Mechanisms and roles of mitophagy in neurodegenerative diseases

Abstract Mitochondria are double‐membrane‐encircled organelles existing in most eukaryotic cells and playing important roles in energy production, metabolism, Ca2+ buffering, and cell signaling. Mitophagy is the selective degradation of mitochondria by autophagy. Mitophagy can effectively remove damaged or stressed mitochondria, which is essential for cellular health. Thanks to the implementation of genetics, cell biology, and proteomics approaches, we are beginning to understand the mechanisms of mitophagy, including the roles of ubiquitin‐dependent and receptor‐dependent signals on damaged mitochondria in triggering mitophagy. Mitochondrial dysfunction and defective mitophagy have been broadly associated with neurodegenerative diseases. This review is aimed at summarizing the mechanisms of mitophagy in higher organisms and the roles of mitophagy in the pathogenesis of neurodegenerative diseases. Although many studies have been devoted to elucidating the mitophagy process, a deeper understanding of the mechanisms leading to mitophagy defects in neurodegenerative diseases is required for the development of new therapeutic interventions, taking into account the multifactorial nature of diseases and the phenotypic heterogeneity of patients.

Mitochondrial dysfunction has been implicated in numerous neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by the degeneration of dopaminergic neurons in the midbrain. Mitochondrial dysregulation can lead to PD. [8][9][10][11] Mutations in PINK1 and Parkin genes are associated with autosomal recessive early-onset PD. 12,13 PINK1 and Parkin genes exert synergistic effect on mitochondrial maintenance-related functions, such as mitochondrial motility, proteasomal degradation of mitochondrial proteins, and mitophagy. Additionally, Parkin also participates in removing mitochondria during the progression of Alzheimer's disease (AD), and the overexpression of Parkin can alleviate symptoms of AD. 14 Supporting a role of mitophagy in AD, amyloid beta-derived diffusible ligands (ADDLs) can induce the fragmentation of mitochondria and mitophagy. [15][16][17] Mitophagy is also altered in HD, and the mutant huntingtin may induce selective autophagy. 18 In a cell culture model of HD, excessive mitochondrial fission partially mediates cytotoxicity. 19 Mitochondrial dysfunction has also been observed in ALS, and reduced targeting of ubiquitinated mitochondria to autophagosomes may contribute to the pathogenesis of ALS. 20 Mitophagy thus plays a multifaced role in neurodegenerative diseases.

| S ELEC TIVE AND NON S ELEC TIVE AUTOPHAGY
Under certain conditions, organelles, together with bits of cytoplasm, will be sequestered and degraded by lysosomes for hydrolytic digestion in a process termed autophagy. 21 Generally, autophagy can be classified into nonselective autophagy and selective autophagy, with the former being primarily a starvation response, while the latter eliminating damaged organelles and remodeling cells to adapt to environmental changes. 22 Defects in selective autophagy can result in a range of human pathophysiologies, including certain types of neurodegenerative diseases. Macroautophagy is an evolutionarily conserved process that allows cells to degrade and recycle cytoplasm. Whereas nonselective macroautophagy can randomly engulf a portion of cytoplasm into autophagosome and subsequently transfer it to the vacuole or lysosome for degradation, selective macroautophagy can specifically identify and degrade specific substances, such as protein complexes, organelles, or invading microorganisms. 23 The morphological hallmark of macroautophagy is the formation of an initial sequestering compartment, the phagophore, which can expand into the double-membrane autophagosome, and the initial sequestration occurs in a compartment separate from the degraded organelle. Typically, selective macroautophagy can be classified into mitophagy, pexophagy, reticulophagy, ribophagy, etc.

| MITOPHAGY: S ELEC TIVE AUTOPHAGY OF MITOCHONDRIA
The by-products of mitochondrial metabolism can induce DNA damage or genetic mutation. 25 Therefore, mitochondrial health is found to play a vital role in cellular health. Mitophagy, a selective autophagy process, is essential for mitochondrial health. 26 ATP is the product of oxidative phosphorylation, which can result in ROS production within mitochondria. Excessive mitochondrial ROS can cause cytotoxicity and lead to cell death under some conditions. Mitochondria are particularly vulnerable to ROS damage due to the "naked" nature of their DNA and limited antioxidant capacity inside mitochondria. If not properly repaired or cleared from cells, the unhealthy mitochondria will cause further production of ROS and release of proapoptotic proteins into the cytosol, finally leading to a high risk of cell death. 27 Mitophagy, first discovered by Lewis and Lewis 28 in cells, is a kind of selective autophagy that handles dysfunctional or damaged mitochondria, which tend to accumulate following mitochondrial damage or stress. Ashford and Porter 29 employed electron microscopes to observe the mitochondrial debris in 1962, and it was suggested that functional alterations of mitochondria would trigger their autophagy. 30 The term "mitophagy" came into use in 1998. 31 Mitophagy is required to remove the unhealthy mitochondria timely and to maintain steady mitochondrial turnover, as well as cellular development and differentiation. 32 Notably, selective degradation of the surplus or dysfunctional mitochondria by autophagy has been observed in organisms ranging from yeast to mammals. In yeast, mitophagy is mediated by Atg32, which is related to NIX and its regulator Bcl2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3) in mammals. Numerous studies in metazoans suggest that mitophagy is mostly regulated by PINK1 and Parkin, which are not present in yeast, suggesting species-specific difference in mitophagy regulation. Mitophagy is one of the most extensively investigated types of "organelle autophagy," which can be partially ascribed to the connection between mitophagy and disease.

| MITOPHAGY IN MAMMAL S
In mammalian cells, mitophagy is activated by two distinct pathways, one is dependent on receptors while the other one relies on ubiquitin ( Figure 1). Although both types of mitophagy, namely the receptormediated and PINK1/Parkin-mediated mitophagy, have been well studied recently, it remains unknown about how these two types of mitophagy differ in expression among different tissues or in output of mitochondrial degradation. 33 In mammals, both NIX (also known as BNIP3L) and SQSTM1/p62 have been implicated to function as the receptors linking mitochondria with the autophagy mechanism in different cell types. During erythrocyte maturation, NIX is essential for mitochondrial clearance, where mitophagy plays a developmental role. Autophagosome can recognize target mitochondria through LC3 adapters (in ubiquitin-dependent and ubiquitin-independent manners) and the direct interaction of LC3 with its receptors. 34

| Mitophagy receptors in mammals
The molecular mechanisms of mitophagy in mammals appear to be quite distinct from that in yeast. The mammalian homolog of yeast Atg32 has not been identified. Some functional counterparts of Atg32, such as BNIP3, NIX, and FUN14 domain-containing protein 1 (FUNDC1), have been suggested to serve as the mitophagy receptors in mammals. 35 38 with NIX being implicated in reticulocyte maturation. 39 Atg7 and ULK1 are also involved in mitochondrial removal in reticulocytes. Moreover, nonautophagic mechanisms may also promote mitochondrial removal during reticulocyte maturation. 40 NIX/ BNIP3L and BNIP3, which are regulated by hypoxia-inducible factor (HIF) or forkhead homeobox type O (FOXO), also participate in the hypoxia-induced mitophagy. 41 While the upregulation of NIX or BNIP3 can enhance their activities in mitophagy, the interplay between BNIP3 and LC3 may also act at the level of phosphorylation of BNIP3. When Ser17 and Ser24 adjacent to the LIRs of BNIP3 are phosphorylated, the interplay between BNIP3 and LC3 is enhanced, suggesting a possible role of kinases or phosphatases in regulating mitophagy. Additionally, the interaction between NIX and a small GTPase Rheb is shown to initiate mitophagy. 42 can not only inhibit the PGAM5 phosphatase activity, but also interact with PGAM5 and block the dephosphorylation of FUNDC1. 46 Under hypoxia condition, ULK1-mediated phosphorylation at Ser17 can promote the interaction between FUNDC1 and LC3. 47 Additionally, the mitochondrial E3 ligase MARCH5 can regulate hypoxia-induced mitophagy through ubiquitinating and degrading FUNDC1. 48 The receptor-interacting serine/threonine-protein kinase 3 (Ripk3) can suppress FUNDC1-mediated mitophagy and promote mitochondrial apoptosis in cardiac ischemia/reperfusion injury. 49 Unexpectedly, it is also suggested that knockdown or overexpression of FUNDC1 has insignificant influence on starvation-or hypoxia-induced mitophagy.

| FUNDC1-mediated mitophagy
The cause of such divergent results remains to be fully clarified.
In mammalian cells, FUNDC1 can recruit LC3 through its LIR motif, thereby activating mitophagy; it can also interact with DNM1L/DRP1 and OPA1, regulating mitochondrial fission or fusion, and mediating mitophagy. OPA1 can interact with FUNDC1 through its Lys70 (K70) residue, and mutation of K70 to Ala (A) will eliminate the interaction between OPA1 and FUNDC1, and promote mitochondrial fission and mitophagy. 50 Therefore, FUNDC1 can coordinate the dynamics and quality control of mitochondria.

| BCL2L13 and FKBP8
Bcl2-like 13 (BCL2L13), which is located on the OMM and can bind to LC3 via the LIR motif, contains one transmembrane region and one LIR on its N-terminal facing the cytoplasm. BCL2L13 has been identified as one of the functional counterparts of Atg32, since exogenous BCL2L13 expression can partially rescue a mitophagy defect in the atg32Δ yeast. Similar to the case of Atg32, the phosphorylation of Ser272 on BCL2L13 can also stimulate the binding of BCL2L13 to LC3. 51,52 FK506-binding protein 8 (FKBP8) is an LC3-interacting protein located on the OMM, which can effectively promote mitophagy in a Parkin-independent manner. 53 In particular, FKBP8 migrates from mitochondria into ER following treatment with CCCP, a chemical ionophore. The FKBP8 N412K mutant that cannot translocate to ER is defective in suppressing apoptosis during mitophagy, suggesting that not all mitochondrial proteins are degraded during mitophagy, and that the subcellular localization of FKBP8 can regulate cell survival during mitophagy. 54

| PHB2 and cardiolipin
Although prohibitin 2 (PHB2) is located in the IMM, Parkin-mediated degradation of OMM proteins can trigger the rupture of OMM, exposing PHB2 to LC3 and finally inducing mitophagy. In C elegans, PHB2 plays a vital role in the removal of damaged mitochondria. 55,56 Cardiolipin is a kind of membrane lipid existing in the IMM, which can also serve as a receptor of LC3 in mitophagy. Cardiolipin can be transferred from IMM to OMM during mitochondrial depolarization to induce mitophagy. Cardiolipin and Parkin can independently respond to CCCP treatment and regulate mitophagy at different levels of mitochondrial depolarization. 57,58 Externalized cardiolipin can directly interact with the N-terminal helix of LC3, which is specific to the LC3 subtype. In rotenone-treated cells, cardiolipin can directly interact with gamma-aminobutyric acid receptor-associated protein (GABARAP), one of the LC3 family members, but it would not recruit GABARAP to mitochondria. Thus, cardiolipin can interact with GABARAP during different autophagic processes. These findings indicate that the IMM components also participate in mitophagy. 59

| Ambra1
Ambra1, a Beclin 1 interactor, is another mitophagy receptor. After autophagy induction, Ambra1 can gradually transfer from the cytoskeleton to ER and regulate autophagosome nucleation. 60 The LIR region in Ambra1 can directly bind to LC3 during mitophagy induction. Targeting Ambra1 on mitochondria can promote mitophagy in either Parkin-dependent or Parkin-independent pathways. In Parkindeficient cells, Ambra1 is subject to ubiquitination when Ambra1 is targeted to mitochondria. 61,62 Ambra1 also promotes ubiquitination of ULK1 through the E3 ligase tumor necrosis factor receptor-associated factor 6 (TRAF6), indicating that Ambra1 is probably the adaptor for E3 ligases. 63 It is demonstrated that Parkin can interact with Ambra1, and prolonged mitochondrial depolarization will further enhance their interaction. Moreover, Ambra1 can be recruited to depolarized mitochondria and eventually promote mitophagy, and Parkin translocation can trigger mitophagy through the activation of Ambra1 and the ubiquitination of OMM proteins. 64 PINK1 and Parkin mutations are the most common pathogenic factors of recessive familial PD. PINK1 is a mitochondria-targeted serine/threonine kinase, while Parkin is a cytoplasmic E3 ubiquitin ligase. PINK1 and Parkin function in the same pathway and to mediate mitophagy in metazoans, with PINK1 acting upstream of Parkin. [65][66][67][68][69] PINK1 is also regarded as a mitochondrial stress sensor whose function depends on the mitochondrial membrane potential. [70][71][72] The PINK1-Parkin pathway may be responsible for regulating the heterogeneity between the healthy and damaged mitochondria and thus mitochondrial homeostasis in cells. Parkin and PINK1 can promote mitochondrial health through several mitochondrial quality control mechanisms, including the turnover of OMM proteins by the proteasome, the generation of mitochondrial-derived vesicles, and organellar degradation through mitophagy. Under healthy mitochondrial condition, PINK1 is maintained at a low level through complex processing and degradation. But PINK1 is stabilized on OMM with decreased mitochondrial membrane potential, where PINK1

| PINK1/Parkin-mediated mitophagy
can recruit Parkin to damaged mitochondria. 73 Thereafter, PINK1 can phosphorylate Parkin at Ser65 and stimulate Parkin's E3 ligase activity 74 ; PINK1 also phosphorylates ubiquitin at Ser65, which would then activate Parkin upon binding. [75][76][77] It is thought that the ubiquitination of OMM proteins by Parkin can initiate the mitophagy process. Parkin can stimulate the attachment of the ubiquitin chain to its substrates through K48 and K63 linkages. Normally, protein degradation can be initiated by K48-linked ubiquitination, which can initiate passive mitochondrial degradation. The autophagy-associated adaptors LC3/GABARAP are generally recruited by K63-linked ubiquitination, which is involved in mitophagy. 78 After being recruited to the mitochondria, Parkin directs the ubiquitination of various OMM proteins, which can mediate mitochondrial sequestration through interaction with the adaptor proteins on the separation membrane. Several substrates of Parkin have been identified, including mitofusin (Mfn), TOM70, Miro, and Drp1, suggesting that Parkin plays multifaceted roles in the dynamics and biogenesis of mitochondria. p62 is suggested to be recruited to mitochondria along with Parkin; however, p62 is reported to be unnecessary for removing the damaged mitochondria, challenging the role of p62 in the PINK1/Parkin-mediated mitophagy. [79][80][81] The mitochondrial fusion GTPase Mfn2 is also a Parkin substrate.

Mfn2 can interact with the Miro-Milton complex on mitochondria
and is therefore also related to the transport of mitochondria. 82 Mfn2 can be phosphorylated by PINK1 and subsequently ubiquiti-

| LC3 adapters: p62, NBR1, OPTN, NDP52, and TAX1BP1
The autophagy adaptor proteins, including SQSTM1 (also called p62), NBR1, optineurin (OPTN), NDP52 (also called CALCOCO2), and TAX1BP1, possess the ubiquitin-binding domains, which can interact with the ubiquitinated mitochondrial proteins, and the LIR motifs, which can recruit a separation membrane through interacting with LC3, in the selective autophagic degradation of mitochondria. [86][87][88] Among them, OPTN is one of the most studied adapters for the recruitment of phagophore to the mitochondria mediated by the PINK1/Parkin pathway. 89 OPTN can be recruited to damaged mitochondria through binding with ubiquitinated OMM proteins, thereby inducing mitochondrial isolation by autophagosome through its interaction with LC3. 90 TBK1 is activated after recruitment by OPTN, which can then promote mitophagy. 91,92 Though TBK1 can phosphorylate various adapters, only OPTN and NDP52 have been regarded as its primary substrates. 93,94 PINK1 has also been shown to play a role in the mitophagy pathway in a Parkin-independent manner. PINK1 recruits OPTN and NDP52 onto the mitochondria, as well as the subsequent recruitment of autophagy initiation factors, including ULK1 and double FYVE-containing protein 1 (DFCP1). Interestingly, overexpression of synphilin-1, an alpha synuclein-interacting protein, can induce PINK1 accumulation. Later, the PINK1-synphilin-1 complex can recruit an E3 ubiquitin ligase (SIAH) to enhance mitochondrial ubiquitination, indicating that E3 ligase other than Parkin can target mitochondria for degradation in distinct ways. 95

| MITOPHAGY AND NEURODEG ENER ATIVE DIS E A S E S
Mitophagy often takes place under baseline conditions; however, it can also be promoted under specific physiological conditions. For example, NIX can remove mitochondria from the mature erythrocytes during development, and Parkin and the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) are necessary for the degradation of paternal mitochondria after fertilization in mice. 96 The differentiation state of stem cells can be affected by the PINK1-dependent mitophagy. 97 On the one hand, mitophagy contributes to metabolic changes during the differentiation and the transition from beige to white adipocytes. 98 On the other hand, mitophagy also exerts great roles in activating the NOD-like receptor protein 3 (NLRP3) inflammasome, and the FUNDC1-dependent mitophagy can downregulate platelet activation following acute ischemia/reperfusion injury. 99,100 As reviewed below, defective mitophagy is frequently observed in neurodegenerative diseases (Table 1).  104 Moreover, SREBF1 is also one of the risk factors for sporadic PD, 105 and mitophagy thus may represent a common mechanism linking sporadic and familial PD. [106][107][108] Mutation in of the F-box domain is reported to be associated with the early-onset autosomal recessive PD. FBXW7 TA B L E 1 Major neurodegenerative disease-associated proteins which play roles in mitophagy and their mechanisms  mechanism. These genes are associated with mitochondrial function, and the corresponding gene products are also involved in mitophagy. Therefore, these proteins may provide mechanistic links between PD with mitophagy. Parkin is normally a cytosolic protein, which is recruited to dysfunctional mitochondria in a PINK1-dependent manner. 114 Parkin will broadly ubiquitinate and degrade the OMM proteins through the ubiquitin-proteasome system. 115 On dysfunctional mitochondria, ubiquitinated OMM proteins produced by Parkin can be recognized by the autophagy receptors, such as OPTN, p62, and NDP52, which can initiate the autophagy process. PINK1 appears to play an incontrovertible role in mitophagy, but Parkin seems to amplify and promote PINK1 effect but itself is not obligatory, since the recruitment of autophagy receptor would take place and mitophagy would be initiated in the absence of Parkin activation. 85,116 The Parkin-independent events downstream of PINK1 during the mitophagy process remain largely unknown.

| PINK1 and Parkin
For instance, the critical substrates and the E3 ligases promoting the mitophagy-inducing ubiquitination signals remain to be fully elucidated. Our recent studies revealed that on damaged mitochondria, the recruitment of cotranslational quality control factors Pelo, ABCE1, and NOT4 to stalled ribosomes results in NOT4-mediated polyubiquitination of ABCE1 and that polyubiquitinated ABCE1 (poly-Ub-ABCE1) provides a molecular signal for recruiting autophagy receptors to initiate mitophagy. 119 We provided evidence supporting that the PINK1 pathway is deployed to stimulate translation of nuclear-encoded respiratory chain (nRCC) mRNAs on mildly damaged mitochondria to promote RC biogenesis and thus mitochondrial repair, revealing a new physiological role of PINK1/ Parkin in mitochondrial regulation. However, for severely damaged mitochondria that are beyond repair, the PINK1 pathway is used to direct their clearance by mitophagy. Our results also showed that many of the effects of PINK1 in activating mitophagy, from recruit-

| DJ-1
DJ-1, encoded by the PARK7 gene, is associated with autosomal recessive PD. 131,132 DJ-1 has been regarded as a redox sensor, with potential roles in mitochondrial homeostasis. 133

| LRRK2
Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are as- Expression of mutant LRRK2 negatively affects mitochondrial health. Endogenous LRRK2 can interact with the regulators of mitochondrial fission and fusion (such as Drp1, OPA1, and Mfn). 142 In PD patient fibroblasts with LRRK2 G2019S mutation, there is increased susceptibility to MPP + -induced cellular death. 143 The loss of LRRK2 will disrupt the autophagy pathway and promote the production of autophagosomes. 144,145 Although CMA may mediate the degradation of LRRK2, but the high levels of wild-type LRRK2 will obstruct the translocation of the CMA complex, leading to the deficiency of CMA. 146,147 The MCU-mediated calcium import; consistently, enhancing calcium export from mitochondria is also protective. Therefore, LRRK2mediated neurodegeneration includes enhanced susceptibility to mitochondrial calcium overload. 150 The toxicity of α-Syn may also be mediated through its interaction with LRRK2 kinase. Common protein interactors may regulate or mediate α-Syn and LRRK2 interaction in PD. 151

| Mitophagy and AD
Alzheimer's disease is a progressively developing neurodegenerative disease, which is associated with the main clinical symptoms of memory impairment, aphasia, indiscriminateness, cognitive impairment, visual-spatial impairment, and changes in behavior and personality. Its pathogenic mechanism remains unclear. Extracellular amyloid accumulation will form the senile plaques, and such cellular changes, together with the intracellular hyperphosphorylation of microtubule-associated tau protein, account for the pathological hallmarks of AD. 152 Recent studies have found that the development of AD is closely correlated with mitochondrial autophagy defects. 153,154 In a transgenic mouse model of AD, the amyloid-β protein is accumulated, accompanied by a cascade of upregulated mRNA levels of mitochondrial autophagy-associated proteins, such as p62, PARK2, DNM1L, BECN1, BNIP3, PINK1, and LC3. In humans, mice, and even nematodes, Aβ can induce UPR mt and mitophagy in a conserved manner, but the specific mechanism remains to be fully defined. The increases in UPR mt and mitophagy are beneficial, which have been shown to contribute to delaying disease progression and boosting the survival in nematodes. 155 Moreover, Disrupted-in-Schizophrenia-1 (DISC1) has been identified to act as a newly discovered mitophagy receptor. The downregulation of DISC1, as well as Aβ accumulation in AD, will be mutually reinforced, thus creating a vicious circle. Additionally, DISC1 has been shown to protect synapses from Aβ accumulation-associated toxicity through promoting mitophagy. 156

| PINK1/Parkin
The PINK1/Parkin pathway is a hotspot in research concerning the effect of mitophagy on AD pathology. 157,158 In neurons expressing the human amyloid precursor protein (hAPP), the AD-related mitochondrial stress is found to markedly promote Parkin-dependent mitophagy. Under normal conditions, more Parkin would be recruited to mitochondrial membrane in AD neurons than in healthy neurons, which has been confirmed in AD patient brain samples. This pheno-

| Tau
In the case of human wild-type full-length Tau (hTau), its intracellular accumulation will affect the mitochondrial membrane potential, thereby inducing mitochondrial maladjustment and dysfunction.
Besides, it was recently found that hTau can prevent the intracytoplasmic translocation of Parkin to the mitochondrial membrane and inhibit mitophagy in nematode models, while such effect is independent of the changes in membrane potential. On the other hand, the NH2-terminal fragment of tau can regulate Parkin and UCHL-1 through inhibiting the ANT-1-dependent ADP/ATP exchange, suppress mitochondrial autophagy, and mediate synaptic degeneration in AD. 160

| Sirtuins
There are also other genes and proteins that can act as a bridge between mitophagy and the progression of AD. The sirtuin family is comprised of seven sirtuins. Three of them have been confirmed to be the mitochondrial sirtuins that are closely related to the mitochondrial performance. 161 Resveratrol has been indicated to upregulate

| Mitophagy and HD
HD is an autosomal dominant neurodegenerative disease, which is frequently seen in middle-aged people. Its clinical symptoms primarily feature dance-like movements, followed by cognitive impairment, regression. [166][167][168] Increasing evidence suggests that mutant Htt can damage mitochondria, leading to energy metabolism disorders, oxidative stress, and release of proapoptotic factors. 18,169 The aberrant morphology of mitochondria can be observed in the Drosophila model of HD. In addition, mutant Htt is found to cause formation of spherical mitochondria in a nonapoptotic state, and it can also repress mitophagy, resulting in the impaired mitochondrial clearance. 170

| GAPDH
Polyglutamine expansion is an important pathogenic mechanism of HD. Recent studies revealed that the amplified polyglutamine can cause mitochondrial dysfunction, which is mainly characterized by morphological abnormalities, blockade of respiratory function, reduced ATP production, and the release of proapoptotic factors.
Such mitochondrial damage is at least partly ascribed to the inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on mitochondria. This is followed by the inhibition of GAPDH-mediated mitophagy and the accumulation of impaired mitochondria in cells.
GAPDH selectively binds to mitochondria, and overexpression of GAPDH can restore mitochondrial function. 171

| Transglutaminase type 2
Transglutaminase type 2 (TG2) is closely related to mitochondrial clearance and homeostasis. Its expression in the brain of HD patients is elevated, and it cross-links with Htt, resulting in mitochondrial membrane potential loss, accumulation of abnormal proteins, and neuronal apoptosis in the brain. On the other hand, pharmacological inhibition or gene knockdown of TG2 can protect against neurodegeneration in HD, and TG2 may be one of the causes of progressive death of HD neurons. 172 Impaired axonal transport of autophagosomes has also been suggested to be responsible for the inability of abnormal mitochondria to be cleared in HD. The Htt-associated protein-1 (HAP1) and Htt are shown to regulate neuronal autophagosome transport. 170 Depletion of Htt increases the autophagosomes containing mitochondrial fragments, indicating that mitochondrial clearance is reduced. However, the authors found that Htt would not alter the flux of mitophagy.

| Valosin-containing protein
Inhibiting the α-tubulin deacetylase HDAC6 is also considered a potential avenue for treating HD, but the mechanism remains unclear, and studies suggest that HDAC6 may block mitophagy. 174 Various studies have revealed reduced mitophagy in the brain of HD patients, which lead to subsequent dysfunction. 171 Currently, developing treatment strategies for HD by rebuilding stable mitophagy balance has attracted great interests.

| Mitophagy and ALS
ALS is a neurodegenerative disease that develops in adulthood, accompanying the loss of motor neuron disease (MND) and upper motor neuron (UMN). The pathological hallmark of ALS is progressive motor dysfunction, but the sensory function is found to be unaffected. Nonetheless, it has been found in recent years that the neurodegeneration in ALS is not limited to the motor system; instead, it has involved the sensor, linguistic, behavioral, and other cognitive domains. Some patients may have mild cognitive impairment and even significant frontotemporal dementia.  169,183,184 In addition, VCP also exhibits some properties similar to OPTN; for instance, it relies on the Parkin-mediated ubiquitination to be recruited to the mitochondria and is involved in mitophagy. The ALSrelated VCP mutations will disrupt the mitophagy balance through the PINK1/Parkin pathway, thereby affecting the clearance of abnormal mitochondria. 185 At present, the pathogenesis of ALS remains to be fully understood, and the existing hypotheses include oxidative stress, mitochondrial dysfunction, excitotoxicity, and neuroinflammation. [186][187][188] Notably, research on mitophagy, inflammation, and their interaction has become a hotspot. Improving mitochondrial autophagy and restoring mitochondrial homeostasis may offer potential treatment for ALS.

| CON CLUS IONS
Neurodegenerative diseases are associated with protein turnover, and protein aggregation is involved in the cellular pathology of many neurodegenerative diseases. Mitophagy can partially account for the mechanisms of cellular homeostasis; therefore, appropriate mitophagy level is of great significance to reduce the aggregation of abnormal proteins and to stimulate organelle removal. For the sake of protecting neurons, it is necessary to maintain the mitochondrial function and promote the degradation of damaged mitochondria. The regulation of mitophagy is suggested to be one of the therapeutic strategies for some neurodegenerative diseases.
The activation of mitophagy has been shown to improve neurodegenerative diseases phenotypes and offer neuroprotection. Several therapeutic tools have been confirmed to increase mitophagy, such as the regulators of PINK1/parkin, metformin, and resveratrol. 189 In recent experiments, some drugs are also thought to delay the disease progression, which target the sirtuins family. Sirtuin activating compounds (STACS) or NAD precursors such as NR/NMN can increase mitophagy through regulating sirtuins. 189,190 Many natural compounds with bioactivation are gradually being discovered.
Some antibiotics or plant ingredients also induce mitophagy, such as actinomycetes. 191 It is associated with mitochondrial autophagy in neural stem cells, which may be mediated by ribosome depletion. 192 Nevertheless, both excessive and reduced mitophagy may be harmful, making mitophagy a double-edged sword. Mitochondrial autophagy plays an important role in the self-maintenance of the nervous system, but only the role of PINK1/Parkin pathway in the regulation of neurodegenerative diseases has been well specified so far, and some results are still controversial. The current results only partially reveal the role of mitophagy mediated by the PINK1/Parkin pathway in PD, while there are few studies on AD and HD, and many questions have not been clarified. 193,194 NIX/BNIP3L and FUNDC1 mainly regulate mitochondrial autophagy under hypoxic conditions, but the regulation of mitochondrial autophagy under hypoxic and ischemic conditions needs to be further explored. 195 Consequently, simply elevating the mitophagy level is not a feasible approach, and there are still some future challenges; for instance, how to optimize the mitophagy activity for neuron protection to treat neurodegenerative diseases, how to elucidate the common molecular mechanisms regarding the pathogenesis of neurodegenerative diseases, and how to clarify the interactions among mitophagy, mitochondrial metabolism, and mitochondrial dynamics. Researches on the molecular mechanisms related to mitophagy should be strengthened, and technologies that can visually and real-time monitor mitochondrial morphological changes should be developed. In addition, more specific agents that target mitophagy can be applied to cultured neurons or animal models of neurodegenerative diseases to observe the relationship between mitophagy and diseases.

ACK N OWLED G M ENTS
Due to space limitation, we cannot cover all the literature in the enormous field of autophagy. We apologize to those investigators whose

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.