FOXO4-DRI Peptide Potential Interactions with Aged Cell Cultures

FOXO4-DRI is a synthetic peptide that is actively researched regarding its potential to induce programmed cell death in aged cells, and thus reduce the burden that aged cells pose on different tissues. Apparently, the peptide achieves that by mimicking a part of the so-called FOXO4, which is a transcription factor switching genes on and off in the DNA of cells. Notably, FOXO4 appears to be at higher levels in aged cells and may deactivate another protein called p53 that works to trigger programmed cell death.

According to researchers such as Baar et al., FOXO4-DRI peptide appears to be designed to imitate a small binding region of FOXO4 that otherwise may interact with p53 and mitigate it from triggering apoptosis.⁽¹⁾ The “DRI” part stands for D-retro-inverso, suggesting it was synthesized using D-amino acids and arranged in a reversed order compared with the original L-peptide fragment from FOXO4. This apparently works to make the binding region similar to the original, while making the whole molecule much more stable. Specifically,  FOXO4-DRI peptide has been researched as a decoy that may compete with FOXO4 for p53 binding, so it may disrupt the FOXO4–p53 interaction in laboratory systems. This may leave p53 unbound, so that it may target senescent cells for apoptosis.

 

Research

FOXO4-DRI Peptide Potential for Selective Apoptosis

Researchers such as Huang et al. have investigated the apparent selectivity of FOXO4-DRI peptide in regard to specifically targeting aged cells for apoptosis.⁽²⁾ To investigate this, the researchers studied in depth the apparent disruption of FOXO4–p53 binding and suggested that by disrupting it, the FOXO4-DRI peptide may shift p53 trafficking toward the mitochondria. Mitochondrial p53 is apparently linked to intrinsic apoptosis signaling, so this relocalization is posited to increase susceptibility to apoptosis specifically in senescent cells. Consequently, the authors suggest that the peptide may engage a caspase-3/7-dependent route downstream of mitochondrial stress.

The selectivity of the peptide is further discussed by the researchers, who suggest that senescent cells may depend more strongly on FOXO4-mediated control of p53 localization and activity, so disrupting that node might preferentially remove cells that are already operating close to an apoptotic threshold. In contrast, cells with lower senescence burden may have less FOXO4–p53 dependence, or may buffer p53 relocalization through other regulators, which might explain the limited apoptosis in younger cultures compared to aged cell cultures.

After evaluating the peptide on aged cell cultures, the researchers apparently observed reduced senescence-associated staining and lower protein levels of p16, p21, and p53 in the remaining cell population, consistent with the removal of cells that highly expressed these markers. For example, the researchers conducted experiments with cartilage cells and noted that “FOXO4-DRI [may] remove the senescent cells in PDL9 chondrocytes”.

At the same time, it appeared that some transcript readouts moved in different directions, which suggests that FOXO4-DRI peptide may also act as a stressor for surviving cells, potentially triggering compensatory transcriptional responses even as senescent cells are eliminated.

FOXO4-DRI Potential Interaction with Hormone-Producing Cells

FOXO4-DRI peptide has also been evaluated in hormone-producing cell cultures, and studies suggest that by inducing programmed cell death in the aged cells, this experiment may support the overall capacity for hormone production.

For example, research by Zhang et al. suggests that aging testosterone-producing cells may become senescence-like with age and then produce less testosterone, and the process does involve the FOXO4 gene.⁽³⁾ The researchers compared younger and older cells and reported that, apparently, the total amount of FOXO4 protein did not clearly change, but its location did. In younger cells, FOXO4 appeared to be mostly in the cytoplasm, while in older cells it was more often in the nucleus, which may be considered its more “active” form.

The researchers also observed that testosterone-producing cells with nuclear FOXO4 staining appeared to have lower expression of 3β-HSD, which is an enzyme needed for testosterone synthesis. Furthermore, the researchers attempted to induce a senescence-like state in a testosterone-producing cell line using oxidative stress, FOXO4 shifted into the nucleus, and senescence pathway markers increased. At the same time, proteins that support testosterone synthesis apparently decreased.

Following FOXO4-DRI peptide evaluation, the researchers noted an apparent increase in markers such as 3β-HSD and CYP11A1. FOXO4-DRI was apparently linked to lower levels of senescence markers (p53, p21, p16) and lower levels of several Senescence-Associated Secretory Phenotype factors in such cell cultures, especially IL-1β, IL-6, and TGF-β.

The authors ultimately posited that “FOXO4-DRI [may have] [supported] the testicular microenvironment and alleviated [cellular] age-related testosterone secretion insufficiency”. They speculate that a less inflammatory, less senescence-driven environment may help steroidogenic function or support progenitor-like testosterone-producing cells. Moreover, reducing the senescent testosterone-producing cell burden may leave a higher proportion of cells that still express the enzymes needed to make testosterone, or may allow remaining testosterone-producing cells to restore some of that enzyme expression.

FOXO4-DRI Peptide Potential Support for Fibrosis in Cell Cultures

Based on research by Han et al., FOXO4-DRI may support cellular fibrosis by shifting the balance away from senescent, pro-fibrotic cell states and away from myofibroblast-driven matrix production.⁽⁴⁾ The researchers investigated cell culture models of fibrosis, which also expressed senescence markers such as more β-gal–positive cells and higher p16 and p21.

After FOXO4-DRI peptide exposure, those senescence readouts dropped. Since senescent cells may release a senescence-associated secretory phenotype, the authors suggest that lowering the senescent cell burden may reduce pro-fibrotic signaling in the surrounding cells. In line with that, FOXO4-DRI peptide was associated with reduced expression of several inflammatory and remodeling factors linked to fibrosis.

The study also suggests that FOXO4-DRI peptide may have reduced collagen accumulation, lowered hydroxyproline content, and lowered expression of multiple collagen genes. At the protein level, FOXO4-DRI apparently reduced markers tied to myofibroblasts and matrix deposition, including α-SMA and Col1a1.

The researchers also conducted RNA-sequencing, which apparently pointed to reduced activity of pathways involved in ECM–receptor interaction and focal adhesion, alongside lower levels of key ECM proteins such as fibronectin, tenascin C, laminin, thrombospondin-2, and Hmmr. Consequently, the researchers posited that FOXO4-DRI may reduce both the production of extracellular matrix and the cell–matrix signaling loops that may lead to a fibrotic state.

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References:

1. Baar MP, Brandt RMC, Putavet DA, Klein JDD, Derks KWJ, Bourgeois BRM, Stryeck S, Rijksen Y, van Willigenburg H, Feijtel DA, van der Pluijm I, Essers J, van Cappellen WA, van IJcken WF, Houtsmuller AB, Pothof J, de Bruin RWF, Madl T, Hoeijmakers JHJ, Campisi J, de Keizer PLJ. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. Cell. 2017 Mar 23;169(1):132-147.e16. doi: 10.1016/j.cell.2017.02.031. PMID: 28340339; PMCID: PMC5556182.
2. Huang Y, He Y, Makarcyzk MJ, Lin H. Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Human Chondrocytes. Front Bioeng Biotechnol. 2021 Apr 29;9:677576. PMID: 33996787; PMCID: PMC8116695.https://doi.org/10.3389/fbioe.2021.677576
3. Zhang C, Xie Y, Chen H, Lv L, Yao J, Zhang M, Xia K, Feng X, Li Y, Liang X, Sun X, Deng C, Liu G. FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice. Aging (Albany NY). 2020 Jan 20;12(2):1272-1284. doi: 10.18632/aging.102682. Epub 2020 Jan 20. PMID: 31959736; PMCID: PMC7053614.
4. Han X, Yuan T, Zhang J, Shi Y, Li D, Dong Y, Fan S. FOXO4 peptide targets myofibroblast ameliorates bleomycin-induced pulmonary fibrosis in mice through ECM-receptor interaction pathway. J Cell Mol Med. 2022 Jun;26(11):3269-3280. doi: 10.1111/jcmm.17333. Epub 2022 May 5. Erratum in: J Cell Mol Med. 2024 Aug;28(16):e18502. doi: 10.1111/jcmm.18502. PMID: 35510614; PMCID: PMC9170815.