Growing old is associated with an increased incidence of constipation, fecal impaction and incontinence, with reduced quality of life. These changes have been associated with age-related lifestyle changes, disease and use of medications which affect gastrointestinal (GI) functions (O’Mahony et al., 2002).

The effects of aging on intestinal functions have been widely studied in animals, especially rodents; many report reduced intestinal motility with loss of enteric neurons, although the latter remains controversial (e.g., Saffrey, 2004). However, compared with humans, rodents have high metabolic rates, high rates of aging and key differences in GI anatomy, neuronal functions, receptor pharmacology and molecular structures, compounded by genetic variation between different strains of laboratory rodents capable of influencing, for example, how advancing age affects GI functions (Wu et al., 2003; Keane et al., 2011; Sanger et al., 2011; Richardson et al., 2016). Accordingly, it is important to study the effects of aging on human GI functions. In healthy aged human intestine, major functions remain unchanged, including epithelial resistance, nerve-evoked secretion (Krueger et al., 2016), muscle tension developed during contraction, and numbers of myenteric neurons within different regions of the colon (Broad et al., 2019). Nevertheless, in many non-GI tissues, changes in several molecular pathways are associated with aging, including those involved with development of chronic senescence and inflammation (Bhatia-Dey et al., 2016; Rea et al., 2018). Further, some studies in human colon report loss of neuromuscular functions among the elderly. For example, increasing age (20–75 years) reduced the ability of distal colon muscle to stretch (Watters et al., 1985). In addition, cholinergic function was found to be reduced with increasing age in ascending colon, associated with increased immunostaining of myenteric cell bodies for choline acetyltransferase (ChAT). Since ChAT is synthesized within nerve cell bodies and transported to axon terminals for acetylcholine synthesis, these findings suggest an age-related decline in cholinergic axon transport (Broad et al., 2019). More recently, an age-related decline in mesenteric nerve nociception was found in human colon (Cibert-Goton et al., 2020). Finally, expression of mRNA for the senescence markers p16 and p21, and for elements of the senescence-associated secretory phenotype (SASP) were increased within mucosal biopsies from aged human colon, especially in individuals consuming normal western diets (argued to increase cellular senescence) and not under calorie restriction (Fontana et al., 2018).

p14, p16, and p21 are markers for cellular senescence. The p21 protein, encoded by the CDKN1A gene, is associated with acute senescence in many tissues in organisms of all age (Stein et al., 1999), including upregulation in myenteric neurons of the mouse intestine following short-term caloric restriction (Jurk et al., 2012). This involves exit of the cell from the cell cycle and secretion of inflammatory mediators, with affected cells usually rapidly cleared by the immune system. The expression of p21 may be facilitated by p14, encoded by the CDKN2A gene (Sherr, 2006; Bieging et al., 2014). However, with increasing age, cells can enter chronic senescence, involving chromatin remodeling and transcription changes (van Deursen, 2014), thought to be potentially causative for different aspects of aging (Bhatia-Dey et al., 2016). The p16 protein, also encoded by the CDKN2A gene, is a marker for chronic cellular senescence (Stein et al., 1999). p16 is a cell cycle regulator usually expressed in the nucleus, where it interacts with CDK4 to prevent the G1/S transition (Serrano et al., 1993; Ciesielska et al., 2017).

Aging of the human bowel is poorly understood, but a decline in enteric cholinergic function (Broad et al., 2019) and perhaps other functions, raises questions about the existence and roles of age-related degenerative pathways in the aging bowel. A senescent-like state has previously been noted in mature post-mitotic enteric neurons in the intestine of elderly mice (Jurk et al., 2012) but similar studies have not yet been conducted for the human enteric nervous system. Additionally, regional differences in the age-related decline of enteric cholinergic function (Broad et al., 2019) highlighted the importance of considering ascending and descending colon as separate regions. The current study began as a pilot investigation which used qPCR to explore the possibility that senescence might be upregulated in the mucosa-free wall of human ascending and descending/sigmoid colon (containing multiple cell-types including muscle and enteric neurons) of the “elderly” (≥70 years), compared with younger “adults” (25–60 years). Analysis of the data generated, highlighted a potential age-related increase in expression of the gene for p16, particularly in the ascending colon. The involvement of p16 with senescence, often considered as the exit of a proliferating cell from the cell cycle (Serrano et al., 1993; Ciesielska et al., 2017), created the hypothesis that potentially proliferative cells within the myenteric plexus had become senescent in the aged human colon. We focused on the enteric neurons (because a senescent-like state may exist in enteric neurons of the aged mouse; Jurk et al., 2012) and glial cells (because of their loss within the myenteric plexus of aged rats and potential to undergo neurogenesis in mice following damage to enteric ganglia; Phillips et al., 2004; Laranjeira et al., 2011). Immunofluorescence was used to confirm the change and localize the upregulation of the p16 protein. The results identified p16 within the cytoplasm of myenteric nerve cell bodies, not the nucleus, and not in glial cells, implying a role for post-mitotic cellular senescent-like mechanisms in declining enteric neuron functions during aging.

This study demonstrated increased p16 expression within myenteric neurons of the aged human colon. An initial search for age-related changes in gene expression in the intact colon (mucosa removed but containing multiple cell-types including muscle and enteric neurons) highlighted the possibility of small increases in CDKN2A (when using primers for p16^INK4A and p14^ARF together but not for p14^ARF alone) and certain other genes. Notably the latter were more associated with increased expression of CDKN2A rather than temporal age. On this basis, p16 immunostaining was performed, to examine the potential for increases in p16^INK4A expression and if confirmed, determine the location and level of protein expression.

A hallmark of senescence is the exit of a proliferating cell from the cell cycle. This involves interaction of p16 with CDK4, so p16 is typically considered a nuclear protein (Serrano et al., 1993; Ciesielska et al., 2017). Thus, the second part of the study began with the hypothesis that upregulated CDKN2A would be reflected by p16 staining in the nuclei of cells undergoing proliferation within the aged human colon, The focus was on enteric neurons and glial cells (see section “Introduction”). However, S100-positive glial cells exhibited little-or-no p16 staining. Rather, high levels of staining were found in nerve cell bodies of the myenteric plexus, but within the cytoplasm and not clearly within the nucleus. Antibody fluorescence was measured as a representative of protein expression. Although inter-individual variability was high, the data showed that cytoplasmic fluorescence was increased in the neurons of ascending but not descending colon of the elderly, suggesting region-dependent upregulation of p16 during aging within enteric neurons.

Understanding the mechanisms of declining neuromuscular functions could identify ways of promoting healthy aging of the bowel. However, experiments are hampered by lack of routinely available primary cultures of multiple human cell-types. Accordingly, this study investigated the intact colon (mucosa removed) and began by using qPCR to look for age-related expression of genes in degenerative pathways, including cellular senescence, mitochondrial dysfunction and low-grade inflammation (Lopez-Otin et al., 2013; Bhatia-Dey et al., 2016; Franceschi et al., 2017). This approach has several weaknesses, including the limited number of genes examined, together with any unintended selection bias. Further, the use of intact colon means that relatively small amounts of extracted mRNA are likely to be derived from neurons and other cells embedded within the much larger muscle mass. Thus, if genes of interest are in neurons at low copy number, statistically significant increases are difficult to detect, compared with genes which have a high intensity of expression in neurons (e.g., the present study detected TUBB3). An additional weakness was the use of primers for CDKN2A encoding p16^INK4A and p14^ARF, although this was mitigated to some extent by additional experiments with primers specific for p14^ARF alone. Nevertheless, although only an exploratory study, the results suggested an increase in expression of CDKN2A (encoding p16^INK4A and p14^ARF, not p14^ARF alone) and several other genes within the ascending colon of the elderly, with increased expression of a smaller number in descending colon.

CDKN2A (p16^INK4A and p14^ARF, not p14^ARF alone) was the only gene tested in which expression was upregulated in both ascending and descending colon from the elderly (although fold change was greater in ascending colon). By contrast, CDKN1A (encoding the senescence protein p21), showed no changes with age in either region of colon. Although not an exclusive divide (Krishnamurthy et al., 2004), the latter is often thought to be involved mostly with “acute” senescence at all ages, whereas p16 is important for “chronic” senescence, being associated with aging and a failure of the immune system to clear senescent cells (Stein et al., 1999). Indeed, chronic senescence may be a better marker of how tissue ages in response to genetic and environmental influences over time (Bhatia-Dey et al., 2016). For this reason, the gene expression data from the present study were reanalyzed according to the degree of expression of CDKN2A. The results showed that high levels of CDKN2A were associated with increased expression of a greater number of genes, compared with the number associated with advanced age, especially in ascending colon. Interestingly, the qPCR data showed some association between CDKN2A and components of the SASP, including TNF, VEGFB and genes involved with oxidative stress (expression of IL-6, often associated with SASP, was unchanged). Notably, the SASP may be context-dependent with different profiles, depending on cell type and cause of senescence (Gorgoulis et al., 2019).

To explore a possible association between chronic senescence and the aged colon, an immunofluorescence study was undertaken with p16. As a control to examine the integrity of the tissue, an immunohistochemistry study with CD45 demonstrated an absence of confounding increases in hematopoietic cells in each of the age groups and regions of colon investigated. Although a greater number of samples is now needed to make definitive conclusions, the subsequent p16 immunofluorescence study (n = 12–14 each age group and region of colon; N = 52 total) showed increased staining in myenteric nerve cell bodies of ascending colon from the elderly. Interestingly, this was not replicated in descending colon, indicating that the age-related increase in CDKN2A expression in this region of colon did not translate to increased p16 protein staining; the reasons for this lack of translation are not clear but could be related to the relatively small increase in CDKN2A in descending colon.

To investigate the biology of human GI neuromuscular and other functions it is important to use human GI tissues, often removed at surgery for disease. This means that to make progress such studies must confront any influence of the disease on the data generated, in addition to variations in patient age, gender and genetic background (Sanger et al., 2013). As in the present study, the use of an optimal sample size and an understanding of the clinical history of the tissue donors are part of the methods used to minimize variability. Thus, if properly controlled, a strength of the use of human tissues is that this can highlight differences between animal and human GI functions, especially important when conclusions are based on particular strains of rodents with unproved translational significance (Sanger et al., 2013). An investigation into the role of p16 in the aging human bowel presents its own challenges because although all tissues, irrespective of colon region and age, were taken as “macroscopically normal” sections 5–10 cm away from the tumor in cancer patients (see section “Human Tissues”), the possibility remains that the cancer itself could have influenced the expression of p16 and not aging. Thus, p16 may occur in the cytoplasm of carcinoma cells (colorectal, head and neck squamous cell, melanoma, astrocytomas; Straume et al., 2000; Zhao et al., 2003, 2012; Arifin et al., 2006), perhaps to inhibit nuclear function (Nilsson and Landberg, 2006). Nevertheless, the present data are striking because although all tissues were removed from cancer patients the region- and age-dependent increase in p16 staining argues in favor of an association with aging. Such a conclusion finds consistency with a previous report of an age-related decline in cholinergic function in the same ascending colon region, not in the descending colon (Broad et al., 2019).

It has conventionally been thought that senescence occurs in mitotic cells, involving upregulation of cell cycle-related genes (including p16), accompanied by exit from the cell cycle (Carnero, 2013). Nevertheless, Post-Mitotic Cell Senescence (PoMiCS) has been described, in which post-mitotic cells activate p16 and/or the p53/p21 axis, accumulate lipofuscin (“age pigment”; another indicator of senescence; Moreno-García et al., 2018) and secrete components of the SASP (Jurk et al., 2012; Sapieha and Mallette, 2018). This has been found in intestinal myenteric neurons of mice, increasing in frequency during advancing age (Jurk et al., 2012). PoMiCS cells exhibited severe DNA damage and oxidative stress, expressed markers of senescence, including p16, and demonstrated morphological changes typical of senescent cells, e.g., accumulation of lipofuscin, increased size of mitochondria, and reduced heterochromatin (Tan et al., 2014; Wan et al., 2014; Ishikawa and Ishikawa, 2019). Ishikawa and Ishikawa (2019) showed immunofluorescence images demonstrating cytoplasmic p16 protein staining in long-term cultured post-mitotic rat neurons. The present study now suggests that PoMiCS could occur within human myenteric neurons of the elderly.

Staining of the neuronal cytoplasm by the p16 antibody, with little-or-no nuclear staining, was not anticipated since p16 acts as a negative regulator of cell cycle progression at the G1 checkpoint in the nucleus (Serrano et al., 1993). Nevertheless, p16 has been reported to have roles in non-cell cycle-related processes including anoikis, matrix-dependent cell migration, protection from DNA damage and neuroprotection in aging mice (Fahraeus and Lane, 1999; Plath et al., 2000; Sen et al., 2017; Micheli et al., 2019), and in addition, has been shown to bind to cytoplasmic proteins (e.g., cytoplasmic β-actin and γ-actin, and Rin2; Souza-Rodrigues et al., 2007). Further, two forms of p16 are proposed, one in both the nucleus and cytoplasm, the other specific to the cytoplasm: Nilsson and Landberg, 2006). This evidence could suggest cytoplasmic roles for p16 unrelated to the cell cycle.

It is not clear if cytoplasmic p16 protein expression in myenteric neurons is involved with senescence or any of the above-described activities of p16, exerting a pathological or beneficial activity in the aging colon. Confirmatory studies are now needed to determine the presence or absence of other senescence and SASP protein markers in the neurons, and whether such changes are regulated by cytoplasmic p16.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and the study was approved by the East London Research Ethics Committee (REC: 10/HO703/71) and subsequently by the London City Road and Hampstead Research Ethics Committee (REC: 15/LO/21/27). All patients were fully informed of the aims of the study and use of tissue, but were not involved in study design, interpretation of results, or writing/editing of this report. Written informed consent was obtained from all patients undergoing surgery, for use of macroscopically-normal ascending and descending/sigmoid colon. The patients/participants provided their written informed consent to participate in this study.

AP performed the qPCR, the immunohistochemistry and subsequent analysis. SEp, EC, and YX performed immunohistochemistry and subsequent analysis. LG performed immunohistochemistry analysis. AP supervised the immunohistochemistry studies, with interpretation guided by JM. CB provided the p16 antibody and its validation. SEp, AP, EC, SEl, JC-A, and CK were instrumental in obtaining human tissue. ME guided interpretation of qPCR analysis. AP and GS designed the study and wrote the manuscript. GS obtained the funding. All authors had access to the study data, critiqued, read and approved the manuscript.

GS received funding from Takeda Pharmaceuticals. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. ME was involved in some parts of the analysis while she was employed by Takeda Pharmaceuticals, United States. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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