGF-1) deficiency. Targeted disruption of GH receptor gene that causes GH resistance and consequently IGF-1 deficiency was shown by Coschigano and her colleagues to cause comparable extension of life span. These findings, their implications, and the putative mechanisms involved in life extension in these animals were briefly discussed in my recent opinion article prepared for SAGE KE. Dr. Sonntag raises an issue of the utility of these animals (referred to as “dwarf models” in his commentary) to study mechanism of aging and concludes that “transgenic animal models that disregard the developmental effects of GH and IGF-1 and the interactions of these hormones with other hormonal systems will not answer the critical questions in this field”. This conclusion is based on a number of specific arguments which, I believe, require some comments.

Secondary effects of deficiency of GH signaling on insulin levels, on the control of carbohydrate metabolism etc. as well as effects of IGF-1 deficiency on development are viewed by Dr. Sonntag as major limitations of these mouse models. We believe that all of these effects are expected from the actions of GH and IGF-1 that have been known for decades and represent potential and, in some cases, very likely MECHANISMS of actions of these mutations on aging and longevity. IGF-1 exerts a multitude of effects and the impact of mutations that alter circulating IGF-1 levels on longevity should probably not be expected to somehow “bypass” these effects. Also, a correction is needed: these long lived mouse mutants do not maintain normal body temperature and we suspect that reduction in body core temperature may represent one mechanism of delayed aging in these animals.

The suggestion of defects in cognitive development of Ames and Snell dwarf mice is consistent with known actions of thyroid hormones but surprisingly to us is not supported by data available to date from behavioral studies. Instead, old Ames dwarf mice outperformed their normal siblings in tests designed to measure learning and memory (Kinney et al, Hormones and Behavior 39: 277, 2001 and unpublished observations). An interesting footnote is offered by a report that in a human pedigree with mutation of the same gene which is mutated in the Ames dwarf mouse, the educational performance and employment history of the affected individuals argued against mental deficiency (Krzisnik et al. J. Endocr. Genet. 1: 9, 1999).

My statement that “there is no evidence that GH therapy ... has any effect on aging per se or life expectancy” is taken from a paragraph referring to GH therapy in the human. It was meant to refer specifically to these studies and I am sorry if this was not made clear in my article. I am not aware of any published studies which were designed to answer the question whether treatment with GH affects human aging or provided unequivocal evidence on this point. This issue and the controversy surrounding promotion of GH as an “anti-aging agent” was discussed previously by us and by others (Olshansky et al. Scientific American, June 2002, p 92) and key arguments in favor of this potential action of GH are clearly spelled out in my SAGE KE article.

I certainly agree that our interpretation of evidence available to date is speculative and that a direct proof of the importance of IGF-1 in the control of aging and longevity is yet to be obtained and this is stated clearly in the opening sentence of the last section of the SAGE KE article. However, I beg to differ from the conclusion that “hormone deficiency (in Ames dwarf, Snell dwarf and GHR-KO mice) MAY impact biological aging”. It seems to me that the magnitude of the extension of the average and the maximal life span, reduced collagen aging, preserved immune function, and results of behavioral testing leave little doubt that these mutations profoundly affect aging.

Introduction

The recent review on the role of growth hormone and IGF-1 on mammalian aging provides interesting insights into potential genetic manipulations that impact mammalian aging. Nevertheless, the review raises issues related to the interpretation of results using dwarf animal models, the relationship of results using dwarf models to studies of growth hormone and IGF-1 replacement and the future direction of studies related to aging research. Each of these issues merit additional comments.

There is general agreement that there has been remarkable progress in understanding the genetic basis of aging. Studies in C. Elegans and Drosophila provide compelling evidence that in invertebrate models deficiencies in insulin/IGF-1 signaling pathways are closely associated with increased lifespan. The logical question then is whether perturbation of the growth hormone/IGF-1 axis regulates mechanisms of aging and lifespan in mammals. The critical issue raised by the aforementioned review is whether studies using Ames or Snell dwarf animals (as well as other dwarf animal models) provide appropriate and sufficient data to warrant the conclusion that growth hormone and IGF-1 deficiency delays aging or results in increased lifespan in mammals.

Do current dwarf models reveal specific information on the effects of growth hormone/IGF-1 deficiency?

As noted in the review, Ames and Snell dwarfs are deficient in growth hormone, TSH and prolactin and, due to complex interactions between endocrine systems, animals exhibit secondary endocrine deficiencies including alterations in glucose, insulin, IGF-1, and glucocorticoids. In addition, the presence of hormonal alterations during development results in tissue anomalies that further complicate the models. For example, the effects of thyroid hormone deficiency on brain maturation (which effects cognitive development) and IGF-1 deficiency in development of a GI tract (which impairs transport capacity) are well-known examples of the consequences of such interactions. Furthermore, the ability of dwarf animals to maintain a normal body temperature and demonstrate increased lifespan is, in some cases, dependent on housing with wild type controls. Thus, perturbations in the endocrine axis that appear straightforward have complex interactions that complicate subsequent interpretation of results.

The developmental and synergistic effects of these hormones are also evident in mutations that are more specific to the growth hormone/ IGF-1 axis. The GHRHR deficient (lit/lit) mice and growth hormone receptor knockout mice indeed result in more specific or targeted mutations compared to earlier models. Nevertheless, these animals exhibit similar alterations in glucose, insulin and glucocorticoid levels and undoubtedly exhibit developmental anomalies compared to the Ames and Snell dwarf. It is entirely possible that these secondary endocrine, developmental and physiological changes mask the effects of growth hormone and IGF-1 deficiency and, therefore, the consequences of these mutations may, or may not, reveal information relevant to the effects of growth hormone and IGF- 1 deficiency on biological aging. Reconciling the results of dwarf studies with studies of growth hormone replacement to old animals

The comment by the author that 'there is no evidence that growth hormone therapy … has any effect on aging per se or life expectancy' is in error. Without appropriate assessment of the consequences of specific deficiencies in growth hormone and/or IGF-1on lifespan, it is inappropriate to speculate whether the data from these models can, or need to, be reconciled with the numerous studies demonstrating that a specific replacement regimen, e.g. growth hormone and/or IGF-1 replacement, improves cognitive function (Markowska et al., 1998; Thornton et al., 2000; Sonntag et al., 2000), immune function (French et al., 2002), cellular protein synthesis (Sonntag et al., 1985), muscle and bone mass (Rudman et al., 1990) and other endpoints typically associated with the aging phenotype. It is not surprising that long-term consequences of pathological increases in growth hormone decreases lifespan but, unfortunately, these results have little applicability to normal physiology - and probably less applicability to mechanisms of aging. Several years ago, it was reported that modest growth hormone replacement either increases lifespan (Khansari and Gustad, 1991) or has no effect on lifespan when administered to mid-aged animals (Kalu et al., 1998). The results of these studies using specific interventions should not be summarily dismissed in favor of manipulations that are either non-specific or use pathological levels of hormones.

Finally, the author appears to make distinctions between lifespan and other endpoints of aging including frailty, functionality and risk factors for disease. In fact, lifespan is only one marker of biological aging - one that is relatively easy to quantify. Functional endpoints are more difficult to measure. However, most individuals would agree that even functional outcomes are the result of an aging process and contribute to the aging phenotype. The authors' conclusion that 'reduced IGF-1 signaling mediates the action of several aging-related genes while the age-related decrease in growth hormone and IGF-1 contribute to destructive changes in body composition' again is speculation, with little definitive data to support the conclusion.

Conclusions and Future Directions

The current dwarf models that have been used to demonstrate increased lifespan support two conclusions: 1) that genetic manipulation has the potential to influence lifespan in mammals, and 2) that direct or indirect consequences of hormone deficiency has profound effects on a number of biological systems that may impact biological aging. Unfortunately, any additional conclusions related to the effects of these manipulations on the growth hormone/IGF-1 axis are purely speculative. For example, the decrease in IGF-1 in caloric restricted rodents and humans has been known for over a decade, yet it has not been suggested that the effects of moderate caloric restriction are mediated solely through the IGF-1 axis. Similarly, the Ames or Snell dwarf or models that purport to offer 'more specificity' in targeting these endocrine pathways have sufficient endocrine, developmental and physiological pertubations that one cannot make conclusions on the effects of growth hormone/IGF-1 on either mechanisms of aging or lifespan.

Transgenic animal models that disregard the developmental effects of growth hormone and IGF-1 and the interactions of these hormones with other hormonal systems will not answer the critical questions in this field. The questions related to the effects of growth hormone/IGF-1 deficiency on mammalian aging can only be addressed with the development of appropriate models to closely regulate expression of these hormones. Conditional expression of these hormones resulting in physiological changes in hormone levels that do not interfere with normal development are necessary as well as appropriate pharmacological tools to stimulate or antagonize hormone action.

References

French RA, Broussard SR, Meier WA, Minshall C, Arkins S, Zachary JF, Dantzer R, Kelley KW. Age-associated loss of bone marrow hematopoietic cells is reversed by GH and accompanies thymic reconstitution. Endocrinology. 2002;143:690-699.

Khansari DN, Gustad T. Effects of long-term, low-dose growth hormone therapy on immune function and life expectancy of mice. Mechanisms Ageing Development 1991;57:87-100.

Kalu DN, Orhii PB, Chen C, Lee DY, Hubbard GB, Lee S, Olatunji-Bello Y. Aged-rodent models of long-term growth hormone therapy: lack of deleterious effect on longevity. Journals of Gerontology A Biol Sci Med Sci. 1998;53:B452-463.

Markowska AL, Mooney M, Sonntag WE. Insulin-like growth factor-1 (IGF -1) ameliorates age-related behavioral deficits. Neuroscience 1998; 87:559 -569.

Rudman D, Feller AG, Nagraj HS, Gergans GA, Lalitha PY, Goldberg AF, Schlenker RA, Cohn L, Rudman IW, Mattson DE. Effects of human growth hormone in men over 60 years old. N Engl J Med. 1990;323:1-6.

Sonntag WE, Hylka VW, Meites J. Growth hormone restores protein synthesis in skeletal muscle of old male rats. Journals of Gerontology (Biological Sciences) 1985;40:689-694.

Sonntag WE, Lynch CD, Thornton PL, Khan A, Bennett SA, Ingram RL. The effects of growth hormone and IGF-1 deficiency on cerebrovascular and brain aging. Journal of Anatomy 2000;197:575-586.

Reply to Kevin Becker:

I agree with Kevin's main theme: evidence, from array data, that expression level of a specific gene is altered by aging, diet, or (as in our paper) mutation, is not the definitive proof that the mRNA level for that gene really does differ between groups, let alone that the protein levels differ between the groups. Our paper presents a listing of 60 genes for which p < 0.001 for a difference, in liver, between dwarf and normal mice, but anyone who wanted to pursue some of the biochemical leads would do well to start by checking mRNA and protein levels for the gene product in their own set of samples. At this stage of the game, no one really knows how often genes found to be altered in an array screening study will be replicable in the next set of samples, in terms of mRNA or protein. Kevin gives a fine summary of the ways in which array data can mislead: cross-hybridization, mis-identification of the cDNAs used to construct the array of probes, and other biochemical mis-adventures.

There are some points, particularly in regard to the discussion of statistical inference, where I think Dr. Becker’s comments may be off point. The key problem is the notion of a false positive result. The term false positive is used in two distinct ways in discussing array data, and it's important to see and honor the difference. An array experiment that shows a large and consistent difference in expression of the mRNA for catalase when in fact catalase mRNAs do not differ between group has produced a misleading conclusion, a false conclusion. Such a result might come about, for example, because the arrayed cDNA probe for catalase was of the wrong sequence, or because the lysates contained mRNAs that do not code for catalase but which cross-hybridize with it. No statistical approach can estimate or reduce the number of such false conclusions. (And, alas, no statistical approach can reduce the proportion of scientists who accept mRNA data of this kind as evidence for altered protein levels.)

The second use of the term false positive is in connection with Type I error rate, the kind of error that arises when an apparent difference between groups arises by chance, that is by sampling variation, alone. If one measures the height of two Democrats (say Bill Clinton and Janet Reno) and the height of two Republicans (say Frank Sinatra and Antonin Scalia), one might conclude that on average Democrats are taller. This is a Type I error, because we have not measured enough of each group to exclude chance variation as the explanation for our observation of a difference between the groups. Statistical theorists have worked out methods familiar to all of us for reducing this kind of error. When a single hypothesis is being examined, the scientific community often accepts the conclusion offered when there is only a 5% chance (p = 0.05) that the observed difference would arise by sampling error. If a given data set yields p = 0.20 for a comparison, this does not disprove the hypothesis of interest; but as a community we’ve decided not to put much faith in the conclusion when there's a 20% chance that it's due to sampling error.

When 1000 genes are being tested at the same time, reliance on a p = 0.05 criterion greatly increases the likelihood that some of the genes will show inter-group differences by chance, that is by sampling error alone. If the tests are done with high statistical power (lots and lots of samples), then using a criterion of p = 0.05 will produce a list of about 50 apparently acceptable conclusions even if none of the genes differ between groups. These 50 conclusions can be considered false positives in the sense that they represent conclusions at odds with the underlying biological situation.

In a real experiment, we don't know how many genes really are altered by the diet or age or mutation under study. If we use a criterion of p = 0.05, and look at 1000 genes, and get a list of 50 winners, we should not put too much faith in any one of the genes on the winner list, because we'd expect to get a list of about 50 even if there were no real effects. Some of the 50 genes are likely to be true positives, i.e. likely actually to differ in the full populations from which we've taken a sample, though there's no way to know which ones are real without looking at a fresh set of samples. If instead we use a criterion of p = 0.001 on the same data set, and still find 40 genes on our list, we can be a good deal more confident that most of these do not represent sampling error alone, because sampling error would produce only one false positive in a test of 1000 genes that did not really respond to the mutation under study.

Dr. Becker is correct when he states that an increase in the number of genes examined will not alter the false positive rate in the first of these two senses, unsuspected cross- hybridization, or sequence misidentification or other misadventures that produce erroneous inferences. But increasing the number of genes examined really will increase the likelihood of Type I errors, i.e. impressive inter-group differences that arise by sampling error alone. Reducing the chance of accepting an effect that is due to sampling errors can be accomplished by appropriate experimental design (using more samples) and by selecting a significance criterion that produces an acceptably low Type I error rate.

What error rate is acceptable in this case, and how can we calculate the error rate? The community accepts p = 0.05 as an appropriate significance criterion for experiments that test a single hypothesis. For gene mapping experiments, the most conservative criterion is to use an experiment-wise error rate, set so that the entire set of gene mapping conclusions has only a 5% chance of containing one or more errors due to sampling variation. Such an experiment-wise error rate, similar to that of the Bonferroni method, is surely too stringent for gene expression profiling, because there is real merit in a list of a few dozen or hundred genes allegedly altered by diet or aging, even if the list contains a smattering of false positives due to sampling. In our paper we used p = 0.001 as our main criterion, because for our 2352 genes it did a good job of ruling out most, though not quite all, chance effects. In current and future work we're using, in addition, a false discovery rate criterion developed by Tusher et al. (PNAS 98:5116 - 5121, 2001) that takes a more sophisticated, but still user-friendly, approach to adjusting acceptance criteria based on the number of genes examined.

There are a few aspects of Becker's presentation that I'd like to comment on for the record. (1) Our comparison of dwarf to normal mice used data on 8 mice per group, not 4. An unpublished simulation, resampling from our real data set, showed that if we had used only 4 mice per group we would have found compelling evidence for only about half of the 60 genes we reported using 8/group. (2) We do present the results of using a Bonferroni-adjusted significance level, but point out that it excludes too many genes and then employ a less restrictive criterion instead. (3) Becker says that the RT-PCR method confirmed 7 of 11 of the array findings, but in fact only 8 genes gave detectable and consistent RT-PCR results, and in 7 of these 8 cases the RT -PCR findings were consistent with the array findings. It is not surprising that the dwarf/normal ratios differ between array and RT-PCR findings: the array data come from tests of 8 mice/group, and the RT-PCR data from tests of only 3 mice in each group. In addition, each test will have slightly different sources of error, including cross-hybridization for the arrays. (4) More importantly, the RT-PCR data were not presented as evidence that increasingly rigorous statistical selection alone leads to real or reliable microarray results, they are taken as evidence that we had not committed a gross blunder such as using the wrong array or an array with mis-identified sequences.

I would strongly endorse Dr. Becker's suggestion that complete sets of raw array data should be made available to anyone who wishes to re-analyze them by alternate methods. We would be glad to supply our complete raw data set to anyone who requests it (to millerr@umich.edu), in exchange for a promise to inform us of the results of the subsequent analysis. A list of the processed data (i.e. after normalization and with gene-specific average values) will also be supplied to anyone who prefers the results in this form.

Lastly, I want to second Dr. Becker's concluding recommendation that analysis should not rely simply on statistical assessment but also on other equally important experimental practices, such as replicating individual RNA samples; increasing the number of individual biological samples; and, in particular, emphasizing systematic validation of individual results. Acceptance of these principles by a critical mass of array-meisters, and by those who review array-based papers and grant applications, will be an important step towards improving the signal/noise ratio in the published literature on gene expression profiles.

Parental age at conception may also have an influence on human longevity:

Gavrilov, L.A., Gavrilova, N.S. Biodemographic study of familial determinants of human longevity. Population: An English Selection, 2001, 13(1): 197-222.

Gavrilov, L.A., Gavrilova, N.S. Human longevity and parental age at conception. In: J.-M.Robine et al. (eds.) Sex and Longevity: Sexuality, Gender, Reproduction, Parenthood, Berlin, Heidelberg: Springer-Verlag, 2000, 7-31.

Gavrilov, L.A., Gavrilova, N.S. Parental age at conception and offspring longevity. Reviews in Clinical Gerontology, 1997, 7: 5-12.

Gavrilov L.A., Gavrilova, N.S., Kroutko, V.N., Evdokushkina, G.N., Semyonova, V.G., Gavrilova, A.L., Lapshin, E.V., Evdokushkina N.N., Kushnareva, Yu.E. Mutation load and human longevity. Mutation Research, 1997, 377(1): 61-62.

Gavrilov L.A., Gavrilova, N.S. When Fatherhood Should Stop? Letter. Science, 1997, 277(5322): 17-18.

Gavrilov L.A., Gavrilova N.S., Semyonova V.G., Evdokushkina G.N., Kroutko V.N., Gavrilova A.L., Evdokushkina N.N., Lapshin E.V. Maternal age and offspring longevity, Proc. Russian Acad. Sci. [Doklady Akademii Nauk], 1997, 354(4): 569-572. English translation published in Doklady Biological Sciences, 1997, 354(4): 287-289.

Hope it helps.

Best wishes,

Recent research promises easy cancer cures within a few years.

But the destructive processes of aging, especially apoptosis, may prove more refractory.

I was extremely happy to note the conception of the Basel Institute for Diseases of Aging( BIDA ) . There is a global need to augment research and health care of the elderly throughout the world . The aging population, treated properly would be a valuable human resource to that country and to the rest of the world at large. The purpose of the research is not to obviate the inevitable but to reach the inevitable in a fitting and prepared manner.Until that point the wisdom and experience of these " old " people should be turned into a national and global asset.

Rich Miller's classification of "longevity genes" ends with a strange implicit assertion: that we have not yet found any genes responsible for the differences in longevity between different mammals. In fact, inter- and intraspecies comparisons are the strongest evidence we have that mitochondrial free radical production rates are a major player in the rate of aging, and hence that the relatively few (a few dozen at most, probably only half a dozen) genes encoding proteins responsible for that process are members of category 6. Stronger proof will come not from genetic triangulation but from technical advances (such as the manipulation of mouse mitochondrial DNA) and from crystallography (identifying the detailed structure and mechanism of Complex I).

This way of looking at genes whose discovery might worry life insurance agents makes it clear that a seventh category must be added to Rich's classification. We should not restrict ourselves to trying to improve genes that we already possess; rather, we should look at the *process* we're trying to improve and consider whether genes that mammals lack might be effective. Long-standing [1] candidates are nuclear-coded versions of our 13 mitochondrial protein-coding genes, which would completely eliminate the deleterious effects of mitochondrial mutations (such as they may be). This may sound incredibly ambitious at first hearing, but the past few years have seen key breakthroughs [2-4]. The alternative of replacing some or all of our Complex I subunit genes with ones from parrots, for example, seems altogether too timid.

Richard Miller has written a very useful and logically constructed position paper on genes that may influence aging and longevity. He has avoided the use of the term "gerontogenes" in his paper because of, perhaps, the evolutionary reasons against the existence of such real genes for aging. However, I think that using the term gerontogenes (which was first coined by me in 1985, and has been used by several authors over the last 15 years) can have several advantages, including drawing attention to the subject of biogerontology and underlining the focus of discussion.

Recent research promises easy cancer cures within a few years.

But the destructive processes of aging, especially apoptosis, may prove more refractory.

Recent research promises easy cancer cures within a few years.

But the destructive processes of aging, especially apoptosis, may prove more refractory.

Recent research promises easy cancer cures within a few years.

But the destructive processes of aging, especially apoptosis, may prove more refractory.

I want to thank Jay Olshansky for this lovely memoir, especially for his moving tribute to Bernice Neugarten. Back in the early days of NIH- supported research on aging, basic biologists, clinicians and social scientists often sat together on the same panel. I thus came to know and admire Bernice and other social scientists sharing her passionate, scholarly and visionary approach to the problems of older people. A particularly fine example is Matilda White Riley. She is still going strong! Matilda, Bernice and their friends taught me to embrace the life course view of aging. Social gerontologists thus have a lot in common with evolutionary biologists concerned with gerontology.

1. Aging genes and fertility. Because a number of mutations disabling the activity of individual wild-type alleles have been shown to extend life, Mitteldorf posits that the unmutated, wild-type, alleles must be “aging genes,” that is genes which have evolved to cause aging by group selection. He also assumes that the only way these observations might be compatible with traditional evolutionary aging theory is if these mutations depress fertility. His errors are two-fold. First, he assumes that alleles associated with extended life in the laboratory are associated with higher darwinian fitness of those individuals. The fact that such alleles have never been isolated in nature is prima facie evidence against such an interpretation. Second, he assumes that fertility (which indeed is often suppressed in these mutant phenotypes) is the only component of fitness. Of course, there are many fitness components in addition to fertility per se, for instance juvenile viability, competitive ability, resistance to environmental stresses, adult competitive ability for resources or for mates, that might be negatively affected by such mutations. In fact, each of these long-lived mutants, when studied in detail, has been found to have pleiotropic negative effects on growth rate, fertility, or competitive ability – traits in other words that have a more direct relation to darwinian fitness than does longevity (Kirkwood, T.B.L & Austad, S.N. 2000. Nature 408: 233-238). If one wished to characterize the unmutated “wild-type” genes by their adaptive value, they would more properly be called “growth,” or “fertility” or “competitive ability” genes rather than “aging” genes as he calls them and their adaptive value is obvious. Therefore neither the existence of these wild-type alleles, nor extended longevity accompanying their disablement, poses any problem for evolutionary theory. In contrast, the fact that disabling these genes leads to longer life nicely supports Williams’ theory of the evolution of aging by “antagonistic pleiotropy.” The mouse p66shc mutant mentioned by Mitteldorf is notable mainly in that it has been scarcely characterized at all with respect to other life history traits than laboratory longevity. Significantly the increased resistance to chemical stressors exhibited by the p66shc mutant is reminiscent of that reported in calorically-restricted mice, which leads to his second point.

2. Caloric restriction and aging. The crux of the “challenge” of calorically-restricted rodent paradigm is “experiments suggest that the life extension brought about by CR is independent of their effect on fertility.” The implication, it seems, is that the retarded aging rate of calorically-restricted animals could not be part of an adaptive tradeoff among life history traits under conditions of food shortage. First, none of the evidence cited by Mitteldorf supports this assertion. Fertility is reduced or completely suppressed in both male and female calorically-restricted rodents in all studies in which it has been examined. Mitteldorf’s point seems to be that further restriction, even after reproduction has been completely suppressed, leads to an even further increase in longevity when there are no remaining tradeoffs available. As in the previous “challenge,” the interpretive problem is that he assumes that ever increasing laboratory longevity must be associated with higher fitness, and that fertility is the only fitness component other than survival. As previously mentioned, other fitness components such as foraging efficiency, competitive ability, and resistance to environmental stresses might be impacted in addition to reproduction. The reason that fully-fed animals do enter the slower aging rate characteristic of caloric restriction is that their extra energy is better used for other things such as warding off cold, escaping predators, or defending territories. For instance, my laboratory has observed that calorically-restricted mice have a lower sprint speed than fully-fed mice when each is forced to run on a treadmill. Again, as with long-lived laboratory mutants, the extended longevity of caloric restriction should not be interpreted as having relevance for animals in nature. It is painfully obvious, that calorically-restricted rodents would likely have substantially lower survival in nature than better-fed animals. Specifically, calorically-restricted mice are more susceptible to cold stress than ad lib fed mice (Campbell & Richardson, 1988, Exp. Gerontol. 23:417-427) and we know that cold stress is a major cause of mouse mortality in nature (Berry, 1981, Mammalian Rev. 11:91-136). Also, we know that calorically-restricted rodents would be more susceptible to death from short-term food shortage. Like the single gene mutants, calorically-restricted rodents are a useful tool for exploring mechanisms of aging, but they represent a low-fitness alternative states, except in the especially favorable conditions of the laboratory.

3. Conserved mechanisms of aging. All researchers in the field ardently hope that at least some fundamental mechanisms of aging are highly conserved, otherwise research with model organisms is a waste of time. To Mitteldorf, the challenge to evolutionary theory of these mechanisms is that he imagines that general cellular phenomena such as apoptosis have the appearance of deliberate adaptations, hence their adaptive value must be in accelerating aging. Researchers who study these phenomena would likely differ. Apoptosis plays an invaluable adaptive role in development and morphogenesis, as well as in protecting us from genetically-damaged cells in danger of neoplastic transformation. As for replicative senescence, it has been cogently interpreted by Campisi as an instructive example of antagonistic pleiotropy – a process which evolved to limit uncontrolled cell proliferation during early life, but which might have deleterious side-effects later in life.

Since that time, mainstream evolutionary theory has been committed to a paradigm of selection exclusively at the individual level. Aging entails a fitness cost to the individual, who has nevertheless acquired a network of genes that determines and regulates the rate of aging. So the prevailing theories of aging have been built on a presumption that aging has evolved as a side-effect of genes that carry a fitness benefit for the individual. This benefit must be strong and universal to account for the universality of aging; hence there is wide agreement that genes that cause aging must enhance fertility.

But in the two generations since foundations were laid for the current evolutionary theories of senescence, new data have come to light that challenge the theories at their core. Today, the theories survive as a framework in which to interpret data, but their persistence might result from an absence of viable alternatives rather than because there is an excellent fit with experimental results. It might be appropriate to consider whether the data demand of us a new paradigm; recent developments in evolutionary theory point toward origins for this new paradigm.

What are the theoretical reasons for thinking that selection operates exclusively at the individual level? The reasons are cogent, and are well-argued in George Williams's classic, Adaptation and Natural Selection (2). The essence of the argument is that a prerequisite for group selection is the persistence and fixation of a mutation, arising originally in a single individual. But evolution at the individual level is rapid and direct, scaled by the lifetimes of individuals; evolution at the group level proceeds at a slower pace, scaled by the extinction time of populations. Hence it seems unlikely that any trait entailing even a small cost to the individual can persist long enough to test its effect on fitness of the population as a whole.

What are some experimental challenges to the prevailing theories?

1. Aging genes and fertility. Over the last decade, single gene mutations have been discovered that extend life-span in nematodes(3), fruit flies (4), and mice (5). Because deletion of these genes causes the animal to live longer, it seems inescapable to characterize them as "aging genes." It is far from clear, however, that they offer any benefit to fertility. For example, the mouse gene p66shc affects apoptosis in damaged cells, which appears to be upregulated to the point where healthy cells are routinely being killed in older individuals. Experiments show that this substantially shortens the mouse life-span, but it is hard to imagine what benefit it might carry for fertility.

2. Caloric restriction and aging. In calorically restricted (CR) rodents, the aging rate is slowed substantially, while the immune system is more effective and activity levels are enhanced (6). Where does the capacity come from to sustain this effort, and why does the body choose not to extend its life when fully fed? This phenomenon is a particular embarrassment to the reigning "Disposable Soma" theory of senescence, which holds that a tight caloric budget is the root cause of aging. Theorists have responded by emphasizing the large fertility cost entailed in the CR response (7), but experiments suggest that the life extension brought about by CR is independent of the effect on fertility (8): In male rodents, life-span is substantially extended with only modest curtailment of fertility; in females, life-span continues to lengthen as the severity of caloric restriction is increased (9). For example, with caloric restriction at >30%, a regime in which the females are already completely infertile, life-span continues to grow with severity of caloric restriction all the way down to the threshold of starvation, around 60%.

3. Conserved mechanisms of aging. Some mechanisms of aging appear to have been conserved over vast evolutionary time scales (10, 11). For example, evidence is emerging that implicates apoptosis as a senescence mechanism (12-16). Apoptosis has all the appearance of a deliberate adaptation, and hints that it plays a role in aging are problematic for the standard theories. Likewise, replicative senescence through telomere attrition is an independent, purposeful adaptation, and its implication in cellular senescence in the aging of higher organisms belies all theories of pleiotropy. If processes such as telomeric senescence and apoptosis, which we think of as purposeful adaptations, do turn out to play a central role in the aging of higher organisms, conventional theories of aging will face trouble: It would then be difficult to support the notion of aging as a side-effect of processes that primarily enhance fertility, and impossible to maintain the idea that aging derives from a failure to allocate adequate resources to repair and maintenance functions.

What new theoretical developments might hint at an alternative framework?

1. Almost as old as the proscription on arguments from group selection is David Sloan Wilson's work on multi-level selection (MLS) theory (17, 18). In recent years, the body of literature on MLS has swelled, as has the number of recognized mechanisms by which group-level adaptations might emerge (19, 20).

2. Complexity theorists revel in the distinction between smooth, analytical processes that closely track our intuitions and the surprising, chaotic realms of complex systems. There is now a community of Artificial Life specialists, who study evolution with computer models rather than differential equations. They shun the equations and theorems of population genetics, because these have been derived on the assumptions of infinite populations and negligible epistasis, conditions that are rarely achieved in nature. Most of the people in this community hail from backgrounds in physics or computer science rather than evolution and have little reverence for the distinction between individual and group selection. It is arguable that a complex systems approach makes group selection far more plausible than does an analytical approach (21).

3. The evolution of evolvability has been an intriguing field of study since the ground-breaking paper of Wagner and Altenberg (22). Evolvability advocates argue that the structure of chromosomes and the organization of the genome appear to be optimized in ways that promote the efficiency of evolutionary processes. But evolvability is a property of populations, not individuals. Evolvability itself can only evolve via a process of long-term selection on populations, which is exactly the kind of selection that traditional evolutionary theory warns us does not exist. And if evolvability has somehow evaded the theoretical ban on group selection, can senescence be viewed as a population trait that contributes to evolvability? Senescence contributes to the adaptability of a population by reducing the effective generation length and enhancing genetic diversity, as Weismann (23) pointed out over a century ago.

What's next? It's one thing to take pot shots at established evolutionary theory, and quite another to put forward a viable alternative. It remains to be seen whether a plausible model of the evolution of senescence can be constructed on the basis of long-term and widely dispersed benefits of higher population turnover and increased genetic diversity.

References

1. M. Leslie, Aging Research Grows Up. Science's SAGE-KE (2001) http://sage ke.sciencemag.org/cgi/content/full/sageke;2001/1/oa1.

2. G. C. Williams, Adaptation and Natural Selection (Princeton Univ. Press, Princeton, NJ, 1966).

3. C. Kenyon, J. Chang, E. Gensch, A. Rudner, R. Tabtiang, A C. elegans mutant that lives twice as long as wild type. Nature 366, 461-464 (1993).

4. M. Tatar, A. Kopelman, D. Epstein, M.-P. Tu, C.-M. Yin, R. S. Garofolo, A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292, 107-110 (2001).

5. E. Migliaccio, M. Giorgio, S. Mole, G. Pelicci, P. Reboldi, P. P. Pandolfi, L. Lanfrancone, P. G. Pelicci, The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402, 309-313 (1999).

6. R. Weindruch, R. Walford, The Retardation of Aging and Disease by Dietary Restriction. (Thomas, Springfield, IL, 1986).

7. D. Shanley, T. B. L. Kirkwood, Calorie restriction and aging: a life history analysis. Evolution 54,740-750 (2000).

8. J. Mitteldorf, Can experiments on caloric restriction be reconciled with the disposable soma theory for the evolution of senescence. Evolution 55,1902-1905 (2001).

9. L. V. De Paolo, Dietary modulation of reproductive function. In: Modulation of aging processes by dietary restriction, Yu, ed. (CRC Press, Cleveland, OH, 1993), pp 221-246.

10. W. Clark, A means to an end: the biological basis of aging and death. (Oxford University Press, NY, 1999).

11. C. Kenyon, A conserved regulatory system for aging, Cell 105, 165-168 (2001).

12. W. Clark, Reflections on an unsolved problem of biology: the evolution of senescence and death. Unpublished manuscript.

13. S. Cornillon et al., Programmed cell death in Dictyostelium. J. Cell Sci. 107, 2691-2704 (1994).

14. K. Hopkin, Death and aging, together at last. Science's SAGE-KE (2001) http://sageke.sciencemag.org/cgi/content/ full/sageke;2001/4/nf2.

15. J. Luo, A. Y. Nikolaev, S. Imai, D. Chen, F. Su, A. Shiloh, L. Guarente, and W. Gu, Negative control of p53 by sir2 promotes cell survival under stress. Cell 107,137-148 (2001).

16. H. Vaziri, S. K. Dessain, E. N. Eaton, S. Imai, R. A. Frye, T. K. Pandita, L. Guarente, R. A. Weinberg, hSIR2SIRT1 functions as an NAD-dependent p53 deacetylase. Cell 107, 149-159 (2001).

17. D. S. Wilson, The Natural Selection of Populations and Communities. (Benjamin Cummings, Menlo Park, CA, 1980).

18. E. Sober, D. S. Wilson, Unto Others. (Harvard University Press, Cambridge, MA, 1998).

19. D. S. Wilson, Ed. Multilevel Selection. The American Naturalist (1997 suppl.).

20. L. Keller, Ed. Levels of Selection. (Princeton University Press, Princeton, NY, 1999).

21. J. Mitteldorf, D. S. Wilson, Population viscosity and the evolution of altruism. J. Theor. Biol. 204, 481-496 (2000).

22. G. P.Wagner, L. Altenberg, Complex adaptations and the evolution of evolvability. Evolution 50, 967-976 (1996).

23. A. Weismann, Essays Upon Heredity and Kindred Biological Problems. (Clarendon Press, Oxford, U.K. 1889).

Dear Kristen Carlberg, I am sorry but I know too little about Excel or SPSS to help you out here. I searched Google Groups for "Excel lowess" and "Excel loess" and found a few requests but no answers. Try to post your question there

http://groups.google.com/groups?hl=en&group=microsoft.public.excel

http://groups.google.com/groups?hl=en&group=comp.soft-sys.stat.spss

Regards

Dear Richard Miller, I will try to answer your questions to the best of my knowledge. (The references are the same as in the introductory paper Bengtsson et al.)

1. Using an ordinary t-test we found 61 genes where p < 0.001. Because we had looked at 2352 genes, we expected to see only 2 or 3 "false positives" at p = 0.001, and so we concluded that most of the genes on our list were probably ok.

A1. Assuming the validity of the t-tests for each individual gene that you perform, it reasonable to conclude that most of the genes that you have found are "probably ok".

2. The statistical program SAS has a test (MULTTEST) that automatically calculates a "false discovery rate" statistic that adjusts for the number of genes being tested at the same time. We found that 72 of the genes had an FDR p-value < 0.05. But to do this test requires you to know someone who can write SAS programs.

A2. As you say, the PROC MULTTEST function in SAS i a p-value adjustment method and it is doing the same as the procedures described in Dudoit et al. ([14]). If you do not want to use SAS the methods described by [14] are all implemented in the sma package [5] and will later be "lifted up" into the sma add-on package com.braju.sma [4]. All methods in the sma package are accessible from the com.braju.sma package. These package requires the free software R [6,7].

3. VG Tusher and colleagues have written a nice paper on significance analysis of microarray data (it's PNAS 98:5116 - 5121, 2001) that you refer to in your own paper as Citation 17. The approach they suggest makes it very easy to "tune" the calculation to adjust the false discovery rate to one the investigator is comfortable with. (For some purposes you might be happy with a list of 100 genes of which 20 are false; for other purposes you might want to examine the same data set to find the 20 best genes for which only 2 are false positives.) The Tusher group have produced an Excell add-in program (free!) that makes this calculation a snap even for the statistically impaired.

A3. There is an important technical difference between Tusher et al's test procedures [16] and those in [14] and PROC MULTTEST. The method by Tusher et al. only provides what is known as "weak control" of the type I error (a true null hypothesis is rejected). In other words, it controls the error rate under the complete null hypothesis that no genes are differentially expressed and this is unsatisfactory.

4. I've glanced at (but not yet had time to read carefully) your own posted paper linked to the SAGE web site. You say that your favorite statistic (B-statistic) is a bit better than the statistic used by Tusher and company - but is the difference substantial enough to justify the pain needed for us users to master the intricacies of the approach you are recommending for picking the best winner genes from the list ranked by the S-, B-, or t-statistic?

Can you provide some advice on which of these approaches has the best balance between statistical rigor and ease-of-use?

A4. Yes, the B-statistics method by Lönnstedt et al. [17] is somewhat tricky to understand, but the implementation of it in [4,5] is pretty straightforward. However, as everything else in data analysis it is important to understand the methods you apply. In [17] it is shown that the S-statistics does almost perform as good as the B-statistics. The t-stastics is worse and using plain M-values (log ratios) should be avoided.

There are important differences between the approaches which relate to the structure of your experiment. For example, the step-down maxT used in PROC MULTTEST and [14] is available for use when the experiment has replicated treated and control slides, and a common reference, but not when there is (only) a treatment and control target cDNA on the same slides (as in the poster paper). Replicated trt/ctl slides cannot be permuted, and so any formal testing with them must be based on parametric statistical modelling assumptions. As [17] suggests, it is better to avoid making such assumptions, and simply rank one's genes using some sensible criterion. As argued in [17] (downloadable), and in section 5.1 in the posted one, the t-statistics should not be trusted.

I hope this answered your questions.

Henrik Bengtsson, Lund University

In our own data set (based on gene expression levels from liver of 8 young Snell dwarf mice and 8 young control mice for 2352 genes), we compared a number of methods to estimate the "false discovery rate," ("FDR") i.e. the probability that a gene we decided did differ in its expression in the dwarf samples was merely a chance effect. Some of these approaches are easy for anyone who can handle Excell; others required more sophistication and the services of expensive statisticians. I'd like your advice on which of these seem sensible:

1. Using an ordinary t-test we found 61 genes where p < 0.001. Because we had looked at 2352 genes, we expected to see only 2 or 3 "false positives" at p = 0.001, and so we concluded that most of the genes on our list were probably ok.

2. The statistical program SAS has a test (MULTTEST) that automatically calculates a "false discovery rate" statistic that adjusts for the number of genes being tested at the same time. We found that 72 of the genes had an FDR p-value < 0.05. But to do this test requires you to know someone who can write SAS programs.

3. VG Tusher and colleagues have written a nice paper on significance analysis of microarray data (it's PNAS 98:5116 - 5121, 2001) that you refer to in your own paper as Citation 17. The approach they suggest makes it very easy to "tune" the calculation to adjust the false discovery rate to one the investigator is comfortable with. (For some purposes you might be happy with a list of 100 genes of which 20 are false; for other purposes you might want to examine the same data set to find the 20 best genes for which only 2 are false positives.) The Tusher group have produced an Excell add-in program (free!) that makes this calculation a snap even for the statistically impaired.

4. I've glanced at (but not yet had time to read carefully) your own posted paper linked to the SAGE web site. You say that your favorite statistic (B-statistic) is a bit better than the statistic used by Tusher and company - but is the difference substantial enough to justify the pain needed for us users to master the intricacies of the approach you are recommending for picking the best winner genes from the list ranked by the S-, B-, or t-statistic?

Can you provide some advice on which of these approaches has the best balance between statistical rigor and ease-of-use?

It's kind of you to offer tutorials in this format, and I'll look forward to hearing your advice.

Back in the 1960’s, when Charlie Epstein, Arno Motulsky and I first became interested in the Werner syndrome and its possible relationship to the biology of aging, George W. Casarett, a radiation biologist, suggested a set of rather rigid criteria for determining if one could conclude that a given intervention was responsible for altering the rate of aging (Adv Gerontol Res 1:109, 1964). While he was mainly concerned about the degree to which the life-shortening effects of ionizing radiation can be considered to induce premature aging, one could also apply his criteria for the evaluation of agents that can in fact retard the rate of aging. In brief, he argued that a given agent had to alter life span without changing the basic pattern of the mortality curve. It also had to change each and every morphological and physiological manifestation of aging in a synchronous manner and had to alter all diseases of aging and all causes of death in a proportionate manner.

While some degree of synchronization of the wide array of phenotypes associated with aging is clearly to be expected, evolutionary theory does not demand perfect synchronization. Given the substantial genetic heterogeneity and diverse environmental impacts that wild type creatures like us experience, there is ample opportunity for various degrees of asynchrony. Thus, is would be helpful for us to consider the idea that rates of aging may proceed segmentally, at least to some extent. Geriatricians will certainly not be surprised by this proposition. They see this expressed in their patients every day.

Incidentally, Table 5.1 (page 233) of Rick Weindruch’s and Roy Walford’s wonderful monograph on “The Retardation of Aging and Disease by Dietary Restriction” (Charles C. Thomas, Springfield, Illinois, 1988) lists nine age-sensitive phenotypes that do not respond to dietary restriction. I imagine that more parameters can be added to that list by now. Could you respond to that, Rick or Roy? Are you out there in cyberspace?

The question as posed in the title (“Is life span the best measure of aging?”) is too easy, but is closely related to a much tougher question: “can an intervention that does not affect aging lead to impressive increases in life span?”

To deal first with the initial question: there are lots and lots of things that shorten life span by ways that do not involve alterations in aging. These include genes that cause lethal perinatal conditions, deciding to join a brigade of firefighters, or not wearing seat belts. Each of these (and many more from the same shelf) shorten life span, but do not do so by a change in aging using any plausible definition of that many-splendored term.

To my mind, it’s more interesting to talk about whether you can extend life span (a lot) by interventions that do not work by alterations of aging rate. To discuss this carefully requires consideration of some specific examples:

1. Lots of things can improve mean life span by effects on diseases that kill juveniles and young adults; most of the increase in life expectancy at birth in the past century has come from lowering mortality risks in the first 40 years of life (see Olshansky et al., Science 291: 1491, 2001, for real numbers.) This kind of change does not involve changes in aging rate, and that’s why most claims that a gene or drug slows aging usually start with documentation of an increase in the longevity of those adults who reach the last 10% or so of the life span. (This number used to be called the “maximum longevity” before demographers started to raise a fuss about it.)

2. Ok, so you’ve got a chemical that doubles mean life span and also doubles the life span of the longest-lived 10% of the crowd…now can you conclude that the agent is slowing down aging based on the mortality data alone? I suspect that this is usually a safe conclusion, but it’s not too hard to come up with hypothetical (and even a few real) counter-examples. For example, imagine a population of inbred mice in which everybody dies, age 5 months, from hemophilia. A gene (or Medi-mouse HMO plan) that provided the missing clotting factor would lead to major improvements in mean and maximal longevity, not because clotting factors prevent aging, but because this particular mouse stock has its life span limited by one specific syndrome. This scenario is not so far-fetched as it may seem: a famous paper showing the effects of different alleles of the mouse histocompatibility locus H-2 on life span now seems to reflect, at least partly, the effects of some of these alleles on a specific, early-life form of thymic lymphoma.

3. One way to dodge the “one-disease” bullet is to do careful necropsies on every mouse in the treated and control group. If the mice are dying of a range of diseases, and the increase in life span seen in the treated group affects with roughly equal force those mice dying of each of the major causes of death, then I think it’s safe to conclude that the treatment really does slow down aging. But this standard of proof is a lot to ask – necropsies are expensive, and you have to wait a long time to get the mice to die. The whole literature contains only a handful of papers that include terminal necropsies (as opposed to cross sectional data, which tells you little about cause of death) on more than a few dozen animals.

4. So this puts the onus on finding a reasonably wide range of age- sensitive traits that can be used in middle-aged adults to test the idea that the gene or treatment affects not merely life expectancy but also multiple signs of advancing age. If, for example, we found that a specific diet not only extends maximal longevity but also produces 22-month old mice or rats that have the immune responsiveness, clear lenses, impressive biceps, endocrine profiles, and gene expression patterns of a healthy 12 month old, we’d be able to claim that the stuff slowed down aging. It took the calorie restriction folks a generation to accumulate this kind of a dossier on CR rodents, and parallel work on mutations that extend mouse life span is only slowly getting under way (advertisement: see Flurkey et al., PNAS 98: 6736 – 6741, 2001). This is a tough (but not hopeless) problem for worm and fly mavens, whose favorite organisms are short on biceps and T cells.

In practice, I try to take a compromise position: if the treatment extends maximum longevity, I promise to take it very seriously indeed, and vote a good priority score even if the grant application has a few problems here and there. If the longevity data is accompanied by evidence that the treatment or gene alters a handful of age-sensitive traits in a consistent direction, then I add the treatment to the short list of age-retarding regimens. This list is, however, a very short one so far: calorie restriction and dwarf mice.

First, let me applaud your efforts and offer my opinion that the SAGE KE site will be of immense value as a central repository and provider of information to those doing research in aging. A similar type of site devoted to Alzheimer's disease, the Alzheimer's Forum (www.alzforum.org), has already demonstrated the utility of this type of information resource.

Second, it's nice to know that as we researchers age, there will be a home for us. As you stated, "Our goal is to serve as an intellectual home base for the aging research community."

Is the focus of the site basic, molecular science or applied clinical science? We are trying to bridge this gap in our division of Allergy and Immunology at the University of South Florida. We are particularly intested in the effects of aging on the diseases of asthma and related obstructive pulmonary disease. I welcome input and dialogue on this subject.

I have read several articles on this site and I have also done several literature searches. The site consistently ignores the development of near-term solutions to some of the problems of aging in favor of long- winded theoretical discussions. This article is a case in point: it mentions high levels of collagen cross-linking but fails to mention that a drug exists that is currently in 2 phase 2 and one phase 1 trial, and that has already produced promising results in animal studies and in phase 1 and 2 clinical trials. The drug is alt-711. See www.alteonpharma.com for info. Also, see these references: : Kass DA, Shapiro EP, Kawaguchi M, Capriotti AR, Scuteri A, deGroof RC, Lakatta EG. Related Articles

Improved arterial compliance by a novel advanced glycation end- product crosslink breaker. Circulation. 2001 Sep 25;104(13):1464-70. PMID: 11571237 [PubMed - indexed for MEDLINE]

2: Doggrell SA. Related Articles

ALT-711 decreases cardiovascular stiffness and has potential in diabetes, hypertension and heart failure. Expert Opin Investig Drugs. 2001 May;10(5):981-3. Review. PMID: 11424901 [PubMed - indexed for MEDLINE]

3: Ulrich P, Cerami A. Related Articles

Protein glycation, diabetes, and aging. Recent Prog Horm Res. 2001;56:1-21. Review. PMID: 11237208 [PubMed - indexed for MEDLINE]

4: Vaitkevicius PV, Lane M, Spurgeon H, Ingram DK, Roth GS, Egan JJ, Vasan S, Wagle DR, Ulrich P, Brines M, Wuerth JP, Cerami A, Lakatta EG. Free in PMC , Related Articles

A cross-link breaker has sustained effects on arterial and ventricular properties in older rhesus monkeys. Proc Natl Acad Sci U S A. 2001 Jan 30;98(3):1171-5. PMID: 11158613 [PubMed - indexed for MEDLINE]

5: Melton L. Related Articles

Age breakers. Rupturing the body's sugar-protein bonds might turn back the clock. Sci Am. 2000 Jul;283(1):16. No abstract available. PMID: 10881297 [PubMed - indexed for MEDLINE]

6: Asif M, Egan J, Vasan S, Jyothirmayi GN, Masurekar MR, Lopez S, Williams C, Torres RL, Wagle D, Ulrich P, Cerami A, Brines M, Regan TJ. Free in PMC , Related Articles

An advanced glycation endproduct cross-link breaker can reverse age- related increases in myocardial stiffness. Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2809-13. PMID: 10706607 [PubMed - indexed for MEDLINE]

7: Wolffenbuttel BH, Boulanger CM, Crijns FR, Huijberts MS, Poitevin P, Swennen GN, Vasan S, Egan JJ, Ulrich P, Cerami A, Levy BI. Free in PMC , Related Articles

&#65279;Re: Re: Re: The sir2 overexpression and knockout

Increasing Sir-2 expression via transgenic overrepresentation of Sir- 2 genes is only one of the paths suggested by this work.

The other path is to simulate the way caloric restriction increases Sir-2 expression - via an increased pool of NAD/NADH. Let me suggest a quick, easy experiment - with immediate and intimidating implications.

Divide a group of genetically normal mice into three groups, each fed differently. 1) A controlled diet simulating ad libitum eating; 2) a restricted calorie diet known to extend lifespan; 3) a controlled diet simulating ad libitum eating, but supplemented with additional NADH (either IV or orally with enteric coating).

In response to the message from Florian Muller, a Sir-2.1 transgenic worm is in fact shown to have an extended life span. This experiment can be found in Tissenbaum and Guarente, Nature 410:227 (2001). In figure 2C, life span is shown for three different lines carrying multiple copies of Sir-2.1 integrated into the genome. In each case, both mean and maximum life span is longer than the wild type control.

Dear Sir,

It is really great to see that a vigorous debate is starting on this web page. It is with great interest that I read your article summarizing yeast research. It seems to me that the strongest piece of evidence arguing that the clonal aging system in yeast has anything to do with aging in multicellular organisms is the "overexpression" of sir2 in C. elegans leading to increased longevity. The article cited, however, doesn't talk about overexpression but about a naturally occurring duplication in the sir2 gene of C. elegans. The obvious experiment to confirm whether higher levels of sir2 are really the causative agent in that strain is to make a transgenic worm.....is such an experiment planned or perhaps already published?

Most sincerely yours,

In his response to our Perspective, Dr. Gershon states that “yeast do not make a proper paradigm for aging research”. Clearly, we are not arguing that yeast are a perfect model for human aging. No model organism perfectly replicates the aging process in humans – that’s why they are models. Similar criticisms have been raised about the use of worms, flies, and mice to study aging. Some scientists would argue that human aging should only be studied in humans. We disagree.

As pointed out in our review, there are several specific features of human aging that have been, and continue to be, usefully modeled using yeast. These include oxidative stress, mitochondrial dysfunction, metabolic changes under caloric restriction, genomic integrity during aging, and the biology of Werner Syndrome/Bloom Syndrome/Rothmund-Thompson Syndrome. Furthermore, we believe that we have already gained valuable information from studying aging in yeast that suggests avenues of pursuit with respect to human aging. Specifically, the discovery that the SIR2 gene affects aging in both yeast and C. elegans begs the question of whether it also affects human longevity.

We would also like to respond specifically to Dr. Gershon’s criticisms. He claims that yeast lacks a “differentiation system which leads to the development of very specialized cell types with unique and limited repertoire of functions.” In fact, under non-standard growth conditions, yeast can form specialized cell types that are capable of pseudohyphal and/or invasive growth. This developmental switch is accompanied by a dramatic change in gene expression patterns. This example, though not directly related to yeast aging, exemplifies the principle that yeast possess the ability to respond to environmental changes with significant metabolic and morphological change. Multicellular organisms certainly have similar mechanisms for coping with environmental changes and stressful situations; it is likely that some of these can affect longevity. Therefore, the identification of the genetic pathways that modulate these responses in yeast should serve as a starting point for studies in higher eukaryotes.

Dr. Gershon also states that “A review of work from various laboratories that investigate aging in yeast shows many discrepancies and contradictions that are quite hard to reconcile.” This is a misleading argument. Some of these so-called “discrepancies” are due to strain specific differences in genetic background. In other words, genetic predispositions affect life span. The same thing is true in other model systems as well as humans, and should not be used to invalidate yeast aging. Other “discrepancies” have been caused by misinterpretation of results. For example, we and others have failed to detect more ERCs in sgs1 mutants relative to wild type (discussed in our perspective). This has been misinterpreted to suggest that ERCs do not cause aging in wild type cells – which would be a discrepancy. However, the correct interpretation is that sgs1 cells are not dying for the same reason as wild type cells, a hypothesis that has been subsequently verified.

Dr. Gershon’s final criticism, that replicative senescence plays no role in the aging of multicellular organisms, is also misleading. The study he cites does indeed suggest that cells from older individuals fail to show reduced replicative capacity. However, many other studies have found an inverse correlation between replicative capacity in tissue culture and donor age (for a recent example see Yan et al., Mech Age Dev. 122:695). The importance of replicative senescence in the human aging process is still an open issue, and is likely to be cell type specific. Clearly then, it is not appropriate to dismiss yeast as a model system based on this argument.

Matt Kaeberlein Department of Biology Massachusetts Institute of Technology

Compliments for writing a piece which properly ruminates on many of the standard misconceptions about interventive gerontology and on many of the rationalizations about "the joys of decrepitude" without falling prey to any of them. Your article ended on just the right note, i.e., Butler's observation that in a sense, we've been there and done that already. And, as the old saying goes, the best is yet to come.

Medawar pointed out that the ability of selection to eliminate germ-line deleterious mutations will depend on when in the course of life these mutations act. Mutations that act at very late ages will be effectively hidden from selection, and so will accumulate over evolutionary time. This occurs because mutations that reduce survival or fecundity late in life will only affect the relatively few individuals who survive to that age, and among those who do survive, these individuals will have already passed those deleterious genes on to future generations. This pool of late-acting mutations, Medawar argued, would lead to senescence.

As a corollary to Medawar's 'Mutation Accumulation' (MA) theory, George Williams developed his 'Antagonistic Pleiotropy' (AP) theory, whereby genes with late-acting deleterious effects might actually be favored by selection if these same genes are beneficial early in life. Experimental tests of these two theories date back nearly twenty-five years. Pioneering work by Michael Rose and others focused on tests of AP, looking for trade-offs between early-life fecundity and late-life survival. These early studies generally supported AP as a primary evolutionary cause of aging.

It was not until the early '90's that Kim Hughes, at the time a graduate student with Brian Charlesworth, developed the first explicit test of MA. Hughes and Charlesworth argued that under MA, additive genetic variance for fitness traits (that is, the total variation in a fitness trait that is due to genes with additive effects) should increase with age. Surprisingly, no previous studies had directly tested this idea. Hughes and Charlesworth measured mortality rates at different ages in the fruit fly, Drosophila melanogaster, and found that additive variance did, in fact, increase with age. Later studies by Promislow, Tatar, and others suggested that variance components very late in life might actually decline, contrary to what was predicted by either theory of aging. To further complicate matters, it turned out that the predicted increase in variance with age was not unique to Medawar's theory.

In a later study, Charlesworth and Hughes noted that additive genetic variance could also increase if senescence were due to genes with antagonistic pleiotropic effects. In this light, they developed models from which they derived novel predictions that were unique to each theory. According to their models, inbreeding load for fitness traits (i.e., the difference in fitness traits between outbred and inbred genotypes) should increase with age under MA but not AP. Furthermore, dominance variance (variation due to genes with dominant effects) should be relatively constant under AP, but should increase if aging is due to MA. (A third, widely cited evolutionary theory of aging—Kirkwood's 'Disposable Soma' theory—is not addressed by Charlesworth's and Hughes's quantitative genetic models).

In the October 29, 2002 issue of PNAS, Kim Hughes and her colleagues present results from a simultaneous test of these two mutually exclusive predictions. Hughes et al. measured genetic variance components for reproductive success in flies of different ages. Reproductive success, simply measured as the number of offspring produced in a vial by five males and five females, declines steadily with age. Hughes et al. discovered that as reproductive success declines, inbreeding load, additive variance and dominance variance all increase with age. They argue that these results provide strong support for the MA model and are counter to the AP model. An apparent strength of this recent work over previous studies by Hughes and others is that it focuses on reproductive success rather than mortality. The use of mortality rates to bridge the gap between quantitative genetics and experimental gerontology presents enormous statistical challenges. Early in life when mortality rates are low, and late in life when few individuals are still alive, the number of deaths is small and we may underestimate the actual variance for mortality in the population. By focusing on reproductive success, Hughes and her coworkers have attempted to circumvent these problems.

Reproductive success is not without its own challenges, however. As the authors note, this focus on reproduction narrows the window of time over which the aging process can be studied. Whereas many flies in Hughes et al.'s study survived for almost two months, reproduction had virtually ceased in most flies by the end of the first month. Thus, the apparent decline in variance seen in earlier studies could have still occurred among the flies studied by Hughes et al., but at an age later than was observed in the study. Of course, one might then ask whether a decline in genetic variance for reproductive success at an age later than anyone ever reproduces would really matter.

More importantly, although reproductive success has simpler statistical properties than mortality rates, there are still pitfalls for even the most wary. Hughes et al.'s result may be somewhat compromised by the transformation that the authors used. Reproductive success is likely to be Poisson distributed. For Poisson variables, the expected variance is equal to the mean. But we are interested in determining whether the variance changes with age (i.e., does the variance increase as the mean decreases). To eliminate this confounding mean-variance relationship, the authors square-root transformed the data. This equalizes the variance over a broad range of values. Unfortunately, the authors then further 'standardized' these transformed data to a mean value of 1 by dividing each value by the mean for the block and age in which it was found. This second transformation creates a very strong negative relationship between the mean and the variance. For Poisson-distributed random data, over a wide range of mean values (from 0.1 to 50), variance of standardized values increases as true mean decreases, approximately following the relationship: variance (of standardized value) is approximately equal to 1/2 µˆ(-5/4), where µ is the mean reproductive success. Thus, the increase in variance components observed by Hughes et al. may have been an artifact of their transformation process.

Fortunately, the other main comparison in support of MA is not affected by this issue. By comparing the pattern of decline in reproductive success between inbred and outbred flies, the authors demonstrated a marked increase in inbreeding load with age, consistent with MA.

In light of these results, Hughes et al. argue that their data provide strong support for the MA theory of aging, and little or no support for the AP theory, at least in this population. This is certainly supported by the inbreeding results, though we may need to await further analysis, in my opinion, before we can be certain about the variance component analysis.

So what are we to conclude from this study? Is AP an aged theory?

Given previous evidence in favor of AP, and now the accumulating data supporting MA, it is likely that both forces play an important role in the evolution of aging. Why might Hughes et al.'s study have found little evidence for AP? Hughes et al. analyzed reproductive success, whereas previous studies have focused on survival rates. It may turn out, as experiments by Rose and his colleagues have shown, that the genetic architecture of aging differs among traits. Perhaps reproductive traits are more likely to be influenced by MA, while survival is affected by AP. This question is likely to be a continuing focus of research in the coming years. In answer to the question of which theory explains the evolution of aging, if I were a betting man, I'd put money down on both.

I would like to alert you of a series of Neurology articles and communication arising on the role for cholesterol and lipids in Alzheimer's disease and other neurodegenerative diseases [ 1 ].

I particularly enjoyed July 22, 2003 article by Pappolla et al. [ 2 ] that adds important additional information to this hot subject of the biomedical research. This article concludes that "serum hypercholesterolemia may be an early risk factor for the development of AD amyloid pathology."

I feel obliged to extend the discussion of the study by Pappolla et al. by highlighting the missed point. While many scientists continue to believe that amyloid beta protein is "a central pathologic feature of brains with AD" I feel convinced that it is an essential brain chemical [ 3 ].

I am glad that recent articles in two premier neuroscience journals, Journal of Neuroscience and Neuron [ 4 ], add to my confidence. The Journal of Neuroscience article reported "evidence suggesting that loss of endogenous amyloid beta by the pharmacological inhibition of amyloidogenesis results in a severe reduction in the viability of central neurons. In three different neuronal phenotypes, the pharmacological knock-down of amyloidogenic secretase activity resulted in cell death. This study further supports a key physiological role for the enigmatic amyloid beta peptide" while Neuron article reports on the function of amyloid beta at synapse, and that neuronal activity modulates the formation and secretion of amyloid beta peptides in the hippocampus.

Our own research suggests that amyloid beta protein is a functional player in an activity-dependent cholesterol neurochemical pathways and in synaptic structure-functional plasticity [ 5 ]. It also supports our proposed hypothesis that the change in amyloid beta biochemistry in Alzheimer's disease and related disorders is a functional (but NOT pathologic [ 5 ]) compensatory phenomenon aiming to counterbalance impaired cholesterol dynamics and associated break of neurotransmission and synaptic plasticity. Such cholesterol mediated failure of synaptic function and neural degeneration in our view may represent the primary cause of the major sporadic form of Alzheimer's disease [ see scheme in Ref. 5 ].
 

Sincerely,

Alexei Koudinov, MD, PhD
neuroscientist and editor
http://anzwers.org/free/neurology
http://neurobiologyoflipids.org

Footnote: This letter is intended for researchers in all fields of biomedicine (including Neurosciences, lipid research, neurodegeneration and Alzheimer's disease). I will appreciate alerting your colleagues about Neurology articles [ 1, 2 ] and this letter. Inform a colleagues form is provided. This letter was originally prepared  as Neurology correspondence . The link for the Newsweek (July 14, 2003; July 28, 2003 International edition) cover story on cholesterol is provided below.

Competing financial interests: I do not have any competing financial interest. I aim free information dissemination and an unbiased development of Alzheimer's neuroscience. I observe the Society for Neuroscience  Guidelines for Responsible Conduct Regarding Scientific Communication. This comment represents my personal view. The patent of one of the authors of the Neurology article under discussion [ 2 ] may indicate competing interest.
 

References:
 

1. Engelhart MJ  et al. Diet and risk of dementia: Does fat matter? The Rotterdam Study. Neurology 2002 59: 1915-21 [ PubMed ] [ Letter to the editor ]; Jaworska-Wilczynska M et al. Three lipoprotein receptors and cholesterol in inclusion-body myositis muscle. Neurology 2002 58: 438-45 [ PubMed ] [ Letter to the editor ]; Launer LJ et al. Cholesterol and neuropathologic markers of Alzheimer's disease: a population-based autopsy study. Neurology 2001 57: 1447-52 [ PubMed ] [ Letter to the editor ]; Simons M, Keller P, Dichgans J, Schulz JB. Cholesterol and Alzheimer's disease: Is there a link? Neurology 2001 57: 1089-93 [ PubMed ] [ Letter to the editor ]; Fassbender K et al. Effects of statins on human cerebral cholesterol metabolism and secretion of Alzheimer amyloid peptide. Neurology 2002 59: 1257-58 [ PubMed ].

2. Pappolla MA et al. Mild hypercholesterolemia is an early risk factor for the development of Alzheimer amyloid pathology. Neurology 2003 61: 199-205 [ PubMed ].

3. Koudinov AR. Amyloid was never clearly implicated in Alzheimer's disease, so look at Abeta from a different angle. BMJ.  (Nov 30, 2002) [ FullText ] [ Author Related Articles and Scientific Correspondence ].

4. Plant LD et al. The production of amyloid-beta peptide is a critical requirement for the viability of central neurons. J Neurosci. 2 July 2003 23: 5531-5 [ PubMed ]; Kamenetz F et al. APP Processing and Synaptic Function. Neuron 27 March 2003 37: 925-37 [ PubMed ] [ Related Correspondence ].

5. Koudinova NV, Kontush A, Berezov TT, Koudinov AR. Amyloid beta, neural lipids, cholesterol and Alzheimer's disease. Neurobiol Lipids. 1, 6 (2003) [ FullText ].
 

cc: Nobel Foundation, editors of 50+ neuroscience journals, Alzheimer’s neuroscientists worldwide

Editor,

I do not agree with (rather then doubt) your commentary [1] statement (associated with featured Neuron article of May 22, 2003 entitled “Antibodies against b-amyloid slow cognitive decline in Alzheimer's disease”[2]) that i) "the effects of antibody production are impressive” and that ii) “the findings presented are important in providing further evidence for the validity of the prevailing working hypothesis, the Amyloid Cascade Hypothesis”. The doubt is implied in the opinion of a top-level Alzheimer’s researcher David Holtzman [3], and editor-in-chief of the top-level American experimental biology FASEB Journal Vincent Marchesi [4].

Dr. Holtzman found that “the title and some of the conclusions of this study are not yet justified” [3]. He specifies that “the plasma and CSF Ab levels are not interpretable with the technique used here.” Dr. Marchesi is “concerned about the different results that are reported for the ELISA tests and the authors' tissue amyloid plaque assay” (TAPIR) [4]. Dr. Marchesi asks: “Can we rule out some nonspecific immunological reactions that cause improvement independent of the ability of the antibodies to bind to Ab?” No, we can not. Moreover, I did not find experimental evidence that TAPIR assay detects specifically Ab, not some other constituent of the amyloid plaque [2, 5]. Many studies showed great number of proteins in amyloid plaques (while amyloid beta deposition was reported in a number of human disorders including cardio-vascular pathology and several other neurodegenerative diseases [6]). Therefore, biological effects of Ab (that Hock et al. [2] as well as Winblad and Blum [1] are blindly ignoring [7]) may trigger some biochemical changes [8, 9] and cause “nonspecific immunological reactions” [4].
 

I would call Neuron article [2] and associated commentary [1] a bias in favor of the expired amyloid dogma-based Alzheimer’s therapy approach in case of a confidence of the authors' conflict of interest. Zurich group article acknowledgment claims that there is no conflict: “the authors declare that they have no competing financial interests related to Elan/Wyeth-Ayerst.” Prof. Dr. Roger Nitsch institutional web site [10], however, indicates that his research is funded by “AHP-Wyeth-Elan", a reasonable situation for a researchers performing clinical study “to the pharmaceutical companies ELAN and WEYTH multi-center vaccination trial of Alzheimer's disease” [11]. Dr. Nitsch also chaired the symposium on Alzheimer’s disease on July 15, 1999 during the IBRO Congress 1999 in Jerusalem [12]. This symposium was “kindly supported by Elan Pharmaceuticals, NeoTherapeutics, Parke Davis”, indicating a situation, fitting the conflict of interests definition by BMJ [13], a major general medicine journal.
 

The above indicates that there is a conflict by a senior author of the Neuron article by Hock et al. [2]. If so, what is the reason of the dishonesty and breaking the cardinal tenet of academic science to make such conflict public and let readers decide whether the article “speaks for the science or for the company” [14] ?

Neuron readers also must know about the conflict associated with the key reference of the article by Hock et. al. This is “The National Institute on Aging, and Reagan Institute working group on diagnostic criteria for the neuropathological assessment of Alzheimer's disease” [15], a Neurobiol Aging publication that has important follow up competing financial interest disclosure in The Wall Street Journal article “Did ties to Alzheimer's test maker sway NIH report ?” [16] and in the Boston Phoenix article “Science for sale: a Harvard researcher stands to profit from a product he "independently" reviewed for the National Institutes of Health” [14]. It was also covered as Nature Medicine reports [17].

The above is tightly linked with my earlier correspondence with Neuron requesting editorial investigation and disciplinary action to punish Dennis J. Selkoe, (a Harvard professor, recent member of the NIH National Advisory Council on Aging, Athena founder and Elan director [14, 18, 19]), apparent non disclosure of competing financial interests in prior Neuron publication, and while serving Neuron editorial board member [20]. You should be in receipt of my letter emailed to you and Emilie Marcus [21] on August 19, 2002. Shortly thereafter I received a reply from Neuron senior scientist Stacie Weninger. Dr. Weninger wrote to me: “I wanted to thank you for bringing this matter to our attention. We take these issues seriously, and we will look into the matter further” [21]. Since then Neuron did not come to any action [that I expected to be] commensurate with the pattern of Dr. Selkoe misconducting academic nondisclosure dishonesty [22]. Moreover, February 2003 Neuron article by Sharon et al. [23] again hided D. Selkoe (a senior author) competing financial interest, disclosure that is required by the academic ethics and the uniform requirements for the manuscripts submitted to the biomedical journals [24].

For a non disclosure of a competing interest in April 18, 2003 Science report on amyloid oligomers (a brand-new Alzheimer's disease culprit substituting amyloid plaque in the pathogenesis scheme by the amyloid cascade proponents [25]) please see Ref. 26.
 
 

Sincerely,

Alexei Koudinov, MD, PhD
neuroscientist and editor
http://anzwers.org/free/neurology
http://neurobiologyoflipids.org
 

Competing financial interests: I do not have any competing financial interest. I aim free information dissemination and an unbiased development of Alzheimer's neuroscience. I observe the Society for Neuroscience  Guidelines for Responsible Conduct Regarding Scientific Communication.
 

References:

1. Winblad B, Blum KI. Preview: Hints of a Therapeutic Vaccine for Alzheimer's?  Neuron. 38, 517-8 (22 May 2003) [ PubMed ][ SAGE KE Record ].

2. Hock C, et al. Report: Antibodies against b-Amyloid Slow Cognitive Decline in Alzheimer's Disease Neuron. 38, 547-554 (22 May 2003) [ PubMed ][ SAGE KE Record ].

3. Holtzman D, ARF advisor. ARF Commentary on [2]. Alzheimer Research Forum. Published online May 21, 2003 [ FullText ][ Associated ARF news item ].

4. Marchesi V, ARF advisor. ARF Commentary on [2]. Alzheimer Research Forum. Published online May 21, 2003 [ FullText ][ Associated ARF news item ].

5. Hock C, et al. Generation of antibodies specific for beta-amyloid by vaccination of patients with Alzheimer disease. Nat Med. 8, 1270-5 (2002). [ PubMed ].

6. Koudinov AR, Berezov TT, Koudinova NV. Alzheimer's amyloid beta and lipid metabolism: a missing link? FASEB J.  12, 1097-9 (1998) [ PubMed ]; Koudinov AR, Koudinova NV, Beisiegel U. Cholesterol homeostasis failure at neuromuscular junctions and CNS synapses: a unifying cause of synaptic degeneration ? Neurology online.  (Feb 26, 2002) [ FullText ].

7. Koudinov AR. Amyloid was never clearly implicated in Alzheimer's disease, so look at Abeta from a different angle. BMJ online (Nov 30, 2002) [ FullText ].

8. Kamenetz F,  et al. APP processing and synaptic function. Neuron. 37, 925-37 (2003) [ PubMed ] [ Abstract at Neuron ] [ Related correspondence ].

9. Koudinova NV, Kontush A, Berezov TT, Koudinov AR. Amyloid beta, neural lipids, cholesterol and Alzheimer's disease. Neurobiol Lipids. 1, 6 (2003) [ FullText ].

10. Prof. Dr. Roger Nitsch. Neuro Science Center Zurich - Research Groups. University of Zurich web site. Last time viewed: May 26, 2003 [ FullText ].

11. Vaccination study for Alzheimer's disease (14.10.02). Medieninformation. University of Zurich web site. Last viewed: May 26, 2003 [ Acrobat .PDF FullText ] [ HTML Cache ].

12. Symposium on Alzheimer’s disease. Scientific Programme for 15 July 1999. IBRO Congress 1999, Jerusalem, Israel [ HTML Cache FullText ].

13. Smith R. Beyond conflict of interest. BMJ. 317, 291-92 (1998) [ FullText ] [ BMJ declaration of competing interest form ].

14. Ready T. Science for Sale: A Harvard researcher stands to profit from a product he "independently" reviewed for the National Institutes of Health. The Boston Phoenix. (29 April 1999) [ FullText ].

15. The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease. Consensus recommendations for the post-mortem diagnosis of Alzheimer's disease. Neurobiol. Aging.18, S1-S2 (1997) [ PubMed ].

16. Waldholz M, King RT, Jr. Did ties to Alzheimer's test maker sway NIH report? The Wall Street Journal. (30 Nov 1998) [ FullText ].

17. Editorial. Taking more interest in conflict. Nat Med. 5, 713 (1999)  [ PubMed ] [ FullText ]; Birmingham K, Ready T. Conflict-of-interest problems lead to policy changes. Nat Med. 5, 717-8 (1999) [ PubMed ] [ FullText ].

18. Crowley D. Elan Alzheimer's expert in pre-slump share sale. The Sunday Business Post. (18 Aug 2002) [ FullText ]; Koudinov AR. Ethical conundrums: an Alzheimer's case. BMJ. Published  online Sept 12, 2002 [ FullText ]; Koudinov AR correspondence with American Academy of Neurology, Nov, 2002 - March, 2003 [ FullText ].

19. Koudinov AR. Open letter to Public Citizen's Health Research Group on Alzheimer's disease research. BMJ online. (Feb. 21, 2003) [ FullText ]; SAGE KE (Feb. 21, 2003) [ FullText ].

20. Selkoe DJ. Biography. ISI HighlyCited.com. Last updated August 15, 2001 [ FullText ].

21. Stacie Weninger response on Alexei Koudinov letter of August 19, 2002 (27 August 2002) [ FullText ].

22. Introduction. Handling Misconduct. Office of research integrity web site [ FullText ].

23. Sharon U, et al. The formation of highly soluble oligomers of a-synuclein is regulated by fatty acids and enhanced in Parkinson's disease. Neuron. 37, 583-95 (2003) [ PubMed ].

24. Uniform requirements for manuscripts submitted to biomedical journals. International Committee of Medical Journal Editors. Med Educ.  33,  66-78 (1999) [ FullText ].

25. Robinson SR, Bishop GM, Munch G. Alzheimer vaccine: amyloid-beta on trial. Bioessays. 25, 283-8 (2003) [ PubMed ].

Editor ^$
JAMA
515 N State St,
Chicago, IL 60610
 

Over the past decade amyloid hypothesis for Alzheimer's disease nor provided an explanation for the pathogenesis, nor yielded the disease cure [ 1 ].

Likewise, there is no understanding for the difference in the body fluid ratio for the isoforms of the normal soluble Ab peptide, in particular for the major Ab1-40 and present in a trace quantities Ab1-42 [ 1 ].

On the other hand research by many groups provided solid evidence that in the CSF as well as in plasma Ab1-40 is associated with high density lipoproteins (HDL) (reviewed in Ref. 2). This observation integrates the association "for increasing late-life HDL cholesterol (HDL-C) levels and an increasing number of neocortical NP" [ 3 ] (neuritic plaques, composed of Ab and many other constituents, including cholesterol, and present in a number of neurodegenerative diseases and in cardio-vascular pathology, not just in Alzheimer’s [ 2 ]), thus futhering a discussion of the role for cholesterol proper in Alzheimer's disease and a role for Ab in cholesterol dynamics and lipoprotein functional biochemistry [ 2 ].

Importantly, Ab association with lipoproteins has emerged as a key factor in arresting Ab toxicity, a fact missed in the recent experimental articles on oligomeric Ab  [ 4 ], a brand-new Alzheimer’s disease culprit substituting amyloid plaque in the pathogenesis scheme by the amyloid cascade proponents [ 1 ].

The above i) explains why it would be critical for the true disease understanding not to limit the JAMA study by Sunderland et al. to CSF Ab1-42 quantification, and to extend it with the evaluation of the CSF Ab1-40 [ 5 ]; and ii) doubts the appropriateness of the selection of the essay "designed specifically to measure the human b-amyloid 1-42 peptide in CSF in a 10 x excess background of 1-40" despite of the bequest of the Consensus report of the working group on: "molecular and biochemical markers of Alzheimer's disease", a key citation of the JAMA study [ 5 ].

With regard to this consensus report there is a need to inform readers of the missed in JAMA [ 5 ] post-publication disclosure of the competing financial interests by the chairman and the members of the working group advisory committee [ 6, 7, also see Refs. 8, 9, 10 ].
 

Sincerely,

Alexei Koudinov, MD, PhD
neuroscientist and editor
http://anzwers.org/free/neurology
http://neurobiologyoflipids.org

Footnote: The earlier 312 words 5 citations version of this letter was submitted to JAMA  (Letter # JLD30273, July 1, 2003; rejected July 12, 2003).

Competing financial interests: I do not have any competing financial interest. I aim free information dissemination and an unbiased development of Alzheimer's neuroscience. I observe the Society for Neuroscience  Guidelines for Responsible Conduct Regarding Scientific Communication.
 

References:

1. Robinson SR, Bishop GM, Munch G. Alzheimer vaccine: amyloid b on trial. Bioessays.25, 283-8 (2003) [ PubMed ] ; Koudinov AR. Amyloid was never clearly implicated in Alzheimer's disease, so look at Abeta from a different angle. BMJ.  (Nov 30, 2002) [ FullText ].

2. Koudinov AR, Berezov TT, Koudinova NV. The levels of soluble Ab in different HDL subfractions distinguish Alzheimer's and normal aging CSF: implication for brain cholesterol pathology? Neurosci Lett. 314: 115-118 (2001) [ PubMed ] [ Author Related Articles and Scientific Correspondence ]; Koudinova NV, Kontush A, Berezov TT, Koudinov AR. Amyloid beta, neural lipids, cholesterol and Alzheimer's disease. Neurobiol Lipids. 1, 6 (2003) [ FullText ].

3. Launer LJ, White LR, Petrovitch H, Ross GW, Curb JD. Cholesterol and neuropathologic markers of AD: a population-based autopsy study. Neurology. 57, 1447-52 (2001) [ PubMed ] [ Correspondence ].

4. Koudinov AR. Alzheimer's amyloid beta oligomers versus lipoprotein Abeta. Science SAGE KE. (May 1, 2003) [ FullText ].

5. Sunderland T, Linker G, Mirza N, et al. Decreased b-Amyloid 1-42 and Increased Tau Levels in Cerebrospinal Fluid of Patients With Alzheimer Disease. JAMA. 289, 2094-103 (2003) [ PubMed ].

6. Growdon JH, Nitsch RM, Wurtman RJ, inventors; Massachusetts Institute of Technology, assignee. Antemortem diagnostic test for Alzheimer's disease. US patent  5,631,168. 1997 May 20 [ USPTO Record ].

7. Thies B, Truschke E, Morrison-Bogorad M, Hodes RJ. Consensus report of the Working Group on: molecular and biochemical markers of Alzheimer's disease. Letter to the editor. Neurobiol Aging.20, 247 (1999) [ PubMed ] [ Article under discussion Neurobiol Aging. 19(2), 109-16 (March-April 1998) [ PubMed ]; Erratum in: Neurobiol Aging. 19(3), 285 (May-June 1998) ].

8. Koudinov AR. Open letter to Donald Kennedy: AAAS, Science, Alzheimer’s disease and academic dishonesty. Science SAGE KE. (June 16, 2003) [ FullText ] ;

9. Barinaga M. Confusion on the cutting edge. Science. 257, 616-8 (1992) [ PubMed ] ;  Koshland DE Jr. Conflict of interest policy. Science. 257, 595 [ PubMed ].
 
 

In September 1991 four men and four women entered the Biosphere 2, a 3+ acre ecological chamber containing miniature biomes (rain forest, savannah, desert, ocean, marsh, agricultural station, and living quarters), with the express purpose of remaining completely self-sufficient over a two-year stay. It quickly became apparent that the "Biosphereans" were not going to be able to produce as much food as they had anticipated, and so began the only long-term experiment in human caloric restriction with a nutrient-dense diet.

Roy Walford, one of the Biosphereans, and colleagues have now published the analysis of blood and urine samples taken at intervals during the entire experiment, as well as similar measures from before and after, so that these these results could be compared to similar measures in laboratory rodents (in which caloric restriction clearly slows aging) and monkeys (in which the effects of caloric restriction on aging are not yet established).

The first thing to note is that there are substantial differences between the human and animal studies. First of all, the humans were highly variable in size hence were calorically restricted to various degrees in that they all consumed the same diet, which ranged from about 1750 to 2100 kcal/day over the course of the experiment. Thus, the subjects were calorically restricted to varying degrees. In fact, the largest person lost nearly one-third of his body mass over the first six months of the experiment while the smallest lost about 11% of hers. The body mass index of some of the individuals even reached levels that would be consistent with anorexia nervosa in a clinical setting. Second, the atmosphere in the Biosphere did not remain at equilibrium, so that at times the oxygen levels were equivalent to living at 14,000 feet elevation. And finally, unlike laboratory animals, the Biosphereans were forced to assume a brutal work (i.e. exercise) level – 70-80 hour work week, often entailing heavy manual labor.

Several aspects of the results are of note. The Biosphereans were in excellent health at the beginning of the experiment, with blood pressures averaging 108/77 mm Hg and total blood cholesterol of about 190 mg/dL. These values (as previously reported for the first six months of the experiment) dropped even further during the experiment, but notably returned to pre-experiment levels within eight months after the experiment ended. Many of the physiological changes observed were consistent with both rodent and monkey experiments, although the interpretation of these changes aren’t always obvious. For instance, total white cell count fell by nearly one-third, which is consistent with rodent and monkey experiments, but lowered white cell count is also a predictor of increased mortality probability in the elderly. So whether the Biosphereans were immunostimulated or immunosuppressed isn’t clear. Also, somewhat surprising is the fact the women did not miss menstrual cycles nor did the men exhibit declines in testosterone. A robust results of caloric restriction in rodents is a suppression of reproductive ability, which did not seem to occur in these humans. One interpretation would be that even at this level of caloric intake, the humans were not subjected to anything like the degree of restriction in most rodent studies. The last intriguing result is that neither growth hormone, nor insulin-like growth factor-1 (IGF-1) was significantly altered, which is in contrast to most rodent studies. In fact, reduced IGF-1 is now a primary candidate for a causal hormonal change associated with extended life, since it is observed universally under caloric restriction in rodents, and also in all four genetically-altered dwarf mice which live up to 50% longer than controls.

No one has ever faulted Roy Walford for cowardice, either personal or scientific. A career that starts in the professional theater, moves to the fore of research on immunological aging, pioneers the rediscovery of caloric restriction as an essential strategy for investigation of the aging process, and then takes a leave of absence from our home planet's biosphere can hardly be considered typical, or recommended to the faint of heart. Walford's 1967 monograph on the aging immune system was the first hardcover work I purchased on biogerontology, and his magisterial review, with Rick Weindruch, on the effects of caloric restriction is the book I take down to consult most frequently. Tired (I suppose) of the vicissitudes of academia, sated (one can assume) with documenting the palatability of low calorie, high broccoli diets and the effects of radical tonsure on body temperature, and proud (we conjecture) to see hoards of new scientists prying apart the calorie restricted rodent he helped to bring to prominence, Roy in 1991 locked himself up for two years in his own semi-private biosphere, at age 67 the one quasi-geriatric outlier in a crowd of 8 astronaut explorer types and an unexpectedly abundant menagerie of cockroaches, from which he has now emerged, much thinner, with the first comprehensive data set in which to explore the effects of long-term caloric restriction on human physiology.

His newly published paper (J. Geronotol. Biol. Sci. 57A:B211-B24, 2002) provides much to ponder and to admire. By mischance, the Biospherians found themselves with a good deal less to eat than they would have wished to eat given their druthers, and (like the average CR rodent) no way to do anything about it. They spent the next two years doing more hard physical work than your average reader of the Journal of Gerontology, and eating a nutritionally adequate, low fat, mostly vegetarian diet containing about 70% - 80% of their pre-entry caloric intake. Each of the eight volunteers lost about 15% - 21% of their starting body weight, maintained this weight, perforce, until they emerged two years later, and then promptly regained the weight they'd lost, all the while donating tubes of blood as frequently as most of us check our e-mail. Although the Biosphere experiment had not originally been planned as a model for human caloric restriction, Walford and his colleagues quickly realized, in medias res, that they had been offered a serendipitous opportunity to monitor the effects of longish-term restriction on a wide range of physiological and (particularly) blood chemical indices, and took full advantage of their good luck to collect and analyze serial blood samples taken throughout their confinement and for several years thereafter. Analysis of these materials, and the accompanying data on blood pressure, heart rate, temperature, and related clinical information, make up the bulk of this valuable compilation.

Were this paper merely a data dump of this unprecedented set of measurements, it would still be of high scientific interest; but Walford and his colleagues provide many bonuses: an innovative statistical approach that squeezes as much information as possible from the low number of independent subjects, a very valuable table comparing the results with analogous studies of monkey and rodent CR and with the few comparable human studies, and a cogent, focused discussion of the implications of the new findings with respect to postulated mediators of the CR-longevity association, such as alterations in glucose and insulin levels, alterations in hormones on the GH/IGF axis, blood lipids thought to protect against specific forms of cardiovascular illness, and effects on reproductive endocrinology. It is noteworthy that the CR regime did not interrupt the menstrual cycle of any of the four women volunteers, potentially good news for those who might wish to delay pregnancy until their sixth or seventh decade through dietary manipulation of aging rate. The before-and-after pictures of the senior author, in the style of Richard Avedon, should make reprints of this paper highly sought after as collector's items.

This is not a flawless study. If one wished to design such a study from scratch, the design would allow disentanglement of the effects of low caloric intake from the potential confounders of hard labor, low fat intake, and the psychological stresses of moving to new accommodations with new companions and new job assignments and unfamiliar food choices, heavy on the papaya and light on the Fritos. (Such a study would elicit an amused chuckle at the local IRB.) On the other hand, it is not a study likely to be repeated until NASA starts to ship tourists to Mars. I suspect that Roy Walford plans to be in the front of the line for Mars tickets, vacutainer kit in hand. In the meantime we should be grateful for this optimally informative exploitation of a lucky windfall.

I read with interest the articles on Alzheimer's disease and amyloid beta protein in the August 1, 2003 issue of The Journal of Clinical Investigation (JCI). These are articles by Monsonego et al. [ 1 ] and by Eriksen et al. [ 2 ]. Additional "insight into research articles" is provided in the accompanying commentary by Cirrito and Holtzman, in the issue cover image, and in two associated EurekAlert news reports [ 3 ].

The above contribution series in my view represent the road show (a term used in Nature Medicine citation, see below) built on a promise for amyloid beta pathogenic primacy in Alzheimer's disease, a well overdue amyloid hypothesis, and an expired approach to treat Alzheimer's by tackling brain b-amyloid.

What all three articles miss is a single mention that amyloid b protein is an essential brain chemical. This was covered in details in the contribution published elsewhere [ 4, 5, 6, 7 ].

The major flaw of the reported Alzheimer's research is the failure of the article by Monsonego et al. to provide a true competing financial interest declaration for senior authors, Dr. Dennis J. Selkoe and Dr. Howard L. Weiner. The Monsonego et al. article footnotes’ conflict of interest disclosure says that "the authors have declared that no conflict of interest exists." This statement does not seem to be true.

An early report on D. Selkoe financial conflict of interest come from the Science magazine issue of July 31, 1992 [ 8 ]:
 

We later learn that Dr. Selkoe is "a Harvard researcher [who] stands to profit from a product he "independently" reviewed for the National Institutes of Health", and that Dr. Selkoe is an "Elan Alzheimer's expert in pre-slump share sale" [in 2002] [ 10, 11; also see Ref. 9, 12 ].

Two minute search with a web search engine for Howard Weiner uncover three reviews [ 13, 14, 15 ] (including the one in Nature Medicine [ 13 ]) for the book by Quinn entitled "Human Trials: Scientists, Investors, and Patients in the Quest for a Cure". Quoting WebMD review [ 14 ]:
 

Further search for "Selkoe+Weiner" links to a story in Harvard Gazette on how D. Selkoe and H. Weiner "decided to fight Alzheimer's with the same protein that seems to cause it", and how "they have successfully treated Alzheimer's disease in mice by putting drops of vaccine in their noses" [ 16 ].  An additional search for "Elan+Autoimmune" uncovers apparently related to JCI article agreement between Weiner's Autoimmune Inc. and Selkoe's Elan, while the latest news are briefed at Autoimmune Inc. page at Forbes magazine Markets section.

The search of USPTO web site further discloses that Dr. Weiner is an inventor of twenty nine US patents [ 17 ]. All 29 patents list "Autoimmune, Inc. (Lexington, MA)" as patents' "Assignee:" (Please note that the total number of  Autoimmune, Inc. patents is 31 according to the USPTO search performed on August 4, 2003).

The above information indicates the break of the Uniform Requirement for Manuscripts submitted to biomedical journals [ 18 ]. The Uniform Requirements state:
 

As Nature Medicine reports in a book review [ 13 ], "Quinn's version of the Weiner-AutoImmune story is not fashioned to examine how corporate interests and funding influence, distort - and maybe even corrupt - the training of research fellows, the agenda of academic researchers, the integrity of research data and the dissemination of research results."

In light of the provided facts I feel that the above definition also does apply to the August 1, 2003 JCI Alzheimer's contribution [ 1 ].