The role of oxidative stress in Drosophila hypoxia tolerance has been explored and reviewed in Zhou et al. (2011), Zhou and Haddad (2013). Unlike mammals, flies do not exhibit a significant increase in metabolic activity or ROS production following re-exposure to oxygen (reperfusion). However, flies expressing a mutant version of TRAP1 (Table 1), a mitochondrial chaperone belonging to the heat shock protein (HSP) 90 family that inhibits ROS accumulation, demonstrate increased metabolic activity and ROS production after hypoxia which results in decreased survival and motor recovery (Kokott-Vuong et al., 2021). TRAP1 mutant phenotypes are rescued with the antidiabetic and antioxidant drug metformin (Nasri and Rafieian-Kopaei, 2014; Kokott-Vuong et al., 2021). Similarly, decreased infarct size with metformin treatment was observed in the rodent MCAO model (Arbeláez-Quintero and Palacios, 2017) and humans taking metformin for type-2 diabetes have a more favorable outcome following ischemia (Jia et al., 2015).

In addition to TRAP1, other HSPs influence oxidative stress. Specifically, Hsp70 mRNA levels increase during reperfusion following hypoxia in both Drosophila and mammals (Habib et al., 2021; Kokott-Vuong et al., 2021) and may contribute to oxidative stress resistance. Flies overexpressing Hsp70 and 23 experience greater survival in hypoxic conditions (Zhou and Haddad, 2013). Similarly, pharmacological and genetic induction of Hsp70 after MCAO is protective in rodents (Kim et al., 2018). Given the similarities between oxidative stress functions in ischemia and hypoxic tolerance in Drosophila, further exploration into these pathways may reveal promising therapies for ischemia and stroke.

Several members of the insulin signaling pathway have been implicated in hypoxic tolerance in Drosophila, likely acting through the fat body (Table 1). As mentioned above, the Drosophila fat body plays a major role in hypoxic detection and subsequent metabolic suppression (Texada et al., 2019; Noguchi et al., 2022). As a result of this metabolic suppression following hypoxia, larvae exhibit a significant decrease in growth and body size that contributes to their ability to survive hypoxic conditions. Dilp2, a ligand for the Drosophila insulin receptor, accumulates in insulin-producing cells after hypoxia, resulting in a decrease in Dilp2 release and subsequent insulin receptor activation. Overexpression of the insulin receptor reverses this phenotype, suggesting that decreased insulin signaling following hypoxia plays a role in larval growth inhibition (Wong et al., 2014).

Insulin receptor ligand binding results in activation of the TORC1 complex that in turn activates the ribosomal protein S6K. Contrary to mammalian cells where induction of mTOR and its effectors seem to reduce protein synthesis as an adaptation to hypoxic conditions, in Drosophila, downregulation of this pathway appears to be important in the Drosophila hypoxia response (Zhou and Haddad, 2013). For example, suppression of TORC1 in adipose tissue contributes to hypoxia adaptation in larvae by controlling body growth (Lee et al., 2019) while TORC1 induction decreases eclosion rates during hypoxic conditions. Overexpression of S6K significantly reduces survival in hypoxic conditions (Lee et al., 2019). Similarly, inhibiting S6K in mouse MCAO decreases ischemic infarct size (Chi et al., 2019).

FOXO, a transcription factor inhibited by insulin pathway activation, mediates tolerance to 5% hypoxia in both adult and larval flies reared in those conditions (Barretto et al., 2020). Adult foxo mutants experience significantly decreased survival when exposed to severe hypoxia. In mammals, increasing FOXO3 using adenovirus in rats (Zhou et al., 2019) and inhibition of miRNA targets of FOXO in mice (Yan et al., 2020) are protective against MCAO. Given the similarities in the insulin system between mammals and Drosophila and that insulin inhibition appears to contribute to hypoxic tolerance, additional research into insulin signaling may reveal potential targets for ischemic therapies.

Hypoxia inducible factors (HIFs) mediate Drosophila’s response to hypoxia (Table 1; Dekanty et al., 2005). There are several HIF human homologs in Drosophila including Sima (similar, HIF1α homolog); Tgo (tango, HIFβ homolog) and Fatiga (fatiga, PHD homolog). Tgo is constitutively expressed regardless of oxygen conditions, while sima expression only occurs following hypoxic conditions (Romero et al., 2007). The HIF pathway in Drosophila has been previously reviewed (Gorr et al., 2010; Zhou and Haddad, 2013) and different Drosophila HIF homologs appear to play distinct roles in hypoxia (Misra et al., 2017; Baccino-Calace et al., 2020).

Sima regulation by insulin and TOR pathways is conserved (Romero et al., 2007), and NF-κB (Bandarra et al., 2014) regulates sima induction. Sima activation seems necessary for hypoxia adaptation as overexpression of sima triggers the inhibition of fat body growth, a critical event in hypoxia tolerance, via activation of Trbl and subsequent inhibition of Akt signaling (Noguchi et al., 2022). In Drosophila, null mutants of sima result in larvae that cannot adapt to hypoxia (Centanin et al., 2005) and cardiac problems in adult flies following acute hypoxia (Zarndt et al., 2015). Interestingly, in adults, the loss-of-function sima mutant does not show differences in survival after anoxia compared to controls (Vigne et al., 2009), suggesting a more pivotal role of sima in larvae than adults. In mammals, HIF function is not consistent, as contradictory results using the MCAO model were observed (reviewed in He et al., 2021).

Loss-of-function fatiga mutants have high lethality in normoxic conditions, which is reversed if sima function is also lost (Centanin et al., 2005). In the MCAO model, inhibiting PHDs, which degrade HIF in normoxic conditions, decreases damage (reviewed in Davis et al., 2019) but this neuroprotection may be independent of HIF1α (Li et al., 2019). Lastly, tgo (Tango) seems important in tracheal development (Emmons et al., 1999) but loss-of-function tgo mutants have not been examined in response to hypoxia. Similarly, no publications directly manipulating HIF1β in mammalian models of ischemia exist. While HIF1α function in mammalian stroke has been investigated, studies investigating the role of HIFβ subunits are necessary as they might be valuable targets for future stroke treatments.

In Drosophila, pathogens trigger the immune response, which is mediated by the NF-κB system (Minakhina and Steward, 2006). NF-κB is released from sequestration by IKK (inhibitor of kB protein kinase), allowing it to bind to promoters of effector genes (Barretto et al., 2020). The cascade involving the release of NF-κB is triggered by the activation of the Toll receptor bound with the Spatzle ligand (Valanne et al., 2011). Although the Toll receptor system in hypoxia has not been examined in Drosophila, toll-receptor 4 expression in endothelial cells decreases following hypoxia, a response mediated by the presence of ROS (Ishida et al., 2002). In support of this finding, inhibiting Toll-like receptor 4 attenuated several hypoxic–ischemic injuries within the brains of neonatal rats (Zhu et al., 2021). Specific subunits of NF-κB, dorsal (p65 homolog), relish (p50/p52 homolog) and dif (p65 homolog), are activated in both adults and larvae following 24 h of hypoxia (Table 1; Bandarra et al., 2014).

Non-specific pharmacological inhibition of NF-κB protects against MCAO damage in mammals (reviewed in Harari and Liao, 2010). Interestingly, studies of MCAO induction in mice lacking p50 demonstrate contradicting data showing both increased and decreased infarct size (Harari and Liao, 2010), suggesting a complicated interplay between factors of the NF-κB signaling pathway in mammals. Currently, few studies exist in Drosophila investigating components of the NF-κB pathway during hypoxia. However, one study using a Drosophila relish mutant observed decreased survival following hypoxia compared to wild type controls (Barretto et al., 2020), supporting MCAO findings in p50 knockout mice (Li et al., 2008). More studies manipulating components of this pathway or tissue-specific expression of Toll-like receptors need to be conducted to elucidate their role in hypoxic tolerance.

The canonical Wnt pathway is highly conserved, playing a role in disease and several developmental events. The pathway is activated following binding of the glycoprotein Wingless (Wg) to the transmembrane receptors Frizzled (Fz) and Arrow (Arr). Once activated by Wg, Fz and Arr recruit the intracellular protein, Dishevelled (Dsh) which, together with other proteins, suppress the activation of a destruction complex, ultimately resulting in the translocation of the transcription factor, Armadillo (arm) to the nucleus and initiation of gene expression (Komiya and Habas, 2008; Bejsovec, 2013).

In Drosophila bred to tolerate hypoxic conditions, polymorphisms of Wnt pathway members may contribute to this generational tolerance (Gersten et al., 2014). Furthermore, a p-element screen revealed several Wnt-related genes as pivotal in hypoxic tolerance (Azad et al., 2012). Indeed, neuron-specific overexpression of Wnt pathway signaling increased rates of eclosion and knocking them down decreased rates of eclosion (Gersten et al., 2014). Likewise, activation of Wnt signaling in mammalian models of stroke seems promising (reviewed in Mo et al., 2022).

Sima (HIF1α homolog) triggers the production of Wg (the Wnt pathway ligand, Table 1) to facilitate Wnt signaling in neurons, suggesting Wnt signaling underlies hypoxic tolerance (Chen et al., 2019). However, this research was conducted with trachealess mutants and not with hypoxic conditions. Supporting the involvement of Wnt in hypoxic tolerance, a down-stream factor of Wnt activation, the Swim protein, is upregulated following adult and larval hypoxia and induces stem cell proliferation in brain injury (Simões et al., 2022). Further exploration of Wnt signaling, especially in adult Drosophila is warranted to understand the contribution of this pathway in hypoxic tolerance as current research is primarily in larva.

The Hippo pathway has Drosophila and mammalian homologs and has been reviewed by Fu et al. (2022) (Table 1). This pathway is activated by stress signals, including hypoxia. There is also opportunity for crosstalk between this pathway and others that are activated during hypoxia, like HIFα and insulin (Fu et al., 2022). A member of the Hippo pathway, homeodomain interacting protein kinase (Hipk), phosphorylates Yorkie, a downstream effector within the pathway, which then translocates to the nucleus to regulate transcription of effector genes (Snigdha et al., 2019; Steinmetz et al., 2021). In the context of hypoxia, Hipk binds to FOXO and regulates low oxygen survival. Indeed, 1% oxygen conditions increased expression of Hipk and other Hippo members, and fewer Hipk knock-down flies survived these conditions (Ding et al., 2022). Moreover, degradation of Hipk using F-box protein 3 in rat MCAO significantly increases infarct size (Gao et al., 2022). This suggests that activation of the Hippo pathway may be a promising target for stroke treatment and additional studies into this pathway are needed.

Involvement of the Notch signaling pathway in hypoxic tolerance has been reviewed extensively for Drosophila (Table 1; Zhou and Haddad, 2013). Notch is a transmembrane protein which, following proteolytic cleavage releases its intracellular domain which regulates transcription (Bray, 2006). Flies with loss-of-function mutations or RNAi-mediated knockdown of Notch are extremely sensitive to low levels of oxygen. Conversely, those with gain-of-function mutations are highly resistant to hypoxia (Zhou et al., 2011). Absence of Notch in excitatory amino acid transporter-positive glial cells decreases eclosion rates under hypoxic conditions, suggesting a possible mechanism for the tolerance (Zhou et al., 2021). Furthermore, Notch signaling elements interact with HIF pathways to regulate hypoxic adaptation, including high altitude adaptation in human populations (O'Brien et al., 2022).

While the Notch signaling pathway appears to be protective in Drosophila hypoxia, this is one instance where the findings in Drosophila are not corroborated by mammalian research. The role of Notch in ischemia has been reviewed (Arumugam et al., 2018) and it appears to be a pro-apoptotic factor, contributing to neuronal death following stroke. Lipoxin A4, an anti-inflammatory agent (Li et al., 2021), has positive effects on stroke and seems to work by suppressing the actions of Notch. Conversely, administration of osthole reduced cerebral infarct following MCAO in rats through activation of the Notch pathway (Guan et al., 2017). Whatever difference exist in these pathways, it appears that the actions of Notch in mammalian models of stroke are not as clear as those observed in Drosophila hypoxia, so further study of Notch in hypoxic tolerance may not be beneficial.