Michael Rae reports from the proceedings at SENS3:

Among the most exciting presentations at the third conference on Strategies for Engineered Negligible Senescence (SENS3) were those in Friday's session on the rescue of mitochondrial mutations. This is a subject to which Conference organizer and Methuselah Foundation Chief Science Officer Dr. Aubrey de Grey has made widely-recognized contributions.

Unlike most other parts of the cell, mitochondria house many of the genes encoding their essential proteins within themselves. These genes are vulnerable to the constant assault of free radicals produced by the mitochondria as a side-effect of their role as cellular power plants. When mitochondrial DNA is damaged, it cannot make the proteins needed to carry on the essential business of generating energy for the cell; the ensuing metabolic damage is the driver of age-related rise in oxidative stress. This oxidative stress fuels free radical damage and interferes with essential signaling pathways in cells far from the original site of the damage.

Dr. de Grey first proposed an 'engineering' solution to this form of aging damage in 1998: the use of allotopic expression -- the creation of 'backup copies' of those genes in the safer confines of the nucleus -- in 1998. In Friday's SENS3 session, several researchers presented their recent work aimed at advancing this and other potentially useful approaches to mitochondrial damage.

PhD candidate Mark Hamalainen of Cambridge University presented the initial success in his Methuselah Foundation-funded work on allotopic expression, showing evidence that his allotopically-expressed genes could encode the relevant proteins and that these were taken up into the mitochondria. In this case, the genes encode healthy and defective versions of the protein that is miscoded in Neuropathy, Ataxia and Retinitis Pigmentosa (NARP), a hereditary mitochondrial disease characterized by blindness and weak and uncoordinated muscles. Well done! It is good to see Foundation-funded research make such solid progress; many thanks go to the generous donors who have made this possible.

Dr. Marisol Corral-Debrinski, of Paris' Quinze-Vingts National Center of Ophthalmology, presented a dramatic step forward with a technique whose effectiveness she had preliminarily confirmed with several allotopically-expressed proteins, that result published in Rejuvenation Research. The technique involves shifting the production site of such proteins closer to the mitochondria themselves, allowing the cell's machinery to thread the proteins through the narrow straits of the mitochondria's import channels as quickly as they are produced. This prevents the proteins from folding back up on themselves, which makes them harder to import. In her recently-published work, she tested her technique by using it to allotopically express several proteins in cells defective for the relevant genes, restoring their ability to generate energy and sustain cellular life. Now Dr. Corral-Debrinski has leapt forward into a living organism, inserting an allotopic version of the defective human gene that causes the mitochondrial disease Leber Hereditary Optic Neuropathy (LHON) into mice retinas. LHON causes blindness in humans, and allotopic expression of the gene caused the same cell loss and abnormal lack of cell communications branching that appears in the disease. Dr. Corral-Debrinski next hopes to take this to the next level, and cure the disease in mice by introducing the healthy gene.

Another way of easing the entry of twisted-up (and thus hard to import) allotopically-expressed proteins is through the introduction of special 'bracing bars' called inteins into the proteins. The inteins hold apart their snarling bends and kinks -- an idea first proposed by Dr. de Grey in a 2000 paper in Trends in Biotechnology. After some preliminary work by Japanese scientists, the University of Zaragoza's Dr. Antonio Enriquez has now picked up the ball, and described his early work with mitochondrial protein inteins during this session.

In addition to the work on allotopic expression, two presentations were given summarizing soon-to-be-published progress on entirely novel ways of overcoming the problem of mitochondrial mutations: the import of whole new mitochondria into the cell by Dr. Volkmar Weissig of Northeastern University. Reviewers initially found his results unbelievable, until he pointed out that it had actually previously been reported - and then forgotten! - by Israeli scientists in 1984. Remarkably, Dr. Enriquez was able to confirm that both he and a Cambridge University lab had independently confirmed this result in recent years, but had never been able to follow through with the additional work required to publish the result in a journal.

Finally, Dr. Samit Adhya of the Division of Molecular and Human Genetics at the Indian Institute of Chemical Biology is pursuing yet another innovative approach. He proposes to dispense with the need for mitochondrial DNA altogether, by instead providing the mitochondrial protein-making machinery directly with the "working instructions" (messenger RNA) that it normally receives in the form of a transcribed copy taken from the mitochondrial DNA originals. This would allow the mitochondria to continue their protein production even if the mitochondrial DNA were completely destroyed: they would still have their marching orders, even if the general himself were incommunicado. Dr. Adhya is accomplishing this goal by borrowing a trick used by a single-celled organism called Leishmania tropica. This organism, unlike mammals, generates another kind of RNA in the main cell body, and uses a specialized protein to move it into the mitochondria. Dr. Adhya reasoned that he could bind copies of our own RNA to the same protein and use it to deliver both kinds of RNA into mammalian mitochondria, bypassing the need for a DNA original. Very clever.

Using a tagging protein, Dr. Adhya first showed that he could in fact move such RNA into mammalian cells. But how to show that it was working? A normal cell already has the needed RNA, so the functionality of such cells would not be improved by adding more. This means that you can't distinguish a cell whose mitochondria has integrated and used such imported RNA to make proteins from one that hasn't.

Like Dr. Corral-Debrinski, Dr. Adhya decided to work backwards as a preliminary test. Instead of delivering more of the functional RNA, Dr. Adhya introduced antisense RNA -- RNA that is designed as a mirror-matched copy of the original, to which it binds and inactivates. He chose antisense RNA that would block the action of the RNA that delivers instructions copied from the gene whose mutations cause a human mitochondrial congenital disorder called Myoclonal Epilepsy with Ragged Red Fibers (MERRF). If Dr. Adhya's protocol could cause mitochondria to take up and translate the antisense RNA, it would interfere with the animals' mitochondrial function in exactly the same way as occurs in the human disease, recreating the muscle-wasting symptoms to which victims are subjected.

While we can't yet draw definitive conclusions, all of Dr. Adhya's results are consistent with success. In some of the most visually arresting presentations of the conference, Dr. Adhya showed how injecting the RIC-linked antisense RNA into the legs of rats quickly caused the same kind of leg muscle degeneration seen in MERRF; when examined under a microscope, muscle cells from such animals showed the death of muscle fibers and the loss of mitochondrial function.

The next step will be to introduce functional RNA into animals with dysfunctional mitochondrial genes. If this restores normal mitochondrial function and blocks the symptoms and pathology associated with the disease, we'll know for sure that the RNA import technology works. This would allow us to sidestep not only the mutations in the mitochondrial DNA of those rare and unfortunate souls who suffer with congenital mitochondrial diseases, but those responsible for the universal mitochondrial failures of aging.

We live in exciting times! You can read the summaries of the conference presentations at the SENS website; references are below:

PhD candidate Mark Hamalainen

Holt IJ, Bokori-Brown M, Hamalainen M.
Allotopic expression: mitochondrial to nuclear gene transfer.
Rejuvenation Res. 2007 Sep;10(Suppl1):S32(Abs53).
http://www.sens.org/sens3/abs/Holt.htm

Dr. Marisol Corral-Debrinski

S. Ellouze, C. Bonnet, S. Augustin, V. Kaltimbacher, V. Forster, M. Simonutti, J-A. Sahel, M. Corral-Debrinski
Allotopic mRNA localization to the mitochondrial surface: a tool for rescuing respiration deficiencies
Rejuvenation Res. 2007 Sep;10(Suppl1):S24(Abs 23).
http://www.sens.org/sens3/abs/Corral.htm

Dr. Antonio Enriquez

J.A. Enriquez
Inteins and allotopic expression of mtDNA encoded proteins
Rejuvenation Res. 2007 Sep;10(Suppl1):S28(Abs 36).
http://www.sens.org/sens3/abs/Enriquez.htm

Dr. Volkmar Weissig

V. Weissig, E. Katrangi, S.V. Boddapati, G.G.M. D'Souza
Manipulating the mitochondrial genome
Rejuvenation Res. 2007 Sep;10(Suppl1):S50(Abs 124).
http://www.sens.org/sens3/abs/Weissig.htm

Dr. Samit Adhya

S. Mukherjee, B. Mahata, B. Mahato, S. Adhya
Use of a parasite-derived protein complex to modulate the function of mitochondria in human cells
Rejuvenation Res. 2007 Sep;10(Suppl1):S19(Abs 2)
http://www.sens.org/sens3/abs/Adhya.htm