feedback requested on ideas(modified)

Iuval clejan clejan at mindspring.com
Wed Feb 14 13:11:37 EST 2001

I modified the last essay a bit. Is there at least one useful idea or
suggested experiment in here? Come on guys, please give me some
feedback. I would like to know if I should work in this field and if so
I would like to know if you think I could convince someone to work with
me on doing experiments such as the ones suggested in the essay. I am
sure much of what I say is naive or even wrong, that's why I need
feedback. I don't know anyone in the field, being a physicist/electrical

In Search of Fundamental Causes of Aging

Differential expression of a particular protein between young and old
cells occurs as a result of :
1 a mutation to some genes (not necessarily the one coding for that
2 decrease (or halting of) replicative capacity or stoping replication
due to e.g. tellomere shortening and lack of tellomerase, and/or
commitment away from stem cells to a more differentiated form.
3 the cell's environment of nutrients and hormones is changed, perhaps
due to other cells (which produce hormones or metabolize nutrients that
go to this cell and the extracellular matrix) experiencing 1 or 2 above.

It is unlikely that aging in all cell types can be explained solely on
the basis of decreased replicative capacity (2) because many organs
which do not contain replicating cells in any significant amount (e.g.
heart and brain), exhibit decreased function with age. Moreover,
ulimately (in a certain organism after maturity) replication is a means
of damage repair, and since another kind of damage repair also happens
in the absence of replication, failure of  this other kind of damage
repair must also be involved in aging.
Decreased replicative capacity of cells in other organs could
indirectrly affect non proliferating cell organs (3). It would be
interesting to determine for various cell types if the population of
stem cells is reduced as a function of age, or if the stem cells simply
stop replicating. In the second case, gene therapy may be used
(introducing hTRT selectively into stem cells) to extend lifespan, with
the associated risks of increased cancer. As far as I know, there is no
study showing decreased replicative capacity in vivo correlating with
decreased organ function for normal aging or premature aging diseases.
In vitro, some studies have found a (inverse) correlation between cell
donor age and replicative capacity, but some (the ones that used better
controls in my opinion) did not.

Gene therapy for improved damage repair (by mechanisms other than
replacing the cell through replication) seems to be more promising than
gene therapy involving stimulation of replicative capacity. Some work in
this area has already started (targeting some repair enzyme normally
found only in the nucleus to mitochondria).

For the reasons above I will focus on mechanism (1) in this essay.

If hormones/nutrients/extracellular matrix change due to decreased
replicative capacity of some cells (3), then the change should be
artificially induced in vitro to see if other cell types (e.g. heart or
brain) react to that change by aging more quickly.

I  Continuous vs binary expression of proteins:

The experiments that have been done so far on differential expression
between old and young tissues (or senescent and young cells for in vitro

systems) mostly look at relative amount of mRNA from a large population
cells of the same type (One purported exception that I need to double
check is  albumin expression in certain liver cells which supposedly
shows continuous changes in expression in each cell, but I don't think
as a function of age, just developmental stage at one time). This does
not allow to distinguish between
mutations that result in a binary (high/low) expression of proteins, vs
mutations that result in a continuous expression of proteins. Such a
distinction is important because if it is determined experimentally that

the continuous class correlates with aging (in a particular cell type,
or even better, in all cell types) then many mechanisms are eliminated
from candidacy for root causes of aging. Actually I think there are
very few non lethal mutations that can cause a continuous change in
protein expression per cell. These include (correct me if I'm wrong)
mutations to
1. chromatin coding genes(coding for histones/scaffold
proteins,methylases and acetylases). Chromatin structure can affect mRNA
through both throughput (ease of transcription initiation by RNA
polymerase and transcription factors) and possibly rate (I think RNA
polymerase might interact with histones, not just DNA, as it moves
along the DNA).
2. enhancer or promoter proximal DNA (TATA, CG and others). Effects on
ease of transcription initiation.
3. hnRNP coding genes, which may be able to function at lower efficiency

in transport out of nucleus and preventing secondary structures from
hapenning if only a few are missing.
4. rRNA coding DNA, since there are many copies of this gene, not just
two per diploid cell. Also, a ribosome may be defective and translation
can still proceed, albeit at a lower rate (?)
5. mtDNA, since there are many? (how many) mitochondria per cell.
6. nDNA coding for mtDNA repair and/or replication enzymes (same reason
as 5).

If the result of the experiment is that differential  protein
expression is binary, one learns less since most of the above mutations
can also cause binary expression (e.g. the histone may not be produced
at all). It would be good to induce each of these separately and
intentionally and see the effect on expression of the protein being
studied, to verify that indeed they produce continuous changes. Actually
I don't think  (if aging occurs because of DNA damage) that protein
expression can vary discontinuously in each cell because, as I show in
the next section, unrepairable damage in non proliferating cells can
only occur by knocking out not only a certain gene coding for a certain
protein, but all repair pathways for that type of damage, and then other
genes that are essential for cell viability will become unrepairable.
But this would mean that unless one of the six cases above occurs, lots
of cells would start dying with age, not just stopping expression of a
few proteins, which is not observed (Is it not true that the cells
producing white hairs are not dead? Or the fibroblasts  producing either
decreased or no collagen are not dead?) . Of course this is a matter of
timescales, as continuous change may also eventually result in cell

Now the question is how does one look at protein expression from 1
cell at a time, and one answer is PCR, but for a protein (whose gene has

already been sequenced so that primers can be obtained) known to be
differentially expressed between young and old cells, such as a collagen
or interferon gamma. This can be done for several different tissue
types. It would be better to do this for hair producing cells which have

(I think) an easily identifiable (single cell specific) phenotype which
differs between young and old ( i.e. a bald spot, or a white hair). One
would look primarily for melanins in the case or greying of hair(?).
FACS can also be used, but I think this is limited to proteins which
bind to the cell membrane. When it was done for albumin, the result was
continuous change in expression, supporting the above arguments (but I
think it was done at one time, not as a function of time, looking at
differences between stem and more differentiated cells).

If aging occurs as a result of changes in nutrients or hormones then it
can also be either abrupt or continuous. Abrupt change might be ruled
out for the arguments above. Continuous change in hormone supply will
cause abrupt change in a cell's protein expression unless it affects one
of the six gene classes above (a hormone may come into the cell at
various time intervals and in various doses but at any one time when the
cell is measured, it would either signal transduce and turn on
transcription of genes or not). If we look at the CAUSE (as opposed to
the effect) of continuous change in hormone production then we don't
learn much because it can be due to abrupt changes in protein production
at the cell level which are continuous on the population level (e.g.
some cells are senescent/non proliferating and some not), not just
continuous changes in protein production .

II DNA repair in replicating vs non replicating cells

Although DNA repair is less efficient in non replicating
cells, there is more time to do it before something is no longer
recognizable as damage (as Syndey Shall pointed out), which is what
happens in replicating cells that
actually fixate a mutation. In a non replicating cell, the only way to
knock out a gene with random damage (due to toxic environmental or
metabolic oxidant species) is if there is only 1 (or no) repair pathway
for that
type of damage (or one enzyme shared between two or more pathways), and
if a gene coding for an enzyme in that pathway (or the shared
enzyme)also gets the
same type of damage. Redundancy makes it almost impossible to knock out
a gene. First consider the simple situation in which different repair
pathways do not share enzymes. Consider a simple example with genes A, B
and C which code for
repair enzymes which can repair damage of type 1,2 and 2 respectively
(with the help of other proteins).
If gene D (which produces some protein necessary for cell function) gets
type 2 damage, (we'll then call it D2), then it can be
repaired by either E2B or E2C (the enzyme from gene B or the enzyme from

gene C). If we have either A2,B2,C2 or D2, then still no problem since
there is still E2 available (either E2B or E2C) to repair the damage. It

is extremely unlikely that we have simultaneous B2 and C2 (simulataneous

meaning B2 happens before E2C has a chance to repair it).

Also no problem if D1 happens, since it can be repaired by E1A. However,

if A1 happens, then it can't be repaired, and eventually D1 will happen
and not be repaired. Unrepairable damage (the no E case above) is also
possible, but I think it is unlikely to be related to aging, because if
unrepairable damage happens to a gene that produces a protein needed for

cell viabilty that cell will die, and the random death of a population
of cells does not have much to do with aging.

Also in the case where an enzyme is essential for two repair pathways,
and if those two pathways repair the same type of damage and no other
repair pathway exists for that type of damage, it is possible to knock
both of these out with a single mutation (of the same type as the repair
pathways) to the gene coding for that enzyme, and in that case
redundancy doesn't help. So we should also look for enzymes that are
shared between repair pathways, as these are candidates for
aging-relevant mutations.

Now the clincher: for diploid organisms, even if there is only 1 repair
pathway (or 1 shared enzyme) for a specific damage type, there are  2
genes coding for it, one
on each chromosome, with the exception of the X for males and females,
and the Y for males. My guess is that there is no repair enzyme coding
genes on Y chromosomes (females would
live shorter lives than males). Indeed, a repair exonuclease has been
found on the X chromosome (of humans, I haven't checked other animals
yet). Now the mtDNA may be the only DNA in mammals for which there is
only one repair mechanism for some some types of oxidative damage. So
the gene coding for this exonuclease, if found in mitochondria, may
satisfy the 3 criteria for a gene involved in aging: uniqueness of
repair path (or equivalently, shared enzyme among two or more sole
repair paths), uniqueness of chromosome, and possible continuous
differential expression of affected genes. I don't think it is known if
this exonuclease is active in mitochondria (this should be able to be
determined experimentally). Maybe aging starts with mutation to this
repair enzyme and everything follows from that. I suppose it is
possible that simultaneous mutations though rare, do happen, and this
kind of system could be simulated (by Monte Carlo) to see if it exhibits

the same linear differential expression observed as a function  of time
(although it is not known, as I said in the first section above, whether

there are just more cells being knocked out, or each cell continuously
expresses more or less protein. Experimental resolution of this would
give the simulation something to compare against)

The situation in replicating cells is totally different. There the trick

is to either repair the DNA before a mutation is fixed, or to have a
good apoptosis/selection mechanism and replace the cell (and in the rare
case of a better cell, let evolution select for that cell) . Redundancy
happens at
cellular level, instead of enzyme level (as it might in non-replicating

III. Selection in sperm and egg in order to keep the germline immortal

Presumably the embryo is young because some selection occured in sperm
and/or egg for a cell without (or with less of) the damage that causes
aging (I won't
consider now the other possibility, that the hormones/nutrients which
may be different in an aged individual compared to a young individual,
and cause differential protein expression in most cells, are not
necessay for sperm or egg function).
Selection for good mtDNA may occur efficiently for eggs since a few
mitochondria are
amplified to many in oogenesis (thus widening, or even bimodaling the
fitness distribution) and this has been proposed a while back and is
known as
the mtDNA bottleneck theory. The bottleneck theory does not explain how
or when selection for eggs low in damaged/mutated mitos occurs. An
obvious example is after fertilization, the high damage fetus
spontaneoulsy aborts. Complete speculation: a selection for good
mitochondria may also occur in either meiosis 1 or 2. TEM observations
suggest two mitochondrial populations prior to meiosis 1 (R Jansen and K
de Boer Mol Cell Endoc 145 (1998) 81-88) . Possibly these segregate into
polar body and oocyte ?) and I'm wondering if these are just selected at
random or not. One population appears to be replicating DNA while the
other is not.

By the argument in section
II, it is not sufficient (for aging to occur in non proliferating cells)
to have damaged DNA (mt or nuc), but a unique repair pathway (or unique
shared enzyme in multiple repair pathways) has to be damaged as well. If
this is the case, then selection on good repair genes in eggs (which
will be passed to non proliferating cells) should happen. Also
there is a role for selection on sperm as well, and indeed selection
does happen on things like motility and ability to penetrate the egg,
which are energy dependent (and
hence ATP and good mtDNA dependent). What is selected on sperm is not
mtDNA (which is not passed to the zygote), but nucDNA coding for mtDNA
repair enzyme(s). And by the argument in the previous section it should
be for the part of the X chromosome coding for the exonuclease used in
repair. More wild speculation: possibly selection in the egg occurs also
on the X chromosome during meiotic division 1: the inactive X may have
less damage and be used as a "spare tire" and segregate to the egg (in
duplicate) while the active X chromosome may segregate to the first
polar body. This should also be easy to test experimentally (I realize
that usually inactive genes are repaired less efficiently, but maybe the
Barr X  is an exception).

Another consequence of these hypotheses is that an
in vitro culture of sperm (is it possible to culture sperm for long
periods?)  will have more vigorous (after damage had a chance to happen
and be repaired more for X than for Y) and longer lived X type sperm
to Y type sperm. In vivo I think we do see a slight selection of females

over males, which means that the time constant for damage is longer than

(but same order of magnitude) as the time sperm spend from meiosis to
fertilization (how long is that on the average?)

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