Like the intestine in other animals, the Drosophila midgut hosts a variety of commensal, food-borne, and pathogenic bacterial populations (Hooper and Gordon 2001; Sansonetti 2004). Flies have evolved a variety of mechanisms to accommodate beneficial flora and defend the intestinal epithelium against pathogens (Ryu et al. 2010). Interestingly, dramatic changes in the intestinal flora occur during aging in mammals (Tiihonen et al. 2010; Woodmansey 2007). Supporting the notion that similar changes in gut flora strongly contribute to the aging process of the intestinal epithelium in Drosophila, maintaining flies in an axenic environment prevents age-induced ISC proliferation and intestinal dysplasia (Buchon et al. 2009a). However, it is still lacking a clear description of age-related changes in the composition and/or size of bacterial populations in the midgut of older flies. In addition, the reasons for these potential changes remain unclear. Several non-exclusive mechanisms may drive these alterations, such as defects in the immune system (i.e., immunosenescence); decay of the peritrophic membrane, a structure essential to protect the intestinal epithelium against bacterial infections (Kuraishi et al. 2011); changes in intestinal function and chemical properties; and selection or expansion of more virulent bacterial strains over time. Importantly, two recent studies have started to address this question and suggest that a collapse of proper immune signaling in the gut itself and the adipose tissue, another essential immune organ in flies, plays a fundamental role in dysbiotic changes occurring in older animals (Chen et al. 2014; Guo et al. 2014). Yet, further investigations will be required to test all the possible factors contributing to age-related changes in microbiota and fully understand the underlying age-related molecular changes.

After establishing that bacteria that populate the intestine play a major role in the age-related degeneration of this tissue, one essential question emerges: how do changes in the gut flora influence intestinal homeostasis and ISC proliferation in older flies? Answers to this question came from the study of the mechanisms that control intestinal regeneration in response to bacterial infection and tissue damage (Fig. 5.2).

Production of reactive oxygen species (ROS) is one of the major responses of the intestinal epithelium to cope with bacterial infection, and specific mechanisms to fight against this oxidative stress have been identified in Drosophila (Ha et al. 2009, 2005a, b). Interestingly, antioxidant molecules, such as N-acetylcysteine and glutathione, prevent induction of ISC proliferation after bacterial infection (Buchon et al. 2009a), while exposure to ROS-generating molecules dramatically increases ISC proliferation (Biteau et al. 2008; Choi et al. 2008), indicating that this oxidative burst directly impacts ISC proliferation.

The increased ROS production and the recognition of bacteria themselves lead to the stimulation of multiple signaling pathways in ECs and the establishment of both an antimicrobial and an inflammatory response, which are essential for the survival of the organism (Buchon et al. 2009a, b; Cronin et al. 2009; Jiang et al. 2009). One major component of this response is the activation of the JNK signaling pathway in ECs, which induces the production of interleukin-like cytokines (unpaired, Upds). This secretion of Upds by ECs leads to the activation of the JAK-STAT signaling pathway in ISCs, directly promoting their proliferation (Buchon et al. 2009a, b; Jiang et al. 2009). At the same time, this inflammatory signal causes the activation of JAK-STAT in the visceral muscle that surrounds the intestinal epithelium. This results in the upregulation of the expression of the EGF ligand vein, which in turn further promotes stem cell proliferation through the activation of the EGFR-MAPK signaling pathway in ISCs (Buchon et al. 2010; Jiang et al. 2011).

Similar to the response to bacterial infection, damage to the intestinal epithelium, such as DNA damage or disruption of the basal membrane, promotes ISC proliferation and tissue repair through the activation of the same stress-responsive signaling pathways (Amcheslavsky et al. 2009).

Over the last few years, it has become clear that during normal aging, similar mechanisms are chronically activated in the intestine. For example, high levels of ROS and increased activity of the JNK pathway are detected in ECs of older flies (Biteau et al. 2008; Hochmuth et al. 2011), and the expression of proliferative signals such as EGF ligand and Upd cytokines is greatly elevated in the aging fly midgut. In addition, the artificial activation of the mechanisms that promote ISC proliferation in response to infection or tissue damage recapitulates the dysplastic phenotype observed in old animals. Finally, recent evidence suggests that the muscles that surround the gut epithelium deteriorate with age (Larson et al. 2012), a process that may indirectly affect ISCs, through the deregulation of muscle-derived proliferative signals (in particular the ligand vein).

Taken together, these studies strongly suggest that chronic activation of the non-cell-autonomous mechanisms that control tissue repair in young animals significantly contributes to the uncontrolled ISC proliferation and intestinal dysplasia observed in older flies. However, it is important to note that the current model remains to be conclusively validated, as the effects of stress signaling pathways in ECs on age-related changes in the ISC regulation and intestinal homeostasis have yet to be directly investigated.

Interestingly, other cell types and tissues have been shown to interact with ISCs and the midgut epithelium to control stem cell proliferation and the function of the intestine. While little is known about the potential effect of aging on these other interactions, it is important to take these into account when trying to elucidate the complex mechanisms that affect ISC in aging flies, as defects in these mechanisms may directly contribute to the ISC hyper-proliferation and differentiation defects observed in the intestine of old flies.

Within the intestinal epithelium, all the different cell types constantly communicate to maintain homeostasis. Undifferentiated EC-committed daughter cells (i.e., enteroblasts, EBs) are capable of repressing ISC proliferation through the maintenance of E-cadherin-mediated cell-cell interactions (Choi et al. 2011). This interaction is directly regulated by the activity of the insulin receptor pathway in EBs, a conserved signaling pathway that is strongly influenced by aging (see below). At the same time, secretory cells (i.e., enteroendocrine cells, EEs) indirectly control ISC proliferation by regulating muscle-derived production of dilp3 (insulin) and vein (EGF) (Amcheslavsky et al. 2014; Scopelliti et al. 2014). Hence, while age-related defects in these feedback mechanisms have not yet been investigated, impaired signaling in EBs and/or EEs may significantly contribute to the uncontrolled proliferation of aging ISCs.

The complex interactions between ISCs, ECs, and the surrounding visceral muscle were described before. In addition to this regulatory network, other neighboring tissues influence the cells of the intestinal epithelium. The tracheal system, which delivers oxygen to all tissues in the fly, also provides an essential factor that promotes EC survival (Li et al. 2013). Loss of this signal, dpp (decapentaplegic, a member of the bone morphogenetic protein family), induces a strong compensatory ISC proliferation. In addition, the intestine is innervated by a complex neuronal network. These neurons control the adaptation of the intestinal function and excretion to nutrient availability and reproduction (Cognigni et al. 2011). Age-related changes in these organs have not yet been identified, but it is conceivable that misregulation of the signals originating from these tissues participates to the decline in intestinal function in older animals.

Finally, ISCs are regulated by systemic factors, including the activity of the insulin signaling pathway and nutrient availability. In Drosophila, insulin-like peptides (dilps) are secreted by multiple tissues. Notably, dilp2, dilp3, and dilp5 are produced by the neurosecretory insulin-producing cells (IPCs) in the brain (Teleman 2010). Relevant to the regulation of ISCs, dilp3 is also expressed in the visceral muscle surrounding the intestine and regulates the proliferation and number of ISCs in the intestine in response to changing nutritional conditions (O’Brien et al. 2011). Importantly, modulation of the Insulin signaling pathways is the one of the most widely conserved mechanisms that affects aging and longevity, from worm to mammals (Fontana et al. 2010; Kenyon 2010; Partridge et al. 2011). Indeed, reducing insulin signaling systematically or specifically in ISCs prevents age-related intestinal dysplasia (Biteau et al. 2010). Similarly, changes in nutrient availability that are known to affect insulin signaling, aging, and longevity modulate ISC proliferation (McLeod et al. 2010). These observations thus support the notion that, in Drosophila, age-related changes in metabolism and insulin signaling impact the biology of ISCs and that alterations of insulin signaling influence aging and extend lifespan, at least in part, by controlling the function of the stem cell pool in the intestine in old flies.

Supporting the notion that ISC themselves are stressed in the aging intestine, high levels of ROS are detected in old ISCs (Hochmuth et al. 2011), and ISCs accumulate damaged macromolecules with age, as shown by the activation of the DNA damage response (γH2AX) and presence of DNA lesions (8-oxo-deoxyguanosine) (Park et al. 2012). Furthermore, overexpression of antioxidant proteins (the peroxiredoxin Jafrac1 and Gclc, an enzyme in the glutathione biosynthetic pathway) in ISCs and EBs is sufficient to reduce ISC proliferation and delay the progression of the intestinal dysplastic phenotype (Biteau et al. 2010; Hochmuth et al. 2011). These intracellular prooxidant conditions lead to the cell-autonomous activation of multiple signaling pathways that promote ISC proliferation, including the JNK and MAPK p38 pathways (Biteau et al. 2008; Park et al. 2009). At the same time, the Nrf2 signaling pathway, which promotes ISC quiescence under normal conditions, is inactivated, further increasing ISC proliferation rate (Hochmuth et al. 2011). Importantly, genetic modulations of these signaling pathways that revert the influence of aging on their activity are sufficient to prevent age-related tissue degeneration in the posterior midgut (Biteau et al. 2010; Hochmuth et al. 2011; Park et al. 2009), demonstrating the critical role of the cell-autonomous activation of stress signaling in the aging of the intestinal stem cell population.

In addition to the response to oxidative stress, ISCs are capable of responding directly to changes in the epithelium integrity and cell-cell interactions, through the inactivation of the Hippo pathway. Decreased activity of this pathway in ISCs leads to the activation of the transcription factor Yorkie and results in increased ISC proliferation (Ren et al. 2010; Shaw et al. 2010). Therefore, age-related changes in the architecture of the intestinal epithelium are likely to lead to the activation of Yorkie and also participate to the establishment of a chronic ISC-proliferative state.

As discussed previously, the increasingly prooxidant ISC microenvironment and the intracellular redox imbalance appear to be the major causes of the increased proliferation of aging ISCs. In addition to the ROS produced by ECs as part of the immune response, it has recently been recognized that mitochondria in aging ISCs themselves are an additional source of ROS (Rera et al. 2011). This recent study suggests that the mitochondrial function in the intestine declines with age. Importantly, reverting these effects of age by increasing the expression of the fly homolog of the PGC1 transcription factors is sufficient to promote mitochondrial activity, decrease the accumulation of ROS in aging ISCs, and prevent ISC hyper-proliferation. These recent findings further confirm that age-related intracellular changes in ISCs directly affect their function in older animals.

Lineage tracing analyses and the use of many cell-specific markers have allowed the elucidation of the ISC lineage in Drosophila posterior midgut (Fig. 5.3). These multipotent stem cells were first described as generating at least three different cell types, nonproliferating undifferentiated progenitors (enteroblasts, EBs), which differentiate in either enterocytes (ECs) or enteroendocrine cells (EEs) (Micchelli and Perrimon 2006; Ohlstein and Spradling 2006). However, it has recently been recognized that the ISC lineage is much more complex than what was originally thought. ISCs primarily asymmetrically divide to give rise to another ISC and a daughter that will undergo differentiation. Yet, ISCs have also been shown to symmetrically divide and generate two ISCs or two daughter cells (de Navascues et al. 2012; Goulas et al. 2012; O’Brien et al. 2011). In addition, it has recently been found that ISCs can give rise to two distinct types of daughter cells: EBs, committed to the absorptive lineage (ECs), and pre-EEs, which differentiate into secretory cells (Biteau and Jasper 2014; Zeng and Hou 2015).

While the intestinal lineage is relatively well characterized, the mechanisms that regulate the balance between self-renewal and differentiation remain largely unclear. In this regard, ISC seems to share some similarities with larval neuroblasts, as suggested by the asymmetric segregation of some components of the Par complex (Goulas et al. 2012), which may specify the EB cell fate during cell division. In addition, it has been suggested that the wingless/Wnt signaling pathway is required for ISC maintenance and may control self-renewal (Lin et al. 2008). Also, the balance between self-renewal and differentiation can be dynamically regulated, in particular during the regrowth of the intestine in response to re-feeding after a period of starvation, a process that may involve the insulin signaling pathway (O’Brien et al. 2011). Several models have been developed to describe the mechanisms that control the decision between self-renewal and differentiation (de Navascues et al. 2012; Kuwamura et al. 2012; Perdigoto et al. 2011). However, it has been recently found that endocrine cells directly control their own production, through the Slit/Robo2 signaling pathway, ensuring that Prospero-expressing daughter cells, which are committed to the secretory lineage, are only produced when EEs are missing in the immediate ISC vicinity (Biteau and Jasper 2014).

In addition to self-renewal signals, multiple conserved signaling pathways have been shown to be critical for the differentiation of ISC daughter cells. Notably, the Notch signaling pathway is absolutely required for the differentiation of EBs into ECs, while the JAK/STAT pathway is involved in the differentiation of EEs (Beebe et al. 2010; Lin et al. 2010; Ohlstein and Spradling 2007

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