Epigenetic Rejuvenation (4)
Dreams of Perpetual Youth (6)
Upon rereading the previous posts in this series it became apparent to me that I needed to postpone consideration of the most sensational report on epigenetic resetting. First more background and context is required for the Yamanaka factors and induced pluripotency, and how they came to be used in anti-aging research.
In his attempts to isolate a few critical factors that could simulate pluripotency (the capacity to become any type of cell) of the sort characteristic of embryonic stem cells, Kazutoshi Takahashi and Shinya Yamanaka of Kyoto University screened 16 candidate molecules. They arrived at a cocktail of four factors, usually abbreviated OKSM, for Oct4, Klf4, Sox3, and Myc, respectively. The latter are abbreviations as well of course, but there is no need to spell that out. Shinya Yamanaka received the Nobel Prize for demonstrating—along with Takashima-- that with these four factors alone, he could induce a fully differentiated cells, such as fibroblasts, to transform into pluripotent stem cell that remarkably resemble the naturally produced pluripotent embryonic stem cells. These transformed cells were called induced pluripotent stem cells, abbreviated to iPSCs. The “i” in iPSCs was inspired by iPhones, a needless affectation that unnecessarily punishes the lay audience and those who want to communicate with them.
Subsequently it was demonstrated that all for of the Yamanaka factors is replaceable by other molecules for pluripotency purposes (10.1016/j.stem.2014.05.00). Which is not surprising when you consider what Yamanaka factors do in actual embryonic stem cells. Yamanaka factors function in a vast and complicated network of molecules in regulating cell potency. Some of these factors are direct epigenetic regulators: DNA Methylation, Histone Acetylation etc. Moreover, the Yamanaka factors themselves are epigenetically regulated, like any other genes. Much to much emphasis has been placed on OMSK as master cell differentiation regulators, as if they and they alone can induce pluripotency. They are just key players, along with 20 or more other teammates in embryonic stem cell behavior. It just doesn’t take the whole team to make something that resembles an embryonic stem cell, if, that is, the concentrations are juiced up.
Once OMSK were deemed sufficient pluripotency regulators, other proteins that control cell fate were largely ignored. Some, such as Nanog, as important as any of the OMSK, were recognized belatedly. Keep in mind that the OMSK quadruplet is sufficient to induce iPSCs, but not necessary. They, along with many other proteins, are necessary for well-functioning embryonic stem cells. IPSCs approximate, but do not fully embody embryonic stem cells. Nonetheless, their discovery was a godsend in the context of religiously inspired opposition to harvesting embryonic stem cells. The benefits of IPSCs were first noticed in medicine.
From Cancer Treatments to Anti-Aging Factors
The Yamanaka factors induced iPSCs are increasingly important in research for medical rehabilitation, from cancers to neurodegenerative diseases. But now their application has expanded to age-reversal and rejuvenation research more generally. To this end the goal is not a full epigenetic resetting to something like embryonic stem cells, just a partial resetting, to the cell’s mature state, but before it becomes subject to the epigenetic modifications that are the vicissitudes of aging. The epigenetic state that is just shy of the loss of cellular identity. This partial reset also originally occurred in the medical context.
Partial epigenome resets with Yamanaka factors were later extended to anti-aging research. It seems that the partial resets of the epigenome occur in two phases. During the first, the cell dedifferentiates to a more stem-like state. In the second stage the cell redifferentiates back into its former cell type. But the redifferentiated cell has been epigenetically reset to a more youthful state.
The Yamanaka factors are introduced in two distinct ways: integrative and non-integrative. In The integrative technique, the Yamanaka genes are physically attached to chromosomes. Several vectors are employed to achieve this, most often viruses. For the non-integrative technique, the Yamanaka factors are not incorporated into the genome. Again, viral vectors are used but viruses of a completely different sort than are employed in the integrative technique.
There are myriad viruses classified in different ways, primarily whether they are single or double stranded nucleic acid sequences, and whether the sequences consist of RNA or DNA. The viral vectors used in the integrative technique are called retroviruses, which are single stranded RNA viruses that attach to the genome and transform themselves from RNA to DNA. HIV is one such retrovirus. This was the original method in age-reversal research to emplace the Yamanaka factors.
For the non-integrative approach a completely different virus is used, one that infects the cell but not the genome. Called adenovirus, because it was first discovered in the adenoids, it is a single stranded DNA virus. The Adeno Associated virus (AAV), is the viral vector of choice. It enters the cell nucleus to replicate but does so independently of the host genome. AAV is generally associated with gene therapy. Pretty soon it will be associated with the quest for a Fountain of Youth.
The non-integrative method is now preferred in anti-aging research, as physically integrating OKSM with the host DNA is less likely to disrupt the genome, generate tumors, and otherwise spoil health. Several different research groups employed this strategy. I will discuss one that is of particular interest to me, as the rejuvenation is in the brain.
The first author on the paper is Steve Hovarth, creator of a widely used epigenetic clock, which I discussed in a previous post. That clock was used as partial validation of the results. The subjects were rats, some young (3.5 months), some old (25.3) months. Some of the old rats got the Yamanaka cocktail, some got the placebo. Their treatments lasted 39 days. The Yamanaka factors and their viral vectors were injected directly into the hippocampus, an area of the brain that ages most rapidly. The hippocampus mediates many forms of memory but is most associated with spatial memory.
The Yamanaka treated old rats showed improvements in spatial memory, relatively to the old rats that didn’t receive the treatment, as measured by the Barnes Test. This suggests that Yamanaka cocktail improved spatial cognition in these rats. In association with the memory improvement, the epigenetic clocks of the hippocampal cells were reset to a more youthful state, but not dramatically so. Oddly, the DNA methylation, on which the epigenetic clocks are based, did not change much in the hippocampus as a whole. I found Hovarth et al’s. explanation for this inconvenient fact unsatisfactory.
In sum, Hovarth’s attempt at hippocampal epigenetic rejuvenation was underwhelming. After 30 + days there was a clear behavioral signal, with respect to performance on the Barnes test, which, to my mind is partly a measure of reaction time. A more sophisticated spatial memory test would be informative.
Hovarth previously used essentially the same methods to test the effects of the Yamanaka cocktail on a part of the brain nearer and dearer to my heart, the hypothalamus. In the next post I will discuss that study, as well as the still unpublished results of another, which could potentially rock the world, if it is validated.