Mitophagy in Cerebral Ischemia And Ischemia/Reperfusion InjuryⅢ

MITOPHAGY IN I/R INJURY 

Mitophagy Is Activated Upon Ischemic Stroke 

Enhanced mitochondrial fragmentation and fission were observed during both the ischemic phase and I/R injury. During OGD in rat cardiomyocyte cell lines (H9C2 cells), massive mitochondria fragmentation was observed (Kim H. et al., 2011). Reoxygenation of mice cardiomyocyte cell lines (HL1 cells) and neonatal primary cardiomyocytes show mitochondria fission (Ong et al., 2010; Disatnik et al., 2013). In vivo experiments using mice, 24-h left anterior descending permanent ligation model show consistent results (Kim H. et al., 2011). The brain tissue has a very similar situation as cardiomyocytes. Following OGD/reoxygenation, enhanced mitochondria fragmentation was observed in mice N2a neuroblastoma and primary rat neurons (Sanderson et al., 2015; Tang et al., 2016), accompanied by Opa1 processing and cytochrome C release. In vivo experiments in CA1 hippocampal neurons of rats have consistent results (Kumar et al., 2016). Later, researchers found that mitophagy pathways are activated during ischemic/reperfusion through multiple signals. 

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During the ischemic phase, ATP production rapidly decreases, which activates AMPK pathways to initiate autophagy via direct activation of the ULK complex through phosphorylating of Ser 317 and Ser 777, or indirect activation of ULK through inhibiting the activity of mTOR, as mTOR suppresses Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK (Kim J. et al., 2011; Zaha and Young, 2012). The activated ULK1 complex will then activate the class III PI3K complex (berlin 1, VPS34, and VPS15) that initiates the nucleation of the phagophore (Kim J. et al., 2011). 

ULK1 has also been found to activate the FUNDC1 receptor, which may collaterally activate mitophagy (Kundu et al., 2008) (Figure 3). During the reperfusion phase, the mechanistic target of rapamycin (mTOR) pathways is inhibited by ROS signals, thereby promoting the autophagosome’s initiation and nucleation (Alexander et al., 2010). ROS has been found to activate mitophagy via BNIP3, while high levels of BNIP3 can induce apoptosis (Scherz-Shouval et al., 2007). PINK1/Parkin-mediated mitophagy is also activated in brain damage induced by cerebral ischemia and reperfusion. Lan et al. (2018) found significant increases in PINK1 accumulation in the outer membrane of mitochondria. They increased Parkin/p62 mitochondrial translocation after reperfusion, along with upregulation of autophagy markers LC3B, Beclin1, and LAMP-1, with a peak at 24 h (Figure 3).

Mitophagy Exerts Its Protective Role Mainly During the Reperfusion Phase 

Various studies have already demonstrated the protective role of enhanced mitophagy in attenuating brain injury after tMCAO in rats (Li et al., 2014; Di et al., 2015). However, whether mitophagy exerts its protective role during the ischemic phase of the reperfusion phase is unclear. Ischemia and reperfusion have different pathophysiology related to mitochondria. Thus, a series of studies have focused on distinguishing the ischemic and reperfusion phase. Intriguingly, mitophagy was identified to have a particular role during the reperfusion phase but may not be in the ischemic phase. Inhibition of Drp1 was neuroprotective in response to OGD in vitro and transient focal ischemia in vivo (Grohm et al., 2012) and cardioprotective in cultured HL-1 cardiomyocytes subjected to OGD and reoxygenation (Dong et al., 2016). 

However, cardioprotection was only seen when inhibition of Drp1 was initiated as a pretreatment. When inhibiting Drp1 during reoxygenation, cell death was paradoxically exacerbated, indicating mitophagy is essential in protecting cells from reperfusion injury. In another study, researchers found that mitochondrial DNA level (which can be used as a marker for mitochondrial mass) does not decrease but significantly increase during the first few hours in the ischemic phase of stroke (Yin et al., 2008), which could indicate that mitochondrial biogenesis, but not mitophagy, is part of the post-stroke repair mechanisms during the ischemic phase. Kumar et al. (2016) also identified distinct phases of mitochondria during OGD and reoxygenation. 

During OGD occurs first round of fission, reoxygenation initially induces fusion but is followed by massive fragmentation. These results have identified the distinct nature of the ischemic phase and reperfusion phase, in which mitophagy seems to have a unique protective role only in the reperfusion phase. However, given these results, which monitor mitophagy accurately to different phases, a fuller picture of the mitophagy dynamic in the ischemic/reperfusion process is emerging, but still with some obscurity. Thus, a more comprehensive and accurate evaluation of different time points is required to fully explain the role of mitophagy in both ischemic and reperfusion injuries.

The Interplay Between Mitophagy and IR Injury 

Mitochondria-Dependent Cell Death in I/R Injury 

During both the ischemic phase and IR injury, mitochondrial-dependent cell apoptosis is one of the critical events defining cell fate. The BCL-2 family proteins, which are a significant regulator of outer mitochondrial membrane permeability and play an essential role in the intrinsic apoptotic pathway (Chao and Korsmeyer, 1998), provide the mitochondria with the function of “sensing” apoptotic stress. The BCL-2 family has been classified into two groups: anti-apoptotic proteins (including Bcl-2, BclxL, and Bcl-w) and pro-apoptotic proteins (including Bax, Bak, etc.) (Oltvai et al., 1993). Under stress conditions such as ischemic insults and IR injury, the anti-apoptotic protein Bcl-2 is phosphorylated and releases Beclin1 to activate autophagy and mitophagy. Bcl-2 prevents the release of pro-apoptotic proteins by maintaining the integrity of the mitochondrial membrane. This process inhibits cell apoptosis (Praharaj et al., 2019). However, pro-apoptotic BCL-3 subgroup proteins are also found to be upregulated after ischemic stroke, which induces the release of cytochrome c from the mitochondria intermembrane into the cytosol. Cytochrome c interacts with the protein cofactor Apaf-1 and procaspase-9 from apoptosome (Gibson and Davids, 2015). Thus, ischemia and IR injury may elicit complex apoptotic effects involving mitochondria. The activities of anti- and pro-apoptotic proteins are correlated with the ROS concentration.

Mitophagy and Ca2+ Overload 

Calcium overload is an essential pathology in I/R injury as it promotes the opening of mPTP, which activates apoptotic factors such as the Bcl-2 family and leads to cell apoptosis eventually. Recent evidence suggests a link between mitophagy and Ca2+ regulation. Many mitophagy proteins are also involved in mitochondrial calcium regulation. For instance, in PINK1/Parkin mediated mitophagy, many Ca2+ sensing proteins are ubiquitinated, which disturbs the function as a Ca2+ buffering system, such as MFNs (de Brito and Scorrano, 2008; Gegg et al., 2010; Filadi et al., 2015), DRP1 (Cereghetti et al., 2008; Wang et al., 2011), VDAC1 (Gincel et al., 2001; Geisler et al., 2010) and BCL2 (Rong et al., 2008; Chen et al., 2010). Moreover, PINK1 phosphorylates mitochondrial rho GTPase 1 (RHOT1), which is an essential regulator of the Ca2+ sensitive characteristics in ER phospholipid exchange (Kornmann et al., 2011) and mitochondrial dynamics (Saotome et al., 2008). Parkin mediates the ubiquitination and degradation of MICU-1, which also affects the stability of MICU2 (Matteucci et al., 2018). Additionally, depending on the calcium level in the ER, Ca2+ can be either prior anti-autophagic (Cárdenas et al., 2010).

Mitophagy and Oxidative Stress 

Oxidative stress is one of the most critical pathological brain damage processes in acute ischemic stroke (Chamorro et al., 2016). ROS and reactive nitrogen species (RNS) are crucial mediators in cerebral ischemia-reperfusion injury. ROS/RNS at a low level is beneficial for facilitating adaptation to stress as a redox signaling, whereas ROS/RNS at a high level is deleterious (Scialò et al., 2016). ROS and RNS have mainly been generated from the electron transport chain (ETC) complexes I and complex III in mitochondria, and excessive ROS/RNS primarily induces mitochondria injury. Under normal condition, less than 10% of oxygen receive electrons to generate superoxide in ETC. However, upon IR injury, the ischemic brains produce a large amount of NO and superoxide anion (O2 −) simultaneously. The rapid reaction of NO and O2 − leads to the formation of ONOO−, a representative/critical cytotoxic factor for both oxidative and nitrosative stress (Marla et al., 1997). ONOO− is a highly active cytotoxic molecule to aggravate neuronal damage, disrupts the blood–brain–barrier (BBB) integrity, and mediates hemorrhagic transformation by triggering numerous cellular signaling cascades, which represents a vital pathogenic mechanism in ischemic stroke (Marla et al., 1997). Mitophagy has regulatory effects on oxidative stress since damaged mitochondria will lead to the release of more ROS. ROS can directly induce mitophagy (Wang et al., 2012), which has been shown beneficial in various disease conditions as mitophagy attenuates oxidative stress. However, multiple studies have demonstrated that ONOO− induces excessive mitophagy activation by tyrosine nitration of Drp1 and mediates mitochondrial recruitment of Drp1, thus aggravating cerebral I/R injury (Feng et al., 2017). This raises the question of whether mitophagy is beneficial or detrimental in response to I/R injury which will be discussed later. Indeed, ONOO−-mediated mitophagy is suspected to be excessive and could be a crucial therapeutic target for IR injury

Mitophagy and Inflammation 

Anti-inflammation is another function of mitophagy. Upon mitochondria dysfunction, leaked mitochondrial ROS can activate inflammasomes, contributing to inflammation (Murakami et al., 2012). Mitophagy has shown a positive effect on kidney disease, ischemic stroke, and I/R injury. For instance, upon encountering I/R injury, kidney tubules rich in mitochondria will enhance autophagy (including mitophagy) to protect cells from ROS-triggered inflammatory response (Kimura et al., 2011; Zhou et al., 2011). In ischemic stroke, overexpression of the ATF4 gene can enhance mitophagy and inhibit NLRP3 inflammasome-mediated inflammatory response (He et al., 2019). Inducing Parkin-dependent mitophagy can improve inflammatory response and prevent cell death in myocardial I/R injury (Yao et al., 2019).

Therapeutic Potential of Mitophagy Regulation in I/R Injury 

Recent years boosts the research of mitophagy in cerebral ischemia and reperfusion, most of them demonstrating the protective role of mitophagy. However, excessive mitophagy, which is probably induced by certain oxidative stress conditions, has also been shown to be deleterious in cerebral ischemia and reperfusion processes. In preclinical studies, several therapeutic agents have been proposed to ameliorate cerebral I/R injury, either through enhancing or inhibiting certain types of mitophagy. These mitophagy-related interventions could be proposed as adjunctive approaches for ischemic stroke management. Studies investigating the role of mitophagy in I/R injury after 2018 are summarized as follows and in Table 1. Earlier studies have been reviewed by Anzell et al. (2018) and Guan et al. (2018). The role of autophagy and potential strategies in ischemic stroke is also comprehensively summarized in a recent review (Ajoolabady et al., 2021).

Protective Role of Enhanced Mitophagy in IR Injury 

Zhang and Yu (2018) found that after reperfusion, NR4A1 was significantly elevated in the brain tissue, inhibiting the activation of protective mitophagy through the MAPK– ERK–CREB signaling pathway. Genetic ablation of NR4A1 reduced the cerebral infarction area and neuronal apoptosis. As demonstrated by functional studies, NR4A1 modulated cerebral IR injury by inducing mitochondrial damage. Higher NR4A1 promoted mitochondrial potential reduction, aggravated cellular oxidative stress, and initiated caspase-9-dependent apoptosis. Mechanistically, NR4A1 induced mitochondrial damage by disrupting Mfn2-related mitophagy. Knockdown of NR4A1 reversed mitophagy activity, sending a prosurvival

signal for mitochondria in the setting of cerebral IR injury (Zhang and Yu, 2018).

Electroacupuncture (EA) has been shown effective in treating ischemic stroke. Recently, Wang et al. (2019) demonstrated that EA ameliorates nitro/oxidative stress-induced mitochondrial functional damage and reduces the accumulation of damaged mitochondria via Pink1/Parkin-mediated mitophagy clearance to protect cells against neuronal injury in cerebral I/R. Later, Mao et al. (2020) also found EA pretreatment has a protective effect on cerebral I/R injury through promoting mitophagy, based on the results that the number of autophagosomes, FUNDC1, p62, and the ratio of LC3-II/I were significantly increased. Still, mitochondrial membrane potential and autophagy-related protein p-mTORC1 significantly decreased in the I/R group (Mao et al., 2020). He et al. (2019) showed that ATF4 overexpression induced by AAV was protective against cerebral I/R injury by upregulating Parkin expression, enhancing mitophagy activity, and inhibiting NLRP3 inflammasome-mediated inflammatory response. 

A series of natural compounds have been found to possess the ability to promote mitophagy and attenuate I/R injuries. Curcumin is a complex extracted from the traditional edible herb which can protect neurons in rats after brain I/R injury (Wang and Xu, 2020). Curcumin reduced the levels of ROS while increasing state 3 respiration to prevent the impairment of mitochondrial function from cerebral I/R. Furthermore, curcumin enhanced the co-localization of LC3B and mitochondrial marker VDAC1, the ratio of LC3-II to LC3-I, suggesting the protective role of curcumin exerts through enhancing mitophagy. Wu et al. (2020) found that garciesculenxanthone B (GeB), a new xanthone compound from Garcinia esculent, can promote the PINK1-Parkinmediated mitophagy pathway and protects the brain from I/R injury. Treatment with GeB dose-dependently promoted the degradation of mitochondrial proteins Tom20, Tim23, and MFN1 in YFP-Parkin HeLa cells and SH-SY5Y cells. GeB stabilized PINK1 and triggered Parkin translocation to the impaired mitochondria to induce mitophagy, and these effects were abolished by the knockdown of PINK1. In vivo experiments demonstrated that GeB partially rescued ischemia-reperfusion-induced brain injury in mice. 

Another two natural compounds named Gerontoxanthone I (GeX1) and Macluraxanthone (McX), were also screened out to possess the ability to enhance mitophagy (Xiang et al., 2020). GeX1 and McX directly stabilized PINK1 on the outer membrane of the mitochondria and then recruited Parkin to mitochondria, suggesting that GeX1 and McX mediate mitophagy through the PINK1-Parkin pathway. GeX1 and McX treatment decreased cell apoptosis and the ROS level in an IR injury model in H9c2 cells. Additionally, a tablet derives from Chinese classical prescriptions of Angong Niuhuang Pills with modified compositions has also been shown to attenuate cerebral I/R injury by improving mitophagy and mitochondrial quality control (Zhang et al., 2020a). The tablets elevated the ratio of Bcl-2/Bax, inhibited apoptosis, decreased the infarction volume, and improved the MCAO rats’ behavioral performance. Although the precise molecular regulatory network of these natural compounds has not been fully addressed, considering the advantages of natural compounds such as low toxicity and safe pharmacokinetic profiles including high utility rate and effective elimination, drugs derived from natural compounds are of high translational value. 

Chen et al. (2020) demonstrated that a sphingosine kinase 2-mimicking TAT-peptide protects neurons against ischemia-reperfusion injury by activating BNIP3-mediated mitophagy. sphingosine kinase 2 (SPK2) interacts with Bcl-2 via its BH3 domain, activating autophagy or mitophagy by inducing the dissociation of Beclin-1/Bcl-2 or Bcl-2/BNIP3 complexes and protects neurons against ischemic injury. Different from cardiac muscle, the long axon is a highly distinct morphology of neurons, which is located in more than half of the total mitochondria content in neurons (Nafstad and Blackstad, 1966). However, under stress conditions, autophagy and mitophagy events are concentrated in the soma (Maday and Holzbaur, 2014), but not in exon, evidenced by concentrated autophagosomes and lysosomes in the soma (Farías et al., 2017). 

Thus, it is unclear how mitochondria in distal axons are cleared in ischemic neurons (Ashrafi et al., 2014). Zheng et al. (2019a,b) have identified unique mitochondria movement in neurons, from axon to soma for degradation. Upon oxygen and glucose deprivation-reperfusion, axonal mitochondria showed loss of anterograde motility but increased retrograde motion upon reperfusion, meaning axonal mitochondria are transported to the neuronal soma for degradation. Anchoring of axonal mitochondria by syntaphilin blocked neuronal mitophagy and aggravated injury. Conversely, induced binding of mitochondria to dynein reinforced retrograde transport and enhanced mitophagy prevent mitochondrial dysfunction and attenuate neuronal damage. Therefore, regulating mitochondria motility in neurons would be another direction for enhancing mitophagy and attenuating I/R neuronal injury.

ONOO− Induces Deleterious Mitophagy 

Feng et al. (2018) found that naringin, a natural antioxidant, could inhibit ONOO− mediated mitophagy activation and attenuate cerebral I/R injury. Naringin possessed strong ONOO− scavenging capability and inhibited the production of superoxide and nitric oxide in IR injury conditions. Naringin inhibited the expression of NADPH oxidase subunits and iNOS in rat brains subjected to 2 h ischemia plus 22 h reperfusion. Naringin can cross the blood-brain barrier, decreases neurological deficit score, reduces infarct size, and attenuate apoptosis in the ischemia reperfused rat brains. 

Furthermore, naringin decreased the ratio of LC3-II to LC3-I in mitochondria. It inhibited the translocation of Parkin to the mitochondria, suggesting naringin prevents the brain from I/R injury via attenuating ONOO−-mediated excessive mitophagy. Rehmapicroside, a natural compound from a medicinal plant, can inhibit ONOO−-mediated mitophagy activation (Zhang et al., 2020b). In vitro, rehmapicroside reacted with ONOO− directly to scavenge ONOO−, decreased O2− and ONOO−, upregulated Bcl-2 but down-regulated Bax, Caspase-3, and cleaved Caspase-3, and down-regulated PINK1, Parkin, p62 and the ratio of LC3-II to LC3-I in the OGD/RO-treated PC12 cells. In vivo, rehmapicroside suppressed 3-nitrotyrosine formation, Drp1 nitration as well as NADPH oxidases and iNOS expression in the ischemia-reperfused rat brains; it also prevented the translocations of PINK1, Parkin, and Drp1 into the mitochondria for mitophagy activation; finally, rehmapicroside ameliorated infarct sizes and improved neurological deficit scores in the rats with transient MCAO cerebral ischemia. Deng et al. (2020) demonstrate that lncRNA SNHG14 promotes OGD/R-induced neuron injury by inducing excessive mitophagy via the miR-182-5p/BINP3 axis in HT22 mouse hippocampal neuronal cells. SNHG14 and BNIP3 were highly expressed, and miR-182-5p was down-regulated in the OGD/R-induced HT22 cells. 

OGD/R-induced HT22 cells exhibited an increase in apoptosis. SNHG14 overexpression promoted apoptosis and the expression of cleaved-caspase-3 and cleaved caspase-9 in the OGD/R-induced HT22 cells. Moreover, SNHG14 up-regulation enhanced the expression of BNIP3, Beclin-1, and LC3II/LC3I in the OGD/R-induced HT22 cells. Furthermore, SNHG14 regulated BNIP3 expression by sponging miR-182-5p. MiR-182-5p overexpression or BNIP3 knockdown repressed apoptosis in OGD/R-induced HT22 cells, which was abolished by SNHG14 up-regulation. Taken together, inhibiting the ONOO−-mediated excessive mitophagy activation exerts neuroprotective effects, with several potential drug candidates being discovered to attenuate cerebral IR injury. Although most studies have demonstrated the possible protective effects of mitophagy upon IR injury, mitophagy is a double-edged sword and requires more studies to test its clinical potential.

DISCUSSION AND FUTURE PERSPECTIVES 

In the treatment of ischemic stroke, reperfusion with thrombolysis and thrombectomy is key to restoring blood flow and improving patient outcomes. However, restoration of blood flow in patients with AIS may result in secondary reperfusion injury. The resupply of oxygen can cause the overactivation of enzymes and pumps that are previously inhibited by ischemia-induced ATP deficiency, thus resulting in the boost of reactive oxygen species (ROS) production and altering calcium homeostasis in both cytoplasm and mitochondria. Such alternations can induce mitochondrial DNA damage and promote the opening of mPTP, which triggers apoptosis-related factors and result in cell death. Mitophagy is an essential cellular process that maintains mitochondrial quality. 

While IR injury primarily induces mitochondrial dysfunction and leads to dysregulation of oxidative stress, calcium homeostasis, and cell apoptosis, regulation of mitochondria dynamics (fission and fusion) and activation of a moderate level mitophagy can contribute to the adjustment of cellular mitochondria quality. Identifying and targeting mitophagy-related pathways molecules may benefit certain subsets of patients with ischemic stroke. And some mitophagy regulators discussed above have already shown great potential in clinical application. For example, for acute ischemic stroke patients, taking some herbal agents before reaching the hospital, and more importantly, during the long recovery stage, may provide vital neuroprotection effects and lead to a better prognosis. To address the clinical potential of mitophagy, further elucidation of mitophagy and its crosstalk mechanism under stroke conditions is required; further discovery of therapeutic targets and drug development for manipulating the mitophagy pathways is needed.

Cistanche neuroprotection effect

Cistanche is a plant extract known for its neuroprotective properties, and its mechanism of action is believed to involve antioxidant, anti-inflammatory, and antiapoptotic effects. There are several relevant tests and application cases related to the neuroprotective effects of Cistanche, which include:

1. In vitro studies: In vitro studies have shown that Cistanche extract protects neurons from stress-induced damage by reducing oxidative stress and inflammation.

2. Animal studies: Animal studies have demonstrated that Cistanche can protect against neuronal damage caused by cerebral ischemia, traumatic brain injury, and neurotoxin exposure.

3. Human studies: There is limited clinical evidence on the neuroprotective effects of Cistanche in humans, but some studies have suggested that it may improve cognitive function and reduce age-related decline in memory.

Luoan Shen1†, Qinyi Gan1†, Youcheng Yang1, Cesar Reis2, Zheng Zhang1, Shanshan Xu3, Tongyu Zhang4 * and Chengmei Sun1,3 * 

1 Zhejiang University-University of Edinburgh Institute, School of Medicine, Zhejiang University, Haining, China, 

2 VA Loma Linda Healthcare System, Loma Linda University, Loma Linda, CA, United States, 

3 Institute for Advanced Study, Shenzhen University, Shenzhen, China, 4 Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, China