Background
Hypoxia-ischemia (HI) in the perinatal period is associated with long-term disabilities affecting 1–4 infants per 1000 births [
2,
13,
40]. The most common cause of HI is intrauterine asphyxia, which may be brought on by placental artery clotting, abruption, or inflammatory processes [
7]. In the event of prolonged abruption and extended period of HI, the neonate develops hypoxic-ischemic encephalopathy (HIE), causing irreversible brain injury [
7].
A main contributing injury mechanism post-HI is the disruption of correct protein folding that subsequently triggers reactive oxygen species (ROS) accumulation, microglia activation, and inflammation [
8,
14,
44]. Previous reports have shown inflammatory cytokine release to be significantly elevated in the full-term infant post-HIE, which is linked with further exacerbating the damage and results in poor neurodevelopmental outcome [
7]. Therefore, inhibition of inflammation is an attractive target for new therapeutic strategies.
Correct folding of transmembrane proteins takes place in the endoplasmic reticulum (ER) which is composed of an elaborate system of chaperones and enzymes [
37]. However, under stressful conditions, such as after HI injury, the number of unfolded proteins exceeds the capacity of the chaperones leading to the accumulation of unfolded proteins and ER stress [
33]. The ER responds to this by activating the unfolded protein response (UPR), which triggers sensor proteins to recognize and ameliorate ER stress [
33,
47,
50]. However, persisting ER stress leads to the over-activation of the UPR, a highly redox-dependent pathway that causes accumulation of ROS [
15]. ROS are a natural byproduct of signaling pathways; however, during stress, ROS production is increased significantly, which causes oxidative stress and damage to the cell [
15].
A major source of ROS production at the ER is from the microsomal monooxygenase (MMO) system which is composed of cytochrome P450 (CYP), NADPH-P450 reductase (NPR), and phospholipids [
12,
15,
32]. Specifically, cytochrome P4502E1, a member of CYP, is associated with the production of large amounts of ROS, due to the leakage of electron transfer between P4502E1 and NPR, thus indicating an important role of these cytochromes during ER stress [
15,
32].
Bax Inhibitor-1 (BI-1) is an evolutionary protein, encoded by the TMBIM6 gene, which mainly resides on the ER membrane. BI-1 is a member of the TMBIM family, associated with cytoprotection [
45,
49], and has been suggested to regulate ER stress-induced ROS production and subsequent inflammatory cytokine production via two essential mechanisms [
19]. First, BI-1 can directly inhibit ROS by disrupting the NPR-CYP complex, a major generator of ROS. BI-1 alters the electron flow from P4502E1 to NPR, thus destabilizing this complex and attenuating ROS accumulation [
12,
15,
17]. The P4502E1 member was found to be significantly reduced and its upregulation attenuated after ER stress in BI-1 overexpressing cells [
15]. Second, BI-1 can directly increase the production of anti-oxidant transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf-2) [
17,
19]. Nrf-2 stimulates the production of antioxidant enzymes, heme oxygenase-1 (HO-1), which in turn blocks ROS, thereby attenuating inflammation and promoting cell survival. Cells that overexpressed BI-1 increased Nrf-2 and HO-1 expression while its protective effects were abrogated after inhibition of HO-1 [
17].
The specific objective of this study was to establish that overexpression of BI-1 protein, with adenoviral-TMBIM6 vector, can attenuate the morphological and neurological consequences postneonatal HI via disruption of the NPR-CYP complex coupled with upregulation of Nrf-2 and HO-1, thus attenuating oxidative stress. HO-1 is a rate-limiting enzyme shown to be able to attenuate ROS accumulation. Increased levels of HO-1 may limit oxidative dysregulation which causes misfolding of ER proteins thereby decreasing the UPR. Our main novelty lies in the ability to upregulate BI-1 protein using an adenoviral vector carrying the TMBIM6 gene to induce overexpression of BI-1 in an in vivo HIE model and the mechanisms involved.
Given the lack of effective treatment options for neonatal HI injury, we hope to establish a novel role for BI-1 protein and ER stress in the pathophysiology of neonatal HI injury and help leverage this new understanding to design interventions that affect the outcome of neonatal HI patients. This work is essential and may help to change the clinical management for HI patients and provide a foundation for future research in other related diseases with similar pathologies.
Discussion
The endoplasmic reticulum (ER) is a major organelle that has an essential role in multiple cellular processes, such as the control of correct protein folding and function [
47,
50]. Hypoxia-ischemia, oxidative stress, calcium disturbances, and inhibition of protein glycosylation may contribute to a disruption in ER homeostasis and lead to stress [
50]. Cells respond to ER stress by activating several pathways, including promoting the ability of proteins to refold correctly, inhibiting protein translation, increasing protein degradation, stimulating the transcription of genes, and enabling self-repair mechanisms [
50]. All these processes are referred to as the unfolded protein response (UPR) which under prolonged activation result in cell death [
47].
Bax Inhibitor-1 (BI-1) is a conserved evolutionary protein that resides on the intracellular membrane of the ER [
12]. BI-1 was first named as Bax Inhibitor due to its ability to suppress cell death in yeast [
49]. More recently, it is also referred to as TMBIM6 as it is part of the transmembrane Bax Inhibitor-1 containing motif 6 family [
19]. Overexpression of BI-1 has been demonstrated to play a protective role in ER stress-induced cell death, and to a lesser extent in other ER related stresses, such as oxidative stress and inflammation. In the present study, we first measured endogenous expression levels of BI-1 and its downstream proteins. Our results showed that endogenous expression of P4502E1 (CYP2E1), a member of the MMO system and a major inducer of ROS generation, increased in a time-dependent manner after HI and remained elevated through 72 h post HI. This is expected as after HI injury, there is a significant increase in ROS production that is regulated mainly through P4502E1 (Fig.
1a, d). Hence, levels of P4502E1 increase during times of injury. On the contrary, BI-1’s role is to reduce ROS by disrupting P4502E1. In our results, we demonstrated an initial increase in BI-1 expression; however, at 72 h post HI, BI-1 expression levels significantly declined (Fig.
1a, b). This initial increase in BI-1 can be explained as a protective response to the injury. BI-1 levels increase with the attempt to reverse the damage caused by HI injury. However, the endogenous levels are not enough to reverse the damage and BI-1 declines significantly by 72 h as seen from our data. Therefore, the main goal in our study was to significantly increase BI-1 levels in the brain, using exogenous means, to be able to reverse damage caused after HI injury.
To demonstrate BI-1’s protective effects after hypoxic-ischemic (HI) injury in the neonatal rat, we overexpressed BI-1 using Adenoviral-TMBIM6 (Ad-TMBIM6) vector. We tested two doses and showed that low dose Ad-TMBIM6 vector significantly reduced the percent infarcted area (Fig.
2a) and improved long-term neurobehavioral outcomes (Fig.
2c–d). In addition, we quantified the amount of viral vector present in the brain using western blot and showed robust expression of Ad-TMBIM6 at 72 h post-HI (Fig.
2b).
ER stress is associated with the production of reactive oxygen species (ROS) through oxidative protein folding by the MMO system, composed of NADPH-P450 reductase (NPR) and cytochrome P450 (CYP) members such as P4502E1 [
27,
28,
32]. It has been shown that BI-1 overexpressing cells can regulate UPR induction and inhibit ROS accumulation under ER stress [
15], thus making BI-1 a crucial regulator of ROS inhibition. Cells respond to ROS by activating genes to encode antioxidative stress enzymes. A key transcription factor activated is nuclear factor erythroid 2-related factor 2 (Nrf-2), which regulates the production of several cytoprotective enzymes, such as heme oxygenase-1 (HO-1), a potent inhibitor of ROS [
15,
17]. Studies showed that in BI-1 overexpressing cells, inhibition of HO-1 attenuated BI-1-mediated protection against ER stress [
17]. However, the role of HO-1 in BI-1’s protective mechanism is mostly unknown and debatable.
Some studies have shown that HO-1 was unaffected in BI-1 deficient mice embryonic fibroblast cells [
24]. In contrast, BI-1 overexpressed cells showed significant inhibition of P4502E1 expression [
15] and suppression of ROS production in human embryonic kidney cells [
16]. The P4502E1 member of the CYP ER heme proteins is a major contributor to ROS production. It acts by metabolizing and activating substrates into more toxic products, thus not only increasing ROS production but also stimulating inflammatory cascades and ultimately worsening HI pathology. Previous studies have shown P4502E1 to be upregulated during ER stress [
15‐
18], thus playing an essential role in HI pathology.
In addition, other studies showed that P4502E1-induced oxidative stress played a role in the translocation of Nrf-2 to the nucleus followed by the upregulation of HO-1. Moreover, increased HO-1 levels may be dependent on P4502E1 [
9], perhaps attempting to counteract P4502E1 effects and act as a survival signal. The same study showed that the inhibition of P4502E1 significantly reduced ROS generation in an acute kidney injury model [
43]. The relationship between P4502E1 and HO-1 needs further research in order to fully understand the specifics covering the apparent connection. Since P4502E1 is a major regulator of ROS production and that Nrf-2 has a role in the response against P4502E1, inhibition of P4502E1 coupled with activation of Nrf-2 is a promising therapeutic target.
Although we are now beginning to understand the importance of BI-1 in the cell and in human physiology, its function and signaling mechanisms remain unknown. Given the importance of P4502E1 and Nrf-2, finding a key molecule to regulate both is of great interest. Here we identified BI-1 as one such potential molecule which may protect the cell against ER stress-induced ROS production as well as the subsequent increase in inflammatory response via two possible mechanisms. First, it may interact with NPR, thus destabilizing the NPR-CYP complex, which directly inhibits the formation of ROS by reducing the activity of P4502E1 [
15]. BI-1 may interact through its C-terminus with NPR and to a lesser extent with P4502E1 [
15]. This interaction induces destabilization of the NPR-CYP complex, thus blocking electron transfer and ROS production [
32]. A recent study showed that BI-1 overexpressing cells caused P4502E1 degradation, leading to ER stress suppression, and subsequent reduction in ROS [
18]. We observed similar findings from our western blot results; overexpression of BI-1 in the neonatal rat significantly upregulated BI-1 levels in the brain, while simultaneously reducing P4502E1 expression levels (Fig.
4a–c). Silencing BI-1 reversed those effects while silencing Nrf-2 did not significantly change P4502E1 levels, which indicates Nrf-2 to be downstream of P4502E1. This data was further supported by our IHC staining that demonstrated across groups that as BI-1 expression increased, P4502E1 decreased; they have converse expression patterns throughout (Fig.
4d, e). Our western blot data also demonstrated a decrease in BI-1 expression levels after silencing Nrf-2 with siRNA (Fig.
4a). This may be explained due to BI-1’s multimodal properties and hence targeting multiple signaling pathways. In our study, we focused on BI-1 and its role on the CYP-NPR complex and Nrf-2 signaling. Specifically, we showed that BI-1 may upregulate Nrf-2, an anti-oxidative molecule, thus attenuating ROS production and subsequent inflammation. However, BI-1 has other roles such as being able to reduce Bax via direct interaction with Bcl-2 [
45,
49]. This interaction with Bcl-2 showed to increase its levels while decreasing the pro-apoptotic protein, Bax [
45,
49]. Like BI-1, Nrf-2 levels may be affected by different signaling pathways as well. In this case, as previous studies have shown an interaction between BI-1 and Bcl-2, studies have also shown a direct connection between Nrf-2 and Bcl-2 [
23,
29,
30,
39,
51]. Specifically, Nrf-2 has been shown to bind to Bcl-2 ARE and regulate the expression and induction of the Bcl-2 gene [
30]. Since both Nrf-2 and BI-1 have a direct interaction with Bcl-2, this may explain as to why the decrease in Bcl-2 levels, due to knockdown of either Nrf-2 or BI-1, will indirectly affect each other’s expression levels. In our study, we knocked down Nrf-2, using siRNA, and saw a decrease in BI-1 expression. This result may be explained, as mentioned above, by the fact that knocking down Nrf-2 affects Bcl-2 levels (Bcl-2 decreases) and since BI-1 and Bcl-2 have a direct interaction as well, it will also affect BI-1 expression levels acting like a negative feedback loop, which resembles the decrease we observed in our experiment (Fig.
4).
Second, BI-1 may upregulate pNrf-2 which in turn triggers HO-1 thus inhibiting ROS production and attenuating inflammation. Lee et al. showed that overexpression of BI-1 in cells increased Nrf-2 transcription factor, which then translocated to the nucleus where it stimulated the production of anti-oxidant enzymes, HO-1. HO-1 is known to block ROS production and accumulation thereby promoting cell survival [
17]. Similar to Lee et al., we found that BI-1 overexpression, after neonatal HI, significantly upregulated pNrf-2 (Fig.
5c) and HO-1 (Fig.
5d) while silencing of either BI-1 or Nrf-2 with siRNAs reversed those effects. In addition, our IHC staining showed changes in Nrf-2 expression patterns among groups that correlated with our western blot findings (Fig.
5f). Furthermore, inhibition of P4502E1 with activation of Nrf-2 and HO-1 was linked with a reduction in ROS accumulation and the subsequent release of pro-inflammatory mediators. Here, we used a ROS marker, Reactive Oxygen Species Modulator 1 (ROMO1), which is a protein-coding gene responsible for ROS generation. There was a significant reduction in ROMO1 expression levels (Fig.
6a, b) as well as in IL-6, TNF-α, and IL-β (Fig.
6c, d). Staining for IL-1β on microglia demonstrated a significantly higher expression and co-localization in the vehicle, BI-1 siRNA, and Nrf-2 siRNA groups compared to sham or Ad-TMBIM6-treated group (Fig.
6f). These results were confirmed to be via BI-1-induced inhibition of P4502E1 or activation of Nrf-2 as either inhibition of BI-1 or Nrf-2 reversed those effects. To further examine BI-1’s inhibitory role on ROS accumulation, we used a dihydroethidium (DHE) dye to detect ROS production [
31]. The dye binds with the superoxide anions, thus illuminating a red fluorescent image which is an indication of ROS presence. Our data indicated a higher amount of ROS accumulation in vehicle and siRNA groups versus sham or Ad-TMBIM6-treated groups, in both cortex region and around ventricles, while overexpression of BI-1 reversed those effects (Fig.
7a, b). To detect whether a reduction in ROS was associated with a reduction in inflammation, we performed immunofluorescence staining for myeloperoxidase (MPO). A similar pattern was observed in the MPO staining where Ad-TMBIM6 significantly reduced MPO positively stained cells compared to vehicle or siRNA groups (Fig.
7c), thus indicating a correlation between a reduction in ROS and the subsequent attenuation of inflammatory processes.
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