A Framework for intestinal barrier dysfunction in aging

Abstract

The intestinal epithelium serves as a barrier that facilitates interaction between intrinsic and environmental factors. Aging is accompanied by the gradual deterioration of this barrier. We postulate that barrier dysfunction results from defects in epithelial membrane trafficking that exacerbate age-related metabolic imbalances. Herein, we integrate barrier integrity, protein homeostasis, membrane trafficking and intracellular lipid sensing into an age-determining mechanism.

The barrier of intestinal epithelium loses structural integrity with age, but the question remains of whether this is causative or merely the consequence of aging. Owing to the recent emergence of this field within the past decade, we are only beginning to understand the molecular details that underlie intestinal barrier dysfunction and its predisposition to pathogenesis and aging(1). Deterioration of the intestinal epithelium has been linked to age-associated metabolic diseases with molecular determinants that include the dissociation of cell adhesion complexes(2). In line with well-established areas of research surrounding age, we propose that healthy aging and pro-longevity are deeply rooted in protein homeostasis maintenance, membrane trafficking and intracellular lipid-sensing pathways whose coordinate functions ensure intestinal barrier integrity and a youthful systemic state later into life. Herein, we highlight emerging research on the intestinal barrier that is at the key intersection of these distinct fields.

Protein homeostasis is essential for the maintenance of intestinal barrier integrity

The intestinal epithelium is one of the largest absorptive surfaces in the human body, measuring approximately 32 m²(3). Consisting of a single layer of polarized epithelial cells with an apical surface and a basal membrane, the intestine interacts with myriad environmental factors, regulates nutrient and water absorption, prevents host bacterial invasion, and houses commensal microbiota(1). The apical surface lines the digestive tract and is overlaid by protrusions termed microvilli, which enhance its absorptive capacity through increased surface area(3). A dense network of actin–intermediate filaments (termed the subapical terminal web) provides structural integrity for the microvilli, as well as anchorage points for cell adhesion molecules to tether neighboring epithelial cells(4,5). Previous studies have demonstrated that age-onset decline in these cell junctions can contribute to deterioration in intestinal barrier integrity(2). At the opposite end of the epithelial cell, the basal membrane regulates the export of nutrients and macromolecules for systemic utilization. As an established hallmark of aging, we sought to understand how maintenance of protein homeostasis within the intestinal epithelium could affect functionality of the subapical actin-rich terminal web and its ability to sustain intestinal barrier integrity throughout the lifespan.

A dye-exclusion technique developed in Drosophila melanogaster has firmly established the loss of intestinal barrier integrity as a highly accurate predictor of mortality(2,6). Termed the ‘Smurf assay’ (owing to the intestinal paracellular diffusion of membrane-impermeable blue dyes into the body cavity), intragenerational variation in barrier integrity predicted overall lifespan in age-synchronized flies more precisely than did chronological age(2,6,7). Further studies correlated the Smurf phenotype with marked age-onset deterioration of intestinal stem cell function, systemic inflammation and pathological microbiome imbalances(2,6,7). Similarly, in age-synchronized Caenorhabditis elegans, diffusion of the same blue dyes or invasion of mCherry-expressing Escherichia coli between enterocytes accurately depict intestinal barrier dysfunction and consequent mortality(7,8). It is important to note that worms lack inflammatory responses, complex microbiomes or intestinal stem cells. However, dye-exclusion assays and pathogenic invasion still predict age-associated mortality, similar to flies(7). This result suggests that simply maintaining a barrier against extrinsic elements and ensuring organismal compartmentalization have an important role in age determination.

In a study investigating the molecular mechanisms that link barrier dysfunction and age, we previously demonstrated in C. elegans that the stability of a low-abundance intestinal-specific actin, ACT-5, is essential for maintaining intestinal barrier integrity(8). Stability of ACT-5 is dependent on heat shock transcription factor 1 (HSF-1), an established regulator of protein homeostasis and longevity(8,9,10). Age-associated loss of HSF-1 expression relieves suppression of the c-Jun kinase KGB-1, which resulted in the hyperphosphorylation, structural destabilization and aberrant condensation of ACT-5(8,10). As a consequence, morphological aberrations manifest as shortened, irregular and decaying apical protrusions. Because ACT-5 is a major component of the subapical terminal web, its aberrant function and mislocalization (caused by loss of HSF-1) promoted adherens junction dissociation and subsequent loss in barrier integrity, aberrant permeability and luminal distension8. Concomitantly, HSF-1-mediated disruption of the intestinal actin network impedes engagement of a myosin–Rab GTPase scaffold with ACT-5 filaments, thus limiting trafficking and recycling of macromolecule transporters at the apical surface(10). Should disruption of the ACT-5 network persist (as reported in the aged intestine) nutrient absorption cannot be sustained; this subsequently triggers metabolic dysfunction and activation of the proposed lipid-sensing response, which alerts the cell of impending starvation and attempts to re-enforce both nutrient absorption and macromolecular catabolism(8,10).

Restoration of membrane trafficking via lipid sensing to regain metabolic homeostasis

Membrane trafficking is the biological process by which proteins and other macromolecules are distributed throughout the cell, taken up by the cell in endocytosis or secreted out of the cell in exocytosis(11). This complex mechanism of cargo sorting and targeting to the correct subcellular locale is coordinated by the largest family of small G proteins: Rab GTPases, of which there are approximately 60 different types in the cell(11). As small monomeric GTP-binding molecules, these Rab proteins associate with transport vesicles and organelles through a covalently bound isoprenoid (a process known as prenylation), which anchors the small G protein to the membrane bilayer. Rab prenylation is predominately dependent on unsaturated 20-carbon geranylgeranyl pyrophosphate (C20:4) that is synthesized de novo via the mevalonate pathway(12). RAB-11.1 (orthologous to Rab11a, Rab11b and Rab25 in mammals) regulates vesicular trafficking through recycling of endosomal compartments and the early endosome to the trans-Golgi network and plasma membrane(11). Although membrane proteins at the cell surface are regularly internalized for regulatory and quality-control purposes, the actions of RAB-11.1 are required to properly recycle these proteins back onto the surface of the cell(11). Thus RAB-11.1 has a critical role in maintaining the surface residency of membrane-associated proteins, including tight junction proteins such as actin, cadherins, claudins and occludins, as well as macromolecular transporters that are responsible for nutrient absorption(10,13).

If endocytic recycling is the actor, we propose that the lipid sensing pathway can be viewed as the director who orchestrates this complex mechanism. Despite seemingly distinct pathways, we propose that lipid sensing and endocytic recycling are linked through the synthesis of geranylgeranyl pyrophosphate and, importantly, in its conjugation to Rab GTPases. First, geranylgeranyl pyrophosphate is primarily synthesized by the body rather than obtained directly through the diet. Second, cells stockpile carbon-rich resources in lipid droplets, which are catabolized to not only provide energy but also carbon precursors for the synthesis of essential molecules such as geranylgeranyl pyrophosphate(12). Upon nutrient deprivation, depletion of metabolizable carbon pools reduce overall synthesis of geranylgeranyl pyrophosphate through the mevalonate pathway(13). With limited levels of geranylgeranyl pyrophosphate, Rab GTPases cannot be prenylated and thus are unable to facilitate membrane trafficking — including endocytic recycling, which depletes membrane proteins, nutrient transporters and cell adhesion molecules on the surface of the cell. This presents a challenging situation as it prevents the starving cell from absorbing systemic or environmental resources that are needed to survive and eventually recover. It also prevents the trafficking and recycling of adhesion molecules that are needed to maintain cell junctions and tissue integrity. We propose that lipid sensing, in essence, enables epithelial cells to indirectly gauge low levels of metabolizable carbon resources by monitoring de novo geranylgeranyl pyrophosphate synthesis and, importantly, its conjugation to the Rab11 family of small GTPases (specifically RAB-11.1, in worms)(13). Upon sensing plummeting levels of geranylgeranyl pyrophosphate, the cell induces the transcription of genes involved in endocytic recycling, geranylgeranyl transferase and nutrient transport(10,13). Through this elaborate stress response pathway, the cell restores metabolic homeostasis by bolstering endocytic recycling and enhancing the cell-surface residency of macromolecular transporters and junctional proteins(13).

Aging originates from metabolic dysfunction caused by a collapsed intestinal cytoarchitecture

Intestinal barrier integrity is a highly accurate predictor of mortality in model organisms. Nevertheless, human intestinal dysfunction is complex and multifaceted, which results in descriptive observations without a means to directly test hypotheses. A dichotomy arises in human studies in which the loss and remodeling of intestinal tight junctions does not necessarily correlate with permeability defects. Colonic biopsies show age-related loss of E-cadherin and occludin in disease-free human individuals(14). Moreover, differences in barrier permeability can be attributed to aging in primates such as baboons(1). Yet barrier permeability cannot be accounted for solely by age in humans(15). To date, clinical research has focused on whether aging increases intestinal barrier dysfunction, leaving untested the opposing hypothesis of whether such dysfunction can accelerate aging. Therefore, despite suggestions that barrier integrity is crucial for a healthy intestine, the extent to which defects in gut permeability drive the aging process in humans remains unclear.

As such, the question arises of whether aging could be a consequence of an unstable actin network, faulty endocytic recycling or a widespread loss of cell adhesion. Central to age-associated barrier dysfunction is the dysregulation of membrane-dwelling components at the apical and lateral membranes, including transport machinery and junctional proteins(2,8,10,13). The inability to maintain geranylgeranyl pyrophosphate production — which is required for prenylation and trafficking of Rab proteins and their associated vesicular cargo — decreases nutrient uptake and dismantles cell junctions. In parallel, age-onset decline in HSF-1 abundance and activity both relieves suppression of the KGB-1 kinase and impairs protein folding machinery, which results in the hyperphosphorylation, destabilization and aggregation of intestinal actin(8,9) — the net result is a collapse of the intestinal barrier and unraveling of the actin terminal web. Although additional studies are required to better determine the precise timing of this cluster of events, we postulate that a major contributing factor to intestinal barrier dysfunction is the inability to regulate endocytic trafficking owing to age-related intracellular metabolic imbalances (Fig. 1).

Figure 1. Barrier integrity, protein homeostasis, membrane trafficking and intracellular lipid sensing synergize to fend off the aging process.

A healthy young cell with an intact actin network can recycle nutrient transporters to the apical surface and adherens junctions to the lateral surface. When the actin network unravels and aggregates during aging or disease, membrane trafficking can no longer function efficiently. The loss of nutrient transporters and adherens junctions leads to nutrient deprivation and paracellular bacterial invasion. As membrane trafficking is diminished, the intracellular lipid sensing pathway is activated to restore endocytic recycling and consequently improve both nutrient absorption and cis-cellular adhesion.

Here we provide an integrated perspective on animal fitness deterioration with age that incorporates intestinal barrier integrity, protein homeostasis, membrane trafficking and lipid sensing, in which the unifying occurrence is dysfunctional endocytic recycling at epithelial barriers provoked by destabilization of the actin network. During aging, endocytic recycling mediated by lipid sensing is as important for nutrient absorption as it is for sustaining tissue barrier integrity through modulating the apical surface residency of any given protein, including macromolecule transporters and cell adhesion proteins. Aging is accelerated when protein surface residency and barrier permeability are compromised, which ultimately increases the risk of mortality. Overall, we hypothesize that animals prevent age-associated metabolic disturbance by fending off intestinal barrier instability and that the proposed lipid sensing pathway has the potential to reestablish both metabolic and barrier homeostasis.