Selective Destruction of Senescent Cells by Interfering in FOXO4-P53 Crosstalk

Today, another research group announced their entry to the field of senescent cell clearance as a means to treat aging, along with the intent to commercialize their novel method of achieving selective destruction of senescent cells in aged individuals. Senescent cells accumulate with age as a result of the normal operation of living tissues: cells become senescent when damaged or when they reach the Hayflick limit on replication. Near all are destroyed, either through the programmed cell death mechanism of apoptosis, or by immune cells attracted by the signal molecules generated by senescent cells. Unfortunately, some linger, resistant. The number of these cells grows over the years, and the signals they generate start to create harmful outcomes in nearby cells and tissue structures, and in addition spur rising levels of chronic inflammation. The increasing presence of senescent cells is one of the root causes of degenerative aging and directly contributes to many specific age-related diseases.

The best and most direct approach to the phenomenon of cellular senescence is to periodically destroy these cells, reducing their numbers to the greatest extent possible. These numbers are never enormous, perhaps a few percent of most tissues in late old age, depending on the details. Removal can proceed as slowly as needed to be safe in older individuals if there are risks of lysis side-effects due to the amount of cell debris generated by senescent cell destruction. While senescence has short-term roles to play in tumor suppression, by shutting down the ability to replicate in potentially cancerous cells, and in wound healing, these cells have no clear and evident long-term use in the body. So a treatment that gets rid of near all of these cells, undergone once every few years, would in fact be a narrow means of rejuvenation. It would make aged tissues less aged. This has been demonstrated in studies of senescent cell removal showing life extension in mice, as well as those that have demonstrated specific improvements and reversals in the pathology of various age-related diseases and aged tissues.

The methodology developed by the researchers noted here is, at the very high level, analogous to that involved in some of the senolytic drug candidates evaluated to date - though it has the merit of having far fewer side-effects per this report. It involves sabotaging one of the mechanisms that lingering senescent cells use in order to resist the fall into apoptosis, but which in normal cells has no important role to play. Thus drug molecules can be delivered everywhere, and will only produce significant effects in cells that are senescent. In the case of drugs like navitoclax, that mechanism involves inhibiting bcl-2 family proteins. The mechanism here is quite different, involving FOXO4's influence on p53, but I wouldn't be surprised to see it turn out to be a part of the same system of inhibition of apoptosis. Almost all cellular mechanisms can be influenced in many ways, by tinkering with the activities and actions of many directly and indirectly involved proteins, and it is not unusual for research groups initially working on a diverse set of proteins to find that they end up in the same place at the end of the day.

Peptide targeting senescent cells restores stamina, fur, and kidney function in old mice

Regular infusions of a peptide that can selectively seek out and destroy broken-down cells that hamper proper tissue renewal, called senescent cells, showed evidence of improving healthspan in naturally-aged mice and mice genetically engineered to rapidly age. The peptide took over four years of trial and error to develop and builds on nearly a decade of research investigating vulnerabilities in senescent cells as a therapeutic option to combat some aspects of aging. It works by blocking the ability of a protein implicated in senescence, FOXO4, to tell another protein, p53, not to cause the cell to self-destruct. By interfering with the FOXO4-p53 crosstalk, the peptide causes senescent cells to go through apoptosis, or cell suicide. "Only in senescent cells does this peptide cause cell death. We treated mice for over 10 months, giving them infusions of the peptide three times a week, and we didn't see any obvious side effects. FOXO4 is barely expressed in non-senescent cells, so that makes the peptide interesting as the FOXO4-p53 interaction is especially relevant to senescent cells, but not normal cells."

Results appeared at different times over the course of treatment. Fast-aging mice with patches of missing fur began to recover their coats after 10 days. After about three weeks, fitness benefits began to show, with older mice running double the distance of their counterparts who did not receive the peptide. A month after treatment, aged mice showed an increase in markers indicating healthy kidney function. "The common thread I see for the future of anti-aging research is that there are three fronts in which we can improve: The prevention of cellular damage and senescence, safe therapeutic removal of senescent cells, to stimulate stem cells - no matter the strategy - to improve tissue regeneration once senescence is removed."

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging

To identify potential pivots in senescent cell viability, we initiated this study by investigating whether apoptosis-related pathways are altered in senescent cells. We performed unbiased RNA sequencing on samples of genomically stable primary human IMR90 fibroblasts and IMR90 induced to senesce by ionizing radiation (IR). As senescent cells are reportedly apoptosis-resistant, we expected pro-apoptotic genes to be repressed. Surprisingly, however, senescent IMR90 showed an upregulation of prominent pro-apoptotic "initiators" PUMA and BIM while the anti-apoptotic "guardian" BCL-2 was reduced. This suggested senescent IMR90 are primed to undergo apoptosis but that the execution of the death program is restrained. We reasoned such a brake could potentially be a transcriptional regulator and focused on transcription factors that have previously been linked to apoptosis, including STAT1, 2, and 4; RELB; NFκB; TP53; and FOXO4.

Interference with JAK-STAT signaling is known not to affect the viability of senescent cells, and we have previously observed similar effects for NFκB and p53 inhibition. Our interest was therefore directed to a factor that has not yet been studied as such, FOXO4. FOXO4 belongs to a larger mammalian family, with FOXO1 and 3 being its major siblings. FOXOs are well studied in aging and tissue homeostasis as targets of insulin/IGF signaling and as regulators of reactive oxygen species. Whereas senescence-inducing IR showed only mild effects on the expression of FOXO1 and 3, both FOXO4 mRNA and protein expression progressively increased. We therefore wondered whether FOXO4 could function to balance senescence and apoptosis. We stably inhibited FOXO4 expression using lentiviral shRNA. FOXO4 inhibition prior to senescence-induction resulted in a release of mitochondrial cytochrome C and BAX/BAK-dependent caspase-3 cleavage. In addition, FOXO4 inhibition in cells that were already senescent, but not their control counterparts, reduced viability and cell density. Together, these show that after acute damage FOXO4 favors senescence over apoptosis and maintains viability of senescent cells by repressing their apoptosis response.

Research on peptide chemistry has shown that protein domains containing natural L-peptides can sometimes be mimicked by using D-amino acids in a retro-reversed sequence. Modification of peptides to such a D-retro inverso (DRI)-isoform can render peptides new chemical properties, which may improve their potency. As a cell penetrating peptide the D-retro inverso (DRI)-isoform of FOXO4, FOXO4-DRI, differs from other senolytic compounds by being designed around a specific amino acid sequence in a molecular target only mildly expressed in most normal tissues. Though a more thorough analysis is required, at least as far as tested here FOXO4-DRI appears to be well tolerated, which is an absolutely critical milestone to pass when aiming to treat relatively healthy aged individuals.

Comments

I have long been accustomed to avoiding too much hope, but...wow. Anti-aging drugs are kind of like recreational drugs: if you have to ask yourself if it's working, you've been sold a load of bunk. These are real, visible, and undeniable results. The real deal. Unbelievable.

Now, I know it may yet fail in some way, and that it's only a piece of the grand puzzle. Maybe it won't work in humans. Maybe it only works a little, or has awful side effects...but this feels like a winner. The effect is so large and obvious it's got to at least somewhat work in humans. Or so I hope :)

One things that's for sure, the FDA is going to stand by and watch foreigners make all the discoveries and bring it to market. When it finally reaches market in a foreign country, they will block it in America because that is their true mission.

And this is good news, because if all those baby boomers have to watch foreigners grow younger before their eyes while the FDA blocks approval, or it gets approved and the drug company with the monopoly gets a million bucks per treatment, it will finally spell the end of their wretched, obstructionist bureaucracy.

Posted by: Paul at March 24th, 2017 12:34 AM

Oh, I see that Dr. Campisi is involved in this research. Maybe we will see Unity develop this approach too?

Posted by: Antonio at March 24th, 2017 4:35 AM

@Antonio: I'd say that UNITY's future is almost certainly that they'll buy someone else's methodology and run with that; I don't see what else they can do given their situation.

Posted by: Reason at March 24th, 2017 7:20 AM

Yep, they were too unambitious when selecting their strategy and now it would be suicidal to continue along that route.

I hope this will happen in other aspects of aging too, when more and more SENS therapies are validated in mice, outcompeting more traditional candidates like sirtuins.

Posted by: Antonio at March 24th, 2017 8:55 AM

PEPTIDES ARE EASY TO MASS MANUFACTURE WE WILL BE BUYING CHINESE FOXO4 LONG BEFORE THE FDA OR ANY ONE HAS PUT THE DOLLAR CUFFS ON IT

Posted by: VAN at March 26th, 2017 9:03 AM

I disagree with an earlier comment re:- outcompeting more traditional candidates like sirtuins.
In my view such approaches are complementary. I believe looking at extending short telomeres is also a potentially useful approach.

Posted by: Russell at March 27th, 2017 8:17 AM

@Russell I'm using you've heard of Epitalon?

Posted by: Joe at April 10th, 2017 11:49 PM

Does anyone have user experience for FOXO4 DRI?

Posted by: martin chitwood at April 16th, 2017 1:40 PM

Prometheist, and anyone who missed it previously: the claim that this vendor is selling FOXO4-DRI is dubious. See this previous comment by Matthias F and following discussion. The blogger endorses both the Buckylabs and the Novopro products.

Posted by: Michael at June 18th, 2017 3:13 PM

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