Episode #70 Peter Attia Podcast

SIR genes and cellular identity

• One of the roles it is most well-known for, is its implication in longevity: In a study by Matt Kaeberlin Sir2 was shown to control the aging process in yeast
• The gene group’s primary roles are to:
• Silence other genes
• Repair damaged DNA

• The SIR gene cannot silence other genes and serve a cellular repair function at the same time
• silenced genes are temporarily “turned on” while the damage is repaired by the enzyme protein
• “turned on” genes help with the repair
• the protein then returns to its “silencing post”

• In aging yeast, the loss of cellular identity results in a sterility phenotype
• With age, our cells lose their “program” but early evidence in mice suggests that the original “hard disk program” can be recovered; restored to the original programming

“We have some early evidence from mice that we can actually find that hard disk drive and reinstall the software so that it’s pristine again and we find that we can actually improve the health quite dramatically in parts of a mouse’s body.” — David Sinclair, Ph.D

• Shannon figured out how to preserve information: make a repository and reset the system

Sirtuins regulate gene expression

• In the embryo, cells begin the same, but gene expression creates different cell types by turning on/off different genes
• The selective gene activity allows for cell differentiation (e.g., it is why an epithelial cell is different to a liver cell)

• Spooled DNA won’t be read by the cell
• If a cell needs to read the gene, SIR proteins will be removed and the bundle will open

• The superficial layer: SIR transient proteins, transcription factors turn genes on and off to be read
• One level below are histones, which chemically modify the spooling proteins
• The deepest layer is where DNA is methylated
• Attaching a methyl group to an end of the DNA makes it so the gene is not expressed
• Some genes in a cell are never turned on

DNA is methylated at the deepest layer of the epigenome

• Attaching a methyl group to an end of the DNA makes it so the gene is not expressed
• Methyl groups on DNA within a genome is the underlying code that tells the cell what type it is

Methylation pattern and determining cellular age

• It’s as though the marbles start to drift into other valleys
• Methyl groups accumulate over time, get removed in certain places, or appear in different places on the DNA genome
• DNA breaks, SIR proteins reorganization and cells can revert back to other kinds of primordial (e.g. skin, liver) cells
• The cell ‘nature’ changes and its ‘identity’ becomes blurry

“it appears very much like a loss of information and interruption and noise because these methyl groups that are laid down in a pristine precise fashion when we’re young, there are other methyls that accumulate over time in different places. What appears to be randomly so it’s a loss of the original pattern. That’s why we talk about entropy.” -David Sinclair, Ph.D

• The compact disc (CD) that is our genome gets scratched overtime (noise) such that cells don’t read the right genes (the epigenome) at the right times

• David’s lab can program cells to “go young again” by correcting the methylation pattern within a cell’s genome
• Hitting the cellular reset button to read the right pattern of methyl groups
• The cell becomes like a stem cell – Yamanka gene reprogramming factors are pre-embryonic cells with fewer methyl groups
• The DNA spools get reset with the original methylation pattern within the epigenome
• Results in a younger phenotype and the mouse’s chronological age is also younger

“But what I think exists in cells and we have some evidence is that, like Shannon suggested for the internet or information, is that if you have a backup copy… and now going back to the genome… there seems to be something in cells that tells them these methyl groups, the programs that were laid down when you were a baby are still there and cells can access that somehow to say: ‘All these other things that have happened since you were born or since you were a teenager, that’s just noise… Ignore that.’” -David Sinclair, Ph.D

• Cellular aging begins at conception and methylation pattern reveals a cell’s age
• Determining chronological age has a 95% fidelity from a blood sample
• This cellular ‘clock’ known as the Horvath Clock tells you how fast you are aging
• The rate can change depending on lifestyle; known mechanisms to increase rate (e.g. smoking) or slow aging (e.g. exercise, caloric restriction)
• When fasting, mechanisms turn on sirtuins – implicated in longevity
• NAD levels increase and sirtuins have more substrate to repair DNA and keep genes in the epigenome that should be silent, silent (i.e. retain cellular identity)
• Telomeres have formerly served as a pretty good clock for chronological age but are not as accurate as the Horvath Clock
• Different tissues divide at different rates
• They are not immutable: A study – that compared the telomeres of twins, one of which spent a period of time in space – revealed that telomere health reversed within 2-3 days after being back on earth

• A published universal clock can use the location of methyl groups – irrespective of cell type – and determine the age (of a mouse)
• David and his colleagues in the field can determine how long a methyl group has been at a given locate on the genome

“There isn’t a program that tells us we must age…but there are processes like I was describing about movement of these SIR proteins in yeast, and movement in mammals like us, that leads to a predictable change on our genome that changes the way genes are switched on and off as we age in very precise locations.”  -David Sinclair Ph.D

• Aging is not a genetic program but processes like the movement of SIR proteins provide predictable patterns of change to the genome
• Genes are switched on and off as we age in very precise locations

Cellular reprogramming

• The stored copy a cell keeps of the original methyl group placements on a gene allows it to reset itself; reprogram itself
• The Belmonte lab showed that if a cell is reprogrammed and too quickly, it will become tumorous
• David’s lab does “partial reprogramming”: the cell regains its youth but does not lose its identity
• Every cell has a master copy to reprogram itself back to a youthful, original state
• The reset is intrinsic to each cell (a neighboring cell could not be reset next to a reprogrammed cell, for example)
• As we get older, genes that were once bundled by SIR proteins and methylated DNA become undone and read by the cell
• Reprogramming signals the cell in the dysfunctional region to re-package: place SIR proteins where they once were, add or remove methyl groups, and switch the gene off again
• Shannon’s backup disc is called the observer: it keeps the original signal (or copy of the original program) and the receiver sense checks the transmission

• We haven’t lost genes due to mutations, as believes in the 1950s
• The information is there but cells don’t know whether to read it or spool it up and hide it away

• Interventions such as rapamycin, NAD booster, Metformin – impact the superficial layer where transcription factors singal which genes to be read or not – slow down aging but they cannot reverse it
• They can change the rate but not the direction [of aging]
• Cellular aging reversal – at the level of DNA methylation and chemical modification of proteins – is achieved with stem cell technology reprogramming factors; Yamanaka Genes

Yamanaka factors to push cells “back in time”

• Three of the Yamanaka genes were packaged into AAV (adenovirus vectors).
• Yamanaka genes program stem cell-like genes. AAV = virus with DNA packed in to insert
• The genes were given a doxycycline antibiotic on/off switch (the gene can be silenced with the antibiotic)
• The fourth Yamanaka, myc gene was excluded – an oncogene that causes cancer, which turned out not to be necessary in order to reprogram cells to be partially young again
• Three scenarios were tested: young mouse with traumitized optic nerve, mouse with glaucoma, old mouse natural vision loss
• nerves regenerated in all three scenarios
• Yamanaka genes in the virus vector were turned on after loss of vision occurred (the treatment needs to reverse and not just prevent the damage)
• Results:
• Nerves were regenerated in all three experiments
• Vision was restored in mice with glaucoma and age-related vision loss
• The AVV treatment mechanism:
• The AVV contains genes that specify the cell during embryogenesis
• A Horvath nerve cell clock was used to determine and restore methylated pattern of genes during early development for such cells
• Nerve cell methylation patterns of young and old mice were compared
• Could determine which retinal genes were on/off in younger mice cells compared to older mice
• Young mice have genes for taste in retinal cells, hypothesized to be relevant for chemical signaling
• When the retina was reprogrammed, genes that went down with aging proportionally corrected:
• Genes that went way down with age went way back up
• Genes that went a little bit down with age went a little bit back up
• One prediction would be that chemical changes map to genes that incur a change with age
• David’s lab have identified hypersensitive regions in the epigenome in response to stress (broken DNA, UV damage)
• Once the DNA unspooled, or opened and methyl groups have been added/removed in the wrong places, the structure remains that way unless reprogrammed: epigenetic noise
• AVV technological limitations:
• How many of the cells can receive the virus vector? Can't do every cell, but may not have to.
• If the cell does not get the virus, it will not get reprogrammed (i.e reprogramming is binary and cell-specific)
• The retina can be restored, the optic nerves re-grown, but it is unknown how much of the animal can be reprogrammed
• Retinal case study advantages:
• The retina has FDA approved drugs for viral delivery
• The eye is insensitive to the rest of the immune system
• A lot of AVV can be delivered to the optic nerve

Human cellular reprogramming

• There is a high demand for making adenoviruses, creating both a long wait time and inflated costs
• Although Peter votes that David’s lab work with the prostate tissue next, other tissues have more of an acute for reprogramming if something goes wrong (e.g. loss of vision, heart failure)
• There is a lot of promise in the gene therapy research area and also a lot of potential economic gain
• Where gene therapy used to be avoided (thought to be risky, dangerous), it is now a notable field for financial investment
• The virus vectors used today are different types of viruses to those used in previous research from decades before
• Viruses used today cannot integrate into the genome and cause mutation (cancer) or cause negative immune reactions

• There are active clinical trials for patients with obvious candidate genes – it won’t be long before these diseases are correctable

Measuring the rate of aging

• To evaluate the extent to which methylation and epigenetic changes in DNA occur

• DNA damage that occurs with chronological age results in senescent cell accumulation
• In early stages of aging: epigenetic noise and the loss of cellular identity
• In the end stages of aging: the cell stops dividing but don’t die – they become senescent
• DNA damage at telomeres is notably persistent and highly efficient in inducing senescence and/or apoptosis
• An important mechanism to prevent cancer proliferation, cells send out chemical signals and disrupt the epigenome

• Contributes to epigenetic interference and DNA methylation of surrounding cells
• Other senescent cells make surrounding cells senescent or tumorigenic
• In one study, implanted senescent cells in mice induced signs of premature aging, such as higher blood sugar

• Senescent cells in the fat of mice or human models of different ages, stained with senescence-associated β-galactosidase (SABG) for identification: Older fat is packed with senescent cells (fat stain is dark blue)
• The most reliable phenotype of senescence is SABG; cells turn blue when stained
• There is no definitive way to identify senescence in a tissue (although SABG is most reliable)

“So, we’ve got a pretty badly scratched CD that we can polish with reprogramming, but if you’ve gouged it so deep [senescent cells] that even reprogramming can’t work, then we need something else probably.” -David Sinclair, Ph.D

• The pre-senescent loss of cellular identity is reversible (mouse model retinal cell experiment results)
• In theory, it should be possible to get a senescent cell to replicate again if its DNA is in tact

Cellular reprogramming for longevity

• The ideal application for longevity and not just disease treatme: A reactive tool to respond to aging phenotypes in specific tissue cells
• One limitation is that the AVV does not infiltrate every cell in the body evenly (liver cells are favoured)
• Different AVV protein variations are used for different parts of the body (e.g. AAV9 is good for muscle cells)
• Reprogramming also holds the possibility to globally ‘turn back the clock’ by restoring pre-embryonic methylation patterns in all cells
• Cells lose genetic information but epigenetic information (the backup drive) remains in tactThe information lost in genetic information (the gauging of the CD) may limit cellular eternal lifespan (immortality)

“I think the problem with what I’m calling the information theory of aging, which is what I wrote about in my book, is that we do lose information. Every cell does experience mutations. It’s not perfect…and so ultimately, if you’re a thousand years old, you may have lost a lot of the genetic information. But epigenetic information, because there’s this backup drive, this observer, that we have found exists in cells somehow, that you can tap into, as long as the genome, the DNA strands, are still largely intact, we can reverse aging.” -David Sinclair, Ph.D

• A couple of years ago, David did not think it was possible to extend human life to 200 years
• If vision can be restored (retinal cellular reprogramming), than other parts of the body can be restored, too
• Even if reprogramming does not work for senescent cells for which too much genetic information has been lost, then perhaps they can be deleted
• Tissues can be regrown and replaced from another organism or in a dish
• David is optimistic about not just being able to slow cell aging, but actually having cells go back in time.. ‘Turning back the clock’…

Compounds David takes for his own longevity

• One of Metformin’s main effects is mitochondrial inhibition, which elicits an upregulated response to make more
• Blunting mitochondria production may not be the best for exercise and recovery
• Peter writes about Metformin and exercise dynamics here

“Put your body in a state of anxiety or fear, adversity, but you don’t want to do it all the time. Your body needs a chance to recover.” -David Sinclair, Ph.D

• Along the same lines, David wouldn’t take Rapamycin (which signals to the cell not to grow) while exercising
• David doesn’t take rapamycin regularly
• Peter doesn’t take rapamycin or metformin while fasting >24h

• He takes a gram with dietary fat, like yogurt
• He doesn’t have any negative effects
• It is not as powerful as rapamycin but it does extend the lifespan of a mouse on a western diet
• This mouse study reported that fat mice taking resveratrol were healthier and lived longer than those that didn’t take the compound
• Similar to metformin, rapamycin seems to be beneficial in metabolically unhealthy individuals but if health is otherwise optimized, there is not as much benefit

• A debated 2013 study found that a SIRT1 mutation blocked resveratrol from activating the sirtuin enzyme
• The new experiment in David’s lab takes a mouse with the protein enzyme mutation (missing an amino acid) that renders it immune to the effects of resveratrol
• The original 2006 study of mice with a high-fat diet with/without resveratrol administered and with/without the resveratrol immunity mutation
• The study confirmed that resveratrol extends lifespan by activating SIRT1

NAD precursors (NR, NMN) and pterostilbene

• Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all cells
• One of the oldest and most important molecules for life
• NAD exists in two forms: NAD+ (oxidized form) and NADH (reduced form)
• NADP, NADH don’t activate sirtuins, only NAD+ will do that
• Sirtuins (protectors of the epigenome) lose their activity overtime and require two things for maximum activity:
• An accelerator (i.e. resveratrol)
• A fuel, or substrate (ie. NAD)
• We now know NAD levels fluctuate and go down with age

• In animal models, more NAD is better (the more there is, the longer the lifespan)
• The idea is that by replacing or boosting NAD, cellular defense and DNA repair is upregulated
• The less healthy someone is, the more they will benefit from more NAD – like is the case for supplementing with metformin, resveratrol
• In studies, the benefits of NAD and resveratrol are seen in obese subjects or older mice that were given a NAD boost
• Young mice that were given a NAD boost did not run further but they did if a boost and exercise was administered at the same time
• In humans and mice, there have been few studies of taking NAD orally but subjects have been given NAD precursors
• Most take NAD intravenously (IV) but there is evidence that cells don’t take up NAD directly
• it first needs to get broken down and then reconstituted once inside of a cell
• There is not convincing evidence that IV NAD makes it into the cell (from plasma)
• NAD goes up and down not only in the cytoplasm but also in the mitochondria
• NAD is made de novo in the cell, but you don’t get to shuttle NAD between cell

• Nicotinamide riboside (NR) is partly a piece of B3 and a piece of DNA
• NR becomes NAD in two steps:

• Once NR it is turned into NAD in the cell, it is recycled and turned into nicotinamide (NAM)
• NAM is a version of niacin, or vitamin B3
• A high dose of supplemented vitamin B3 can raise NAD levels but other components will be missing (riboside/sugar/DNA/phosphate)
• Taking niacin isn’t as effective as taking either NR or NMN
• There are reasons not to take high does of NAM

• Try to avoid NAM while raising NAD levels
• It could be great to have a field definition of NAD:NAM ratio (to give an indication of cellular ‘boost’ to ‘break’)
• A study showed that orally-taken NR got turned into NAD in the liver (while most did not exit the liver)
• The study reported that NAM blood levels were raised but not NR
• Another clinical trial showed that orally administered NR raised NAD levels in the muscle and increased plasma NAM
• High dose of 100mg administered
• Placebo controlled with a high BMI study population in their mid-50s
• The result could imply that the NR is getting past the liver and uptake in muscle cells produce NAD
• There were a few improvements in inflammatory markers (some inflammation decreased in the muscle and mitochondrial markers were lower)
• The interpretation of this finding is not clear
• It could be an NR-specific effect or having to do with the class of molecules
• It is surprising as other research shows that NMN and NR boost mitochondrial activity (in mice)
• It is important that NAD increases in the mitochondria, in particular – which has not yet been demonstrated

• Under the tongue, the substance gets directly absorbed, rather than passing through the liver (via the portal circulation)
• The microbiome will consume some of the NR when ingesting niacin (the nicotinamide comes off easily)
• There is evidence that the gut plays a big role in how much and if NR gets into the rest of the body

• Not all findings are in agreement, but David believes that the microbiome removes a lot of the nicotinamide from NR and NMN before it is taken up by the gut
• It makes sense not to put all of it in the gut
• But if it all passes through the gut, some will get through
• NMN gets broken down by the gut and then remade in the body into NAD
• There is also data that some NMN and some of the NR gets through to the rest of the body and gets beyond the liver into the muscle

• Johan Auwerx says that administering NR to mice does expand lifespan
• David’s lab experiments with NMN and lifespan in mice
• The administering of these molecules are beneficial for mice in health and longevity and research is still being done to answer if the same results occur in humans

• It could be that there are only measurable positive effects if the individual is not healthy
• In terms of sirtuin activators, David’s lab is focused on NAD-boosting drugs