Mitophagy in Cerebral Ischemia And Ischemia/Reperfusion InjuryⅠ

ischemic stroke is a severe cerebrovascular disease with high mortality and morbidity. In recent years, reperfusion treatments based on thrombolytic and thrombectomy are major managements for ischemic stroke patients, and the recanalization time window has been extended to over 24 h. However, with the extension of the time window, the risk of ischemia/reperfusion (I/R) injury following reperfusion therapy becomes a big challenge for patient outcomes. I/R injury leads to neuronal death due to the imbalance in metabolic supply and demand, which is usually related to mitochondrial dysfunction. Mitophagy is a type of selective autophagy referring to the process of specific autophagic elimination of damaged or dysfunctional mitochondria to prevent the generation of excessive reactive oxygen species (ROS) and subsequent cell death. Recent advances have implicated the protective role of mitophagy in cerebral ischemia is mainly associated with its neuroprotective effects in I/R injury. This review discusses the involvement of mitochondria dynamics and mitophagy in the pathophysiology of ischemic stroke and I/R injury in particular, focusing on the therapeutic potential of mitophagy regulation and the possibility of using mitophagy-related interventions as an adjunctive approach for neuroprotective time window extension after ischemic stroke.

Click to check the cistanche dose for ischemic stroke 

Keywords: mitophagy, mitochondrial dysfunction, ischemic stroke, ischemia/reperfusion injury (I/R injury), recanalization therapy, the therapeutic window

INTRODUCTION 

A stroke is a sudden onset of cerebral blood circulation disorders caused by cerebral infarction or hemorrhage (Shi et al., 2021). Depending on the area of the brain affected, patients may present with different symptoms, among which the most common ones are the acute onset of weakness in one side of the body and reduced speaking ability (Nor et al., 2005). Stroke is the fifth leading cause of death according to the American Heart Association (AHA). Around 795,000 people suffer from either new or recurrent strokes each year (Virani Salim et al., 2020). There are two major types of stroke: ischemic stroke and hemorrhagic stroke. In ischemic stroke, blood flow is blocked by thrombosis formed around the ruptured atherosclerotic plaques in the artery, while hemorrhagic stroke usually results from bleeding induced by blood vessel rupture.

Ischemic stroke accounts for about 87% of all stroke cases. Thus it is under great attention in research and clinical practice. Blocked blood flow leads to a lack of oxygen and nutrients, triggering an ischemic cascade in the brain. The production of adenosine triphosphate (ATP) would be disrupted, which is often a lethal situation for vulnerable brain cells that are highly energy-dependent. In detail, failure in ATP generation can result in the weakened activity of ATP-dependent ion channels, including sodium channels, thus causing intracellular hyperosmolarity (Deb et al., 2010). Also, increased anaerobic respiration during ischemia produces the byproduct lactic acid, leading to metabolic acidosis. Severe alternations in the ion balance can cause cytotoxic edema, and disrupt the glutamate receptor activity, which eventually damages DNA and structural proteins, or even leads to cell deaths (Nishizawa, 2001; Deb et al., 2010). Irreversible neuropathological changes in neurons usually occur within 20–30 min after ischemia (Ordy et al., 1993). 

Reperfusion treatments which aim at restoring blood flow and oxygen to the ischemic area before neuronal damage, are the primary management for ischemic stroke patients in the clinic. Intravenous tissue plasminogen activator (IV tPA) is the only FDA-approved thrombolytic agent for treating acute stroke. Previous evidence suggests it only shows significant clinical improvement when given within 3 h after ischemia (Kwiatkowski et al., 1999). Another randomized trial study performed in 2017 indicated that intravenous therapies within 6 h still benefit over safety concerns (Berkhemer et al., 2014). Mechanical thrombectomy, a surgical procedure to remove thrombosis from arteries, is another commonly used reperfusion therapy. Thrombectomy is efficient in reducing post-stroke disability, though its efficacy and safety can only be ensured within 8 h after the stroke onset (Jovin et al., 2015). In recent years, two high-quality clinical trials focusing on delayed recanalization indicate that reperfusion treatment given at 24 h or even later after the stroke onset still shows some improved prognosis in selected patients, thus extending the therapeutic window to 24 h in specific patient populations (Ragoschke-Schumm and Walter, 2018). 

However, all current treatments have the major limitation of increasing the risk of intracranial hemorrhage (ICH) when given outside the therapeutic window, which can further damage the brain tissue. This subsequent injury following reperfusion therapy is termed an ischemia/reperfusion (I/R) injury, a process that involves reoxygenation-induced reactive oxygen species (ROS) production, calcium overload, and tissue damage. Therefore, extending the reperfusion time window after ischemic stroke while providing neuroprotection is extremely important for disease management. Autophagy is a natural process that degrades unnecessary or damaged organelles and proteins to maintain cellular homeostasis. Autophagy can be activated after ischemic stroke when brain cells are exposed to the risk of oxygen and nutrient deficiency. To be more specific, oxygen-glucose deprivation stimulates the increase in AMP/ATP ratio, which is an activator for the AMPK pathway (Oakhill et al., 2011; Jiang et al., 2018). Upregulation of the AMPK pathway can thus 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 (Egan et al., 2011; Kim J. et al., 2011). 

Previous research shows controversial results regarding the role of autophagy after ischemic stroke. Some studies show that autophagy provides neuroprotection and improves clinical outcomes by significantly reducing ischemic damage to neurons, glia, and endothelial cells (Papadakis et al., 2013; Jiang et al., 2015; Dai et al., 2017). Meanwhile, other findings suggest that excess autophagy might be harmful to brain cells (Li et al., 2017; Mo et al., 2020). To sum up, despite the controversial evidence, it is generally agreed that moderate autophagy is protective, while excessive autophagy may contribute to cell death during ischemia (Mo et al., 2020). 

Ischemic preconditioning (IPC), a strategy that uses short periods of vascular occlusion and reperfusion to prevent fetal ischemic events and recanalization, can activate the neuroprotective program in the brain via triggering adaptive autophagy targeting damaged organelles and alleviating oxidative stress in the acute ischemic stroke (Yang et al., 2020; Ajoolabady et al., 2021). In addition, cerebral ischemia postconditioning, which reduces maladaptive autophagy by applying short periods of reperfusion interrupted by ischemia at the beginning of recanalization, has been induced to suppress reperfusion injury, indicating its protective role in treating ischemic stroke (VintenJohansen, 2017; Ajoolabady et al., 2021). Mitophagy, a type of selective autophagy, can remove dysfunctional mitochondria. Mitochondria play a central role in cellular energy production, calcium homeostasis maintenance, and ROS regulation. Mitochondrial dysfunction can increase oxidative stress and cellular damage (Liu, 1999; Indo et al., 2007). 

Mitophagy mainly works as a mitochondrial quality control through the clearance of damaged mitochondria. In mammals, dysfunctional mitochondria can be cleared either via PINK1- Parkin-dependent ubiquitination pathway or via the activation of mitophagy receptors, thus reducing ROS generation from mitochondria (Lemasters, 2005) and protecting cells against unfavorable niches (Huang et al., 2011). 

Under the condition of ischemic stroke, malfunctioned mitochondria increase the release of pro-apoptotic factors including cytochrome c, to induce cell deaths in the affected area (Jürgensmeier et al., 1998; Lemasters, 2005). It is worth noting that mitophagy may have different effects during the first ischemic phase and later the reperfusion phase. Studies have indicated that mitophagy exerts its protective role mainly during the reperfusion phase (Kumar et al., 2016). 

In recent years, the role of mitophagy in acute stroke has been extensively studied. Most studies indicate a neuroprotection role of mitophagy in alleviating reperfusion injury through multiple mechanisms. This review summarizes the role of mitophagy in ischemic stroke and I/R injury, proposing mitophagy-related interventions as an adjunctive approach for ischemic stroke management. Updates regarding delayed recanalization and the potential involvement of mitophagy in it were also discussed.

MITOCHONDRIA AND MITOPHAGY 

Mitochondria are important organelles that are mainly responsible for energy production. However, damaged mitochondria release harmful ROS and other oxidants, such as H2O2 and peroxynitrite, into the cytoplasm and cause damage to the proteins, nuclear acid, and membranes (Zhou et al., 2011). Worse over, cytochrome c, a mitochondrial intermembrane space protein, will be released under severe mitochondrial damage, which triggers caspase cascade and finally apoptosis (Ott et al., 2002). 

Therefore, rapid degradation of damaged mitochondria is necessary for cell survival. Mitophagy is a process during which damaged or aging mitochondria are selectively wrapped by phagophores and undergo lysosomal degradation to maintain cell homeostasis and prevent cell apoptosis. Mitophagy starts with the formation of the phagophore, a membrane structure isolated from the endoplasmic reticulum. Phagophore then recognizes damaged mitochondria through LC3 adaptors or LC3 receptors and engulfs damaged mitochondria for autosomal degradation. Currently, the mitophagy pathways consist of two major types: ubiquitin-mediated pathway and receptor-mediated pathway (Figure 1).

Ubiquitin-Mediated Pathway 

The PTEN-induced putative kinase protein 1 (PINK1) and Parkin-mediated ubiquitination pathway are some of the most well-characterized mitophagy mechanisms. In healthy mitochondria, PINK1, a serine/threonine kinase, is imported from the cytoplasm continuously and undergoes cleavage by the mitochondrial proteases mitochondrial-processing peptidase (MPP) and presenilin associated rhomboid like (PARL) (Greene et al., 2012). Upon ischemic stroke, the mitochondria membrane is depolarized, which prevents the import of PINK1 and results in the accumulation of PINK1 on the mitochondrial membrane. As a result, the kinase activity of full-length PINK1 induces phosphorylation of E3 ligase Parkin, which activates the enzymatic function of Parkin and leads to the ubiquitination of several mitochondrial proteins (Kazlauskaite et al., 2015). Meanwhile, PINK1 phosphorylates ubiquitin and results in the binding of phosphorylated ubiquitin and Parkin, where the ligase activity is further improved (Koyano et al., 2014; Kazlauskaite et al., 2015). 

Once Parkin is activated, it conjugates ubiquitin moieties onto the OMM proteins, and thereby mitophagy is induced. Some ubiquitinated mitochondrial proteins, such as MFN1, are degraded, which is essential for mitochondrial fission and mitophagy (Tanaka et al., 2010). Other ubiquitinated proteins recruit autophagy adaptor proteins such as optineurin (OPTN) and nuclear dot protein 52 (NDP52), which anchors the marked mitochondria to autophagosome by its LC3- interacting region (LIR) motifs, thereby mitophagy is initiated (Lazarou et al., 2015). Moreover, PINK1 can recruit OPTN and NDP52 independently of parkin, which subsequently recruits several autophagy initiation factors, such as unc51-like autophagy activating kinase 1 (ULK1), for mediating phagophore synthesis and elongation (Wong and Holzbaur, 2014; Lazarou et al., 2015). Several other E3 ligases have also been discovered to initiate mitophagy. Some mechanisms are related to PINK1/Parkin-mediated mitophagy and some are independent of Parkin. For example, overexpression of mitochondrial ubiquitin ligase 1 (MUL1) mediates the degradation of mitofusin by ubiquitination, which rescues the PINK1/parkin mutant phenotype (Yun et al., 2014). 

In mature neurons, MUL1 is also important for the contact of ER and mitochondria and the absence of it impairs the Ca2+ homeostasis in mitochondria and reduces the intake of Ca2+ from ER. This leads to the activation of calcineurin, which activates Drp1 and therefore induces mitochondria fission. Fragmented mitochondria lose their membrane potential, and PINK1/Parkin-mediated mitophagy is induced (Puri et al., 2019). Another E3 ligase, seven in absentia homolog (SIAH)-1, is recruited by synphilin-1 when full-length PINK1 is present. SIAH-1 promotes mitophagy through the ubiquitination of mitochondrial proteins independently of Parkin (Szargel et al., 2016). In addition to the benefits of PINK1-Parkin-mediated ubiquitination, deubiquitinases are essential for correct mitophagy. The deubiquitination of Parkin is carried out directly by ubiquitin-specific peptidase 8 (USP8) (Durcan et al., 2014), while USP15 deubiquitinates the substrates of Parkin to inhibit mitophagy (Cornelissen et al., 2014). Several other deubiquitinases, such as USP30, USP35, and USP33 (Bingol et al., 2014; Wang Y. et al., 2015; Niu et al., 2020), counteract ubiquitin-mediated mitophagy by removing ubiquitin chains from the mitochondrial membrane. Therefore, a fine-tuned balance between ubiquitination and deubiquitination is established for the regulation of mitophagy.

Receptor-Mediated Pathway 

An alternative pathway of mitophagy is through mitophagy receptor signaling. Multiple mitophagy receptors are currently identified in mammalian cells (Ren et al., 2018), which contain a least one LIR for the direct binding of autophagy mediator LC3 and the subsequent phagosome engulfment. A critical receptor in the turnover of mitochondria in erythrocytes is an OMM protein named BCL2 interacting protein 3-like (BINP3-L, also known as NIX) (Sandoval et al., 2008). It is transcriptionally upregulated during erythrocytes maturation to clear mitochondria. In conditions of hypoxia, the BINP3-L is induced along with its homolog, BNIP3, promoting mitophagy through OPA1 disassembly and DRP1 recruitment, which is transcriptionally regulated by forkhead box O3 (FOXO3) and hypoxia-inducible factor (HIF), thereby inducing mitochondrial fission and inhibiting mitochondrial fusion (Sowter et al., 2001; Mammucari et al., 2007). 

Moreover, its binding affinity to LC3 is further improved by the phosphorylation of the LIR under stress conditions (Rogov et al., 2017). Another essential mitophagy receptor is the FUN14 domain containing 1 (FUNDC1), which mediates mitophagy under hypoxic conditions (Liu et al., 2012). FUNDC1 activity is regulated by its phosphorylation state. In non-stress conditions, it is suppressed by phosphorylation of Src at Tyr18 and casein kinase II (CK2) at Ser13 (Chen et al., 2014). Under hypoxia, PGAM5 phosphatase dephosphorylates FUNDC1, which activates the LIR motif on FUNDC1 and induces mitophagy. Moreover, FUNDC1 recruits Drp1 and disrupts its physical association with OPA1 under stress (important for mitochondria dynamic), thereby inducing mitochondrial fission and inhibiting mitochondrial fusion (Chen et al., 2016). 

Additionally, there are multiple other mitophagy receptors. On the outer mitochondrial membrane, BCL 2 Like 13 (BCL2L13) and FKBP prolyl isomerase 8 (FKBP8) have been shown to mediate mitophagy by binding LC3 via the LIR motif independently of Parkin (Murakawa et al., 2015; Bhujabal et al., 2017). Some receptors also locate in the IMM, such as prohibitin 2 (PHB2) and cardiolipin. Once the OMM is depolarized or damaged, PHB2 will interact with LC3 to directly promote mitophagy (Wei et al., 2017). However, the depletion of PHB2 upon OMM rupture destabilizes PINK1 through the activation of PARL and therefore leads to the cleavage of full-length PGAM5 (Yan et al., 2020). This abolishes PGAM5-involved PINK1 stabilization and thereby inhibits PINK1/Parkindependent mitophagy. Recently, cardiolipin, a phospholipid, has also been identified as a mitophagy receptor, whose primary synthesis is conducted in the IMM. When encountering OMM rupture, cardiolipin is released to the OMM and interacts with LC3, triggering a signaling cascade that results in the engulfment of the mitochondria (Chu et al., 2013).

Mitochondria Dynamics and Its Relationship With Mitophagy 

To adapt to the external environment, mitochondria fuse both the inner and outer membranes or undergo fission and separate into several mitochondria. These two essential processes in mitochondria dynamics are termed fusion and fission. When confronting cellular stress, fusion is promoted to ensure energy production by repairing partially damaged mitochondria (Youle and van der Bliek, 2012).On the other hand, fission is necessary for mitophagy since it enables the separation of depolarized mitochondria, allowing the preservation of “the healthy part” in mitochondria and reducing unnecessary loss during mitophagy. Depending on the quality of mitochondria, either fusion or fission will be activated along with the inhibition of the other (Twig et al., 2008). 

In mammalian cells, MFN1, MFN2, and OPA1, which are GTPases, mediate the fusion of the mitochondria (Wu et al., 2019). These proteins are often modified post-transcriptionally to control their potency. For MFN1, extracellular regulated kinase (ERK) can phosphorylate it at Thr562 to suppress fusion. MFN1 can also be ubiquitinated by MARCH 5 for degradation (Park et al., 2014). Mitogen-activated protein kinase 8 (MAPK8, also known as JNK) phosphorylates MFN2 at Ser27 under stress for subsequent ubiquitination by E3 ligases Parkin (Gegg et al., 2010), HUWE1 (Leboucher et al., 2012), and mitochondrial ubiquitin ligase membrane-associated RING-CH (MARCH 5) (Sugiura et al., 2013). MFN1 and MFN2 can be deubiquitinated by USP30, where inhibition of it will lead to non-degradative ubiquitination of MFN1/2 (Yue et al., 2014). 

For OPA1, it is regulated by changes in the protease activity of YME1L and OMA1, which is responsive to intramitochondrial signals (Griparic et al., 2007; Head et al., 2009). Mitochondrial fission is regulated mainly by a cytosol protein, DRP1, whose recruitment is mediated by mitochondrial fission factors such as MFF. DRP1 can be phosphorylated by protein kinase A at Ser637 and Ser656, which inhibits its activity. Dephosphorylation of DRP1 is mediated by the calcium-dependent protein phosphatase calcineurin or by protein phosphatase 2A (PP2A) for enhanced fragmentation under stress (Chang and Blackstone, 2007; Cribbs and Strack, 2007). 

Moreover, energy-sensing adenosine monophosphate (AMP)- activated protein kinase (AMPK) phosphorylates MFF under energy stress, which recruits Drp1 and accelerates mitochondrial fission (Toyama et al., 2016). Mitophagy is closely interrelated with mitochondrial dynamics as multiple mitophagy proteins are found to promote fission and facilitate mitophagy. For instance, phosphorylated Parkin can ubiquitinate MFN1 and MFN2 for degradation, which decreases mitochondrial fusion and enhance fragmentation, leading to the initiation of mitophagy (Tanaka et al., 2010). During mitophagy, MFN2 is also phosphorylated by PINK1 to recruit Parkin for further mitophagy (Chen and Dorn, 2013).

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