Balancing Apoptosis and Autophagy for Parkinson's Disease Therapy

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Balancing apoptosis and autophagy for Parkinson’s disease therapy: targeting BCL-2 Jia Liu, Weijin Liu, and Hui Yang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00356 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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ACS Chemical Neuroscience

Balancing apoptosis and autophagy for Parkinson’s disease therapy: targeting BCL-2 Jia Liu, Weijing Liu, Hui Yang* Department of Neurobiology School of Basic Medical Sciences, Capital Medical University, Center of Parkinson's Disease Beijing Institute for Brain Disorders, Beijing Key Laboratory of Neural Regeneration and Repair, Beijing Key Laboratory on Parkinson's Disease, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing 100069, China *Corresponding author: Prof. Hui Yang; Department of Neurobiology, Capital Medical University, 10 Xi Tou Tiao, You An Men, Beijing 100069, China Tel: +86 10 8359 0070; Fax: +86 10 8359 0070; E-mail: [email protected] Keywords: apoptosis; autophagy; BCL-2; BECN1; Parkinson’s disease

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Abstract Apoptosis and autophagy are important intracellular processes that maintain organism homeostasis and promote survival. Autophagy selectively degrades damaged cellular organelles and protein aggregates, while apoptosis removes damaged or aged cells. Maintaining a balance between autophagy and apoptosis is critical for cell fate, especially for long-lived cells such as neurons. Conversely, their imbalance is associated with neurodegenerative diseases such as Parkinson’s disease (PD), which is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Restoring the balance between autophagy and apoptosis is a promising strategy for the treatment of PD. Some core proteins engage in crosstalk between apoptosis and autophagy, including B cell lymphoma (BCL)-2 family members. This review summarizes the role of BCL-2 members in the regulation of apoptosis and autophagy and discusses potential therapeutic approaches that target this balance for PD treatment. Introduction Cell death plays an important role in development and contributes to the maintenance of homeostasis in multicellular organisms. Perturbation of cell death signaling is implicated in many neurodegenerative disorders, including Parkinson’s disease (PD)1. Generally speaking, there are three types of cell death—i.e., apoptosis (type I), autophagy (type II), and necrosis (type III)2. The balance of these systems is important for cell survival, especially for long-lived cells such as neurons1. There is usually more than one type of cell death that is dysregulated in PD patients or experimental models3, 4. There is also some overlap among the molecules involved in the various modes of cell death including B cell lymphoma (BCL)-2 family members. Modulating these factors to restore the balance among the various types of cell death and can in theory protect against neurodegeneration. In this review, we introduce the apoptosis and autophagy pathways and their roles in PD pathogenesis. We also discuss the modulation of BCL-2 family members to restore the balance of apoptosis or autophagy in the context of PD treatment. 1. Apoptosis Apoptosis is a type of cell death characterized by morphological changes such as cell shrinkage and chromatin condensation. This process is critical for metabolism and development. Excessive or insufficient apoptosis is associated with diseases such as neurodegeneration, cancer, and autoimmunity1, 5, 6. In general, apoptosis can be divided into intrinsic (mitochondrial) and extrinsic (death receptor) pathways. Both of these are dependent on the activation of caspases, which are a family of cysteine proteases that specifically target aspartic acid residues7. However, in the intrinsic pathway, caspase-9 and -3 are successively activated whereas in the extrinsic pathway caspase-8 and -3 are activated. As the name suggests, the extrinsic apoptotic pathway is activated in response to external stimuli, a process that is dependent on death receptors of the tumor necrosis factor receptor (TNFR) family including TNFR1, Fas (also known as CD95 or APO-1), TNF-related apoptosis-inducing ligand receptor (TRAILR)1 (also known as death receptor [DR]4), TRAILR2


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(also known as DR5), DR3 (also known as WSL-1), and DR67. Caspases are cleaved and activated upon binding of a ligand to death receptors, thereby triggering the extrinsic apoptosis pathway (Fig. 1). Activation of the intrinsic apoptotic pathway is closely related to changes in mitochondrial outer membrane permeabilization (MOMP)8. Under normal conditions, cytochrome (Cyto) C exists only in the mitochondrial intermembrane space. Stress activates pro-apoptotic members of the BCL-2 family, resulting in an increase in MOMP9. Cyto C is released from mitochondria into the cytoplasm and interacts with apoptotic protease-activating factor 1, which binds dATP/ATP and oligomerizes to form a functional apoptosome10. The apoptosome then activates caspase-9 and -3 to induce mitochondria-dependent apoptosis (Fig. 1). In this review, we mainly introduce intrinsic apoptotic pathway. 1.1 BCL-2 family proteins modulate apoptosis MOMP plays a critical role in the intrinsic apoptosis pathway and is tightly regulated by BCL-2 family proteins, which are characterized by one or more BCL-2 homology (BH) domains (i.e., BH1–4) and can be divided into three classes: pro- and anti-apoptotic and BH3-only proteins11 (Fig. 2. A). BCL-2-associated X protein (BAX) and BCL-2 antagonist/killer (BAK) are pro-apoptotic proteins that contain BH1–3 domains. Although BAK is normally localized in mitochondria, BAX is present in the cytoplasm in an inactive form, and is translocated to mitochondria in response to pro-apoptotic signal such as DNA damage, oxidative stress, and nutritional deficiency12. Both proteins are activated under stress and form a homodimer or heterodimer that is inserted into and disrupts the mitochondrial outer membrane, leading to the release of pro-apoptotic factors and induction of apoptosis13, 14. The pro-apoptotic activities of BAX or BAK are balanced by anti-apoptotic BCL-2 family members including BCL-2, BCL extra-large (BCL-XL), BCL-W, mantle cell lymphoma (MCL)1, A1/BFL1, and BCL-B. Unlike BAX or BAK, these anti-apoptotic members reside in mitochondria and contribute to the integrity of the mitochondrial outer membrane under normal circumstances. Anti-apoptotic BCL-2 members are globular proteins formed by nine α-helices with a hydrophobic cleft known as a BH3 binding-groove at the surface that can accommodate BH3 domains of pro-apoptotic or BH3-only members15. Thus, anti-apoptotic BCL-2 members can interact with monomeric BAX or BAK to prevent their oligomerization or to antagonize BH3-only members by interacting with their BH3 domains16, 17. Pro-/anti-apoptotic proteins modulate MOMP and apoptosis while BH3-only members regulate this balance. These proteins act as upstream sensors that inhibit the activities of antiapoptotic proteins by inserting their BH3-containing helix into the hydrophobic BH3-binding grooves of anti-apoptotic proteins. Additionally, they also bind and activate BAX or BAK directly to induce intrinsic apoptosis18. Bcl-2-like protein 11 (BIM) and BH3-interacting domain death agonist (BID), the most common BH3-only proteins with the most potent inducing properties, can directly activate BAX and BAK19, 20. Although BID is a BH3-only protein, it harbors multiple BH domains and must be cleaved for these to be exposed. The truncated form of BID is inserted into the mitochondrial membrane and activates BAX, leading to permeabilization21, 22. Other BH3-only



members such as p53 upregulated modulator of apoptosis (PUMA) and phorbol-12-myristate-13acetate-induced protein 1 (NOXA) also have activating properties targeting BAX and BAK, albeit weaker than BIM or BID23, 24. Since BH3 domains play an important role in the regulation of apoptosis, BH3 mimetics including ABT-737, ABT-263, and ABT-199 represent a potential therapeutic strategy in various diseases25, 26. As mentioned above, apoptosis is determined by the interaction of BCL-2 members, and differences in the affinities of BCL-2 proteins determine their competitive binding27. The interactions among anti-apoptotic BCL-2 proteins and other family members or BH3 mimetics are illustrated in Figure 2. B. 2. Autophagy Autophagy, which literally means “self-eating”, is an important degradation pathway in the cell for eliminating protein aggregates and damaged organelles. Autophagy was first discovered by and characterized in the early of 1960s by Christian de Duve, who won the Nobel Prize for Physiology or Medicine in 1974 for his work on the discovery of the lysosome. In 2016, Yoshinori Ohsumi won the Nobel Prize in Physiology or Medicine for his elucidation of autophagy mechanisms28. Autophagy plays a key role in maintaining intracellular energy and homeostasis. There are three types of autophagy—i.e., macroautophagy, microautophagy, and chaperonemediated autophagy (CMA)29. In macroautophagy, autophagosomes engulf cargo including aggregate-prone proteins and organelles and deliver them to the lysosome for degragation. In microautophagy, the lysosome engulfs long-lived proteins for direct degredation, whereas in CMA, intracellular cargos bind to a chaperone and are delivered to the lysosome for degradation. In this review, we mainly focus on macroautophagy, which is henceforth referred to as autophagy. A critical step in autophagy is the formation of an autophagosome, which consists of a double-layer membrane structure. This cup-shaped, double-layer sac is known as a phagophore and engulfs cargo along with cytoplasm. Autophagosome maturation occurs in three steps: initiation, nucleation, and expansion30. Initiation involves the formation of a phagophore assembly site and is mediated by the UNC51-like kinase (ULK) complex comprising ULK1 or ULK2, autophagy-related protein (ATG)13, ATG101, and focal adhesion kinase family interacting protein of 200 kDa (FIP200)31. This complex can be activated by mammalian target of rapamycin complex (mTORC)1, adenosine monophosphate-activated protein kinase (AMPK), and p53 signaling pathways32, 33. During nucleation, the ULK complex initiates autophagy by activating the phosphoinositide 3-kinase (PI3K) complex composed of Beclin (BECN)1, ATG14L, and vacuolar protein sorting (VPS)34 and its partner VPS15. Activation of the ULK complex causes BECN1 to phosphorylate and activate the VPS34 complex34. The PI3K complex is required to produce phosphatidylinositol 3-phosphate (PtdIns3P) in nascent autophagosomes. PtdIns3P recruits its binding partners WD repeat domain phosphoinositide-interacting protein (WIPI)1 and WIPI2 to the autophagosome membrane. After binding, the ATG16L1 complex (also known as the ATG12ATG5-ATG16L1 complex) whose formation requires the catalytic activity of ATG7 and ATG10 is recruited to the autophagosome membrane35. During autophagosome expansion, the ATG16L1 complex functions as an E3-like ubiquitin ligase to promote the lipidation of microtubule-


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associated proteins 1A/1B light chain (LC)3, a ubiqutin-like protein, with phosphatidylethanolamine (PE). LC3 can be cleaved by ATG4B to form LC3I, which is transformed into LC3II36. This signals the formation of the autophagosome; therefore, LC3II is considered as a marker of autophagic flux37. The mature autophagosome is retrogradely transported along microtubules to the lysosome-rich perinuclear region by dynein38. Autophagosomes fuse with endosomes to form amphisomes that in turn fuse with lysosomes to form autophagolysosome, which is followed by cargo degradation. 2.1 BCL-2 family and autophagy Although the BCL-2 family is closely related to mitochondria-mediated apoptosis, it plays additional non-apoptotic roles, including autophagy39. Anti-apoptotic BCL-2 members inhibit autophagy by binding to BECN1, which contains a functional BH3 domain that inserts into the hydrophobic groove of these anti-apoptotic BCL-2 members to regulate autophagy40. Posttranslational modifications of BECN1 or BCL-2 contribute to their interaction and autophagy induction. Thr108 phosphorylation of BECN1 promotes the BCL-2–BECN1 interaction41. Additionally, c-Jun N-terminal kinase (JNK)1-mediated BCL-2 phosphorylation stimulates autophagy by disrupting the BCL-2–BECN1 complex42. The dissociation of BCL-2 and BECN1 and autophagy activation is blocked in mice with mutations in BCL-2 phosphorylation sites including Thr69Ala, Ser70Ala, and Ser84Ala43. Anti-apoptotic BCL-2 members including BCL-2 or BCL-XL inhibit autophagy only in the presence of pro-apoptotic BCL-2 members such as BAX or BAK44, although the precise mechanisms of action remain unclear. The effects of the pro-apoptotic proteins BAX and BAK on autophagy are controversial. The deficiency of either protein increases the level of autophagy, indicating a direct or indirect mode of inhibition45. Another study showed that BAX reduced autophagy by promoting caspasemediated cleavage of BECN1 to disrupt the interaction between VPS34 and BECN1, which is required for autophagy46. On the other hand, BAX was also shown to promote autophagy including mitophagy in response to mitochondrial disturbance47. Under normal conditions, BAX mediates mitochondrial fusion and thereby suppresses mitophagy whereas BAX overexpression promotes mitochondrial fission and accelerates apoptosis48. Another study showed that macrophage stimulating-1, another pro-apoptotic protein, promoted BECN1 phosphorylation and the interaction among BECN1, BCL-2, and BAX41. BH3-only proteins such as BIM, Bcl-2-associated death promoter (BAD), and BIK also regulate autophagy by disrupting the BCL-2/BCL-XL and BECN1 complex49-51. BIM knockdown increased autophagy52 since BIM binds to BECN1, which is facilitated by the dynein light chain 1 and mislocalizing BECN1 to the dynein motor complex prevents their interaction to inhibit autophagy. BH3 mimetics targeting autophagy or apoptosis induction53, 54 are promising agents for disease treatment; for instance, the small molecule BH3 mimetic ABT-737 stimulates autophagy by blocking the interaction between BCL-2 or BCL-XL and BECN149. 3. PD PD is the second most common neurodegenerative disease after Alzheimer’s disease, and is



characterized by motor symptoms including bradykinesia, rigidity, resting tremor, and abnormal posture and gait. In addition to these motor deficits as well as non-motor symptoms such as olfactory dysfunction, rapid eye movement sleep behavior disorder, depression, constipation, and dementia. The pathological characteristics of PD include loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the formation of Lewy bodies in remaining neurons55. Although the etiology of PD remains unclear, recent studies suggest that genetics, environment, and aging are contributing factors. Aging is the major risk factor for PD; the rate of PD increases with aging especially after the age of 80 years56. Therefore, as the global population ages, the number of PD cases is expected to increase by 50% by 203057. Since about 10% of cases are heredofamilial, genetics play an important role in the pathogenesis of PD. A large-scale metaanalysis of genome-wide association studies identified 24 gene loci involved in PD58; some which are linked to autosomal dominant PD including SNCA (encoding α-synuclein), leucine-rich repeat kinase 2, vacuolar protein sorting 35, eukaryotic translation initiation factor 4 gamma 1, DnaJ heat shock protein family member C13, and coiled-coil-helix-coiled-coil-helix domain-containing 2; whereas others are related to the autosomal recessive form of the disease including PARKIN, phosphatase and tensin homolog-induced kinase 1, and the protein deglycase 1-encoding DJ-1. Environmental factors, such as exposure to the neurotoxins rotenone, paraquat, 6hydroxydopamine (6-OHDA), and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) also contribute to PD. Treatment options for PD are limited to pharmacotherapy, neurosurgery, gene therapy, and cell-based therapy59. However, these approaches have side effects and most only alleviate motor symptoms without curing the disease. As such, there is an urgent need to develop novel therapeutic strategies for PD treatment60. The balance between apoptosis and autophagy maintains intracellular homeostasis under normal conditions; this is disturbed in PD, which accelerates neurodegeneration61. Therefore, therapeutic strategies that restore apoptosis and autophagy may be effective for treating PD. 3.1 Imbalance of apoptosis and autophagy in PD Although various types of cell death are involved in the pathogenesis of PD, apoptosis is considered to play the most important role4. Apoptosis is necessary for the construction of an efficient neuronal network in the developing brain; however, excessive apoptosis can accelerate disease progression, including PD. In addition, autophagic disturbance is strongly linked to neurodegeneration. On the other hand, autophagy can be considered as a process of self-recovery. Misfolding and aggregation of intercellular proteins such as α-synuclein are implicated in many neurodegenerative diseases including PD. Autophagy is the main pathway for eliminating these proteins to prevent them from injuring cells, which maintains neuronal survival. Autophagy also eliminates aged or damaged organelles to preserve intracellular metabolism. Therefore, autophagy is important in both physiological and pathological contexts. In PD, autophagy is defective and results in an imbalance of homeostasis in neurons, resulting in an increase in the number of autophagosomes in the brain tissue of patients62 and MPTP, 6-OHDA, or rotenone-induced animal models of PD63. It is unclear whether autophagy contributes to or inhibits apoptosis. Under some


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conditions, autophagy protects cells from apoptosis since misfolded proteins or dysfunctional organelles can be eliminated to generate new amino acids, consistent with a pro-survival role. However, excessive or dysregulated autophagy can accelerate apoptosis by releasing pro-apoptotic proteins from the lysosome including cathepsin, which activates caspases and induces apoptosis by increasing mitochondrial membrane permeability. Thus, the balance between apoptosis and autophagy is critical for maintaining normal physiological function and is disturbed in PD, and restoring this imbalance is a promising strategy in PD treatment. 3.2 BCL-2 proteins in PD The degeneration of dopaminergic neurons is the most important pathological change in PD, and there is evidence suggesting that the function of BCL-2 family proteins is closely related to the development of dopaminergic neurons. BCL-2 overexpression from the promoter of tyrosine hydroxylase—the rate-limiting enzyme in dopamine synthesis—increased the number of dopaminergic neurons postnatally and protected dopaminergic neurons from death induced by neurotoxin, indicating that BCL-2 not only influences the number of dopaminergic neurons after embryogenesis, but also protects against toxic insults64. Another study showed that BCL-2overexpressing neurons had more dopaminergic fibers that grew longer distances65, indicating that BCL-2 may promote dopaminergic neuron development. Abnormal expression of BCL-2 proteins may contribute to the pathogenesis of PD, although the molecular details are still debated66, 67. BCL-2 levels were markedly decreased in PD patients, which was negatively correlated to disease duration and severity68, 69. Similarly, pro-apoptotic member BAX accumulation has been observed in SNpc neurons of postmortem brains from PD patients70, 71 and BAX-rich inclusions have been detected in neurons containing Lewy bodies72, 73. Abnormal expression of BCL-2 family members is a characteristic of various PD models74. αSynuclein overexpression decreased BCL-2 and increased BAX levels75, while DJ-1 knockdown resulted in BAX upregulation and dopaminergic neuron death in zebrafish76. The ubiquitin E3 ligase Parkin plays a critical role in mitophagy and intrinsic apoptosis and its mutation can cause autosomal recessive PD77. Parkin exerts an anti-apoptotic function by promoting BAX ubiquitination and inhibiting BAX translocation to mitochondria78. BAX is recognized by and can interact with Parkin through its BH3 domain. However, R275W mutation of Parkin—a diseaselinked missense mutation—failed to affect BAX translocation, suggesting that BAX is a Parkin substrate and that modulating apoptosis by inhibiting BAX translocation is an important function of Parkin in mitochondrial injury and repair79. Similar perturbations of BCL-2 members have been observed in neurotoxin-induced models. In the MPTP-induced model, the Tat-JBD peptide was shown to disrupt JNK and JNK-interacting protein-1 complex formation to further inhibit JNK activation by decreasing its phosphorylation, which in turn reduced BCL-2 phosphorylation and promoted BAX release from BCL-2/BAX dimers, thereby activating apoptosis80. 1-Methyl-4phonylpyridnium (MPP+) treatment increased the expression of MCL-1, while MCL-1 silencing induced apoptosis and neurotoxicity81. BAX accumulation was observed in mitochondria in the SNpc of MPTP-induced PD mice, while BAX knockout had a protective effect against neurodegeneration in this model82. Another study reported BAX activation in MPTP-induced PD



models and suggested that its translocation is dependent on JNK-induced activation of the BH3only protein BIM73. Similarly, in mice lacking BAX, 6-OHDA treatment did not cause dopaminergic neuronal death83 whereas BAX knockdown provided protection against rotenoneinduced PD84, indicating that dopaminergic neuron death induced by these neurotoxins is mediated by Bax-dependent apoptosis. Mice deficient in the pro-apoptotic protein BAK were resistant to paraquat-induced neurotoxicity85. Many of the neurotoxins related to PD target mitochondrial complex I. However, defects in complex I do not induce apoptosis directly but do so via activation of BAX, which thus plays an important role in complex I dysfunction leading to apoptosis86. In addition, BH3-only proteins including PUMA are also involved in 6-OHDA-induced cell death87 and in the paraquat-induced PD model, several BCL-2 proteins are upregulated including BAK, BID, and NOXA85. These lines of evidence suggest that the balance between BCL-2 family members is disrupted in PD patients and animal models. 3.3 Modulation of BCL-2 proteins in PD treatment Given the aberrations in the expression of BCL-2 family members in PD, a potential strategy for disease treatment is to target these proteins. Bcl-2 overexpression protects dopaminergic neurons against MPTP or 6-OHDA-induced neurodegeneration88, 89, while expression of BAX promoted the formation of α-synuclein deposits and neuron death90. Inhibitors of pro-apoptotic BCL-2 and BH3-only proteins are considered as promising agents. Intrastriatal administration of BAXinhibiting peptide V5 prevented dopaminergic neuron loss induced by 6-OHDA through suppression of caspase-dependent apoptosis91. Mesencephalic astrocyte-derived neurotrophic factor, a well-known inhibitor of Bax, improved PD-like symptoms and protected neurons from neurodegeneration in a rat model of 6-OHDA-induced PD92. Various drugs that target BCL-2 proteins are currently being developed. Ursodeoxycholic acid alleviated motor deficits and suppressed dopaminergic neuron degeneration in rats with rotenone-induced PD by increasing BCL-2 and decreasing BAX expression93. Kukoamine A alleviated mitochondrial deficits by decreasing the BAX/BCL-2 ratio as well as α-synuclein expression by stimulating autophagy in MPP+ induced models94. Overexpression of mitochondrial ferritin, which is specifically expressed in brain neurons, reversed the perturbation of iron redistribution induced by 6-OHDA by modulating the BCL-2-BAX ratio95. Jiang and colleagues screened a chemical library of ~200,000 small molecules that block cytochrome c release and found that compound A exerts an antiapoptotic function by activating BAK and BIM. Compound A also prevented dopaminergic neuron loss in a rat model of 6-OHDA-induced PD96. BCL-2 members also contribute to fate determination in glial cells. Paeunol reversed the increase in BAX/BCL-2 ratio in MPP+-treated astrocytes97, and nicotine alleviated MPTP-induced PD symptoms by blocking astrocyte and microglia activation in the SNpc, thereby restoring the BAK/BCL-2 balance98. These results indicate that targeting BCL-2 proteins may be an effective strategy for PD treatment. There are about 20 BCL-2 members in mammals but only two homologs in Drosophila— namely, the pro-apoptotic Debcl and anti-apoptotic Buffy99, 100. Overexpression of Debcl in the dopaminergic neurons of Drosophila decreased survival and caused the loss of climbing ability while aggravating α-synuclein-associated phenotypes101. BAX inhibitor (BI)-1 is a suppressor of


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BAX-induced apoptosis whose silencing in Drosophila shortened lifespan and caused locomotor deficits. These effects were abrogated by overexpressing Buffy and exacerbated by overexpression of α-synuclein102. Buffy overexpression had similar neuroprotective effects in other Drosophila PD models, including those induced by α-synuclein and HtrA2103, 104. BCL-2 family proteins are linked to PD pathogenesis not only through regulation of apoptosis but also by their role in modulating autophagy. The E46K in α-synuclein was shown to be associated with familial PD; overexpression of this mutant form of α-synuclein impaired macroautophagy by blocking autophagosome formation via JNK1 inhibition, which resulted in decreased BCL-2 phosphorylation and the failure of BCL-2-BECN1 to dissociate, which prevented the initiation of autophagy105. α-Synuclein aggregation increased, accompanied with reduction of BCL-2 level and blockage of autophagic flux, in dopaminergic neurons of rotenoneinduced PD rats. While inhibition of BCL-2 could aggravate autophagic insults and increase rotenone-induced α-synuclein aggregation, indicating dysregulation of BCL-2 enhanced rotenoneinduced neurotoxin106. One study found that BAX is translocated to and internalized by lysosomes following MPTP treatment and that inhibiting BAX could restore lysosomal function and reverse autophagosome accumulation, suggesting that BAX contributes to lysosomal disruption in MPTPinduced PD models107. Restoring autophagy through modulation of BCL-2 is another potential therapeutic strategy for PD treatment. Fasudil, a Rho kinase inhibitor, enhanced neurotoxicity and promoted the clearance of abnormal α-synuclein in an A53T mutant α-synuclein-overexpressing model, which stimulated autophagy by promoting BCL-2-BECN1 dissociation through JNK1 activation and BCL-2 phosphorylation108. Deep brain stimulation (DBS) may also be effective for improving PD symptoms; a recent study showed that DBS of the subthalamic nucleus (STN) mitigated motor deficits and loss of dopaminergic neurons in 6-OHDA-induced PD models. DBS of the STN inhibited protein phosphatase 2A activity and enhanced BCL-2 phosphorylation, which induced its dissociation from BECN1 and consequent autophagy109. 3.4 Targeting BCL-2 to restore the balance between apoptosis and autophagy in PD BCL-2 proteins participate in both of apoptosis and autophagy, providing a link between these processes. Anti-apoptotic BCL-2 members modulate apoptosis by binding to the pro-apoptotic protein BAX or BAK110, 111 and inhibit autophagy by binding to BECN1112, 113, indicating that they have dual function as inhibitors of both apoptosis and autophagy. The post-translational modification of BCL-2 is critical for the regulation of apoptosis and autophagy. BCL-2 phosphorylation, which is mainly mediated by JNK1 signaling, plays a critical role in apoptosis or autophagy modulation42. BCL-2 phosphorylation at Ser70 promotes BAX and BAD binding and is essential for its anti-apoptotic activity114, 115, whereas phosphorylation at Thr69, Ser70, and Ser87 cause its dissociation from BECN1 and induction of autophagy116. Interestingly, JNK-1 mediated BCL-2 phosphorylation sequentially induces autophagy or apoptosis. Under a short period of starvation (4 h), BCL-2 phosphorylation promotes the dissociation of BCL-2 from the BCL-2–BECN 1 complex but not from the BCL-2-BAX complex, whereas after long period of starvation (16 h), BCL-2 phosphorylation is maximal, resulting in the dissociation of the BCL-2– BAX complex and caspase-3 activation. This suggests that rapid BCL-2 phosphorylation initially



promotes cell survival by activating autophagy but when autophagy can no longer keep cells alive, phosphorylation leads to apoptosis. Thus, BCL-2 functions as a switch in the balance between apoptosis and autophagy42. Pro-apoptotic BCL-2 members including BAX and BAK induce mitochondria-dependent apoptosis by oligomerizing and accumulating and forming pores on the mitochondrial surface, which mediates cytochrome c release and caspase activation117. Additionally, BAX and BAK are also modulate autophagy; in the absence of these proteins, prosurvival BCL-2 family members cannot participate in autophagy, indicating that anti-apoptotic BCL-2 family members do not inhibit autophagy directly but indirectly through inhibition of BAX and BAK44. We previously reported that piperlongumine, an alkaloid of Piper longum L., exerts therapeutic effects in rotenone-induced PD models by promoting BCL-2 phosphorylation at Ser70 sites, which induces autophagy by promoting the dissociation of the BCL-2–BECN1 complex and inhibiting apoptosis by stabilizing the BCL-2 and BAX heterodimer. Our findings indicating that targeting BCL-2 is a potentially effective strategy for PD treatment since it can restore the balance between apoptosis and autophagy118. Conclusion The pathogenesis of PD is not fully understood and therapeutic options are limited. PD is characterized by a progressive loss of dopaminergic neurons, which is closely related to increasing levels of apoptosis or disturbance of autophagy. Elucidating the crosstalk between apoptosis and autophagy in PD is essential from an etiological standpoint and for developing effective therapeutic strategies. The current evidence suggests that targeting BCL-2 family members— which have dual functions as modulators of both apoptosis and autophagy—are potential targets in PD treatment. Acknowledgments This work was supported by The National Key R&D Program of China (no. 2016YFC1306000), National Natural Science Foundation of China (no. 81870994). Conflict of Interest The authors declare no conflict of interest. Author Contributions All authors contributed to search the references and write the manuscript. Abbreviations 6-OHDA, 6-hydroxydopamine; AMPK, adenosine monophosphate-activated protein kinase; ATG, autophagy-related protein; BAD, Bcl-2-associated death promoter; BAK, BCL-2 antagonist/killer; BAX, BCL-2-associated X protein; BECN1 Beclin 1; BCL-2, B cell lymphoma-2; BH, BCL-2 homology; BCL-XL, BCL extra-large; BID, BH3-interacting domain death agonist; BIM, Bcl-2like protein 11; CMA, chaperone-mediated autophagy; Cyto C, cytochrome (Cyto) C; DBS, Deep


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brain stimulation; FIP200, focal adhesion kinase family interacting protein of 200 kDa; JNK, cJun N-terminal kinase; LC3, microtubule-associated proteins 1A/1B light chain 3; MOMP, mitochondrial outer membrane permeabilization; MPP+, 1-Methyl-4-phonylpyridnium; MPTP, 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine; mTORC1, mammalian target of rapamycin complex 1; NOXA, phorbol-12-myristate-13-acetate-induced protein 1; PD, Parkinson’s disease; PE, phosphatidylethanolamine; PI3K, phosphoinositide 3-kinase; PtdIns3P, phosphatidylinositol 3phosphate; PUMA, p53 upregulated modulator of apoptosis; SNpc; substantia nigra pars compacta; STN, subthalamic nucleus; TNFR, tumor necrosis factor receptor; TRAILR, TNF-related apoptosis-inducing ligand receptor; ULK, UNC51-like kinase; VPS, vacuolar protein sorting; WIPI, WD repeat domain phosphoinositide-interacting protein.

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Table of Contents Graphic (TOC) legend Targeting BCL-2 to restore the balance between apoptosis and autophagy in PD. Mutations in Parkinson’s disease related genes such as those encoding α-synuclein (α-syn), PTEN-induced putative kinase (PINK)1, and DJ-1 and neurotoxins such as rotenone and 1-methyl-4phenylpyridinium (MPP+) increase apoptosis level and block autophagy, result in imbalance of apoptosis and autophagy, which is one of the most important etiology of Parkinson’s disease. Novel drugs restore this balance by modulating crosstalk protein BCL-2 via phosphorylation, it is a potentially effective strategy for PD treatment. Figure legends Figure 1. Intrinsic and extrinsic apoptotic pathways. In the extrinsic apoptotic pathway, death receptors bind their cognate ligand and activate caspase-8 and -10 through dimerization via adaptor proteins such as FAS-associated death domain protein (FADD). Caspase-3/7 are activated by caspase-8 and -10, which triggers apoptosis. The intrinsic (mitochondrial) apoptotic pathway is controlled by the stability of MOMP. BCL-2 maintains the integrity of the mitochondrial outer membrane under normal conditions; under stress, BH3-only proteins induce the oligomerization and membrane insertion of BAX/BAK, which promotes Cyto C release from mitochondria. Cyto C induces apoptosome formation through interaction with apoptotic protease activating factor (Apaf)-1, which activates caspase-9, resulting in caspase-3/7 activation and induction of apoptosis.


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Figure 2. BCL-2 family members and their interaction partners. A. The BCL-2 family consists of anti- and pro-apoptotic members and BH3-only members. B. BCL-2 members and BCL-2 mimetics show distinct affinities. Gray, no interaction; light pink, low affinity; dark pink, high affinity. Figure 3. Overview of the autophagy pathway. The pathway is activated in response to upstream signaling such as mTORC1, adenosine monophosphate (AMP)-activated protein kinase (AMPK), and p53 signaling pathways. The maturation of autophagosomes comprises three key steps: initiation, nucleation, and expansion. Upon maturation, autophagosomes fuse to lysosomes to form autophagolysosome that degrade the cargo. Figure 4. Imbalance between apoptosis and autophagy in PD. Mutations in PD-related genes such as those encoding α-synuclein (α-syn), PTEN-induced putative kinase (PINK)1, and DJ-1 and neurotoxins such as rotenone and 1-methyl-4-phenylpyridinium (MPP+) increase mitochondrial membrane permeability, which promotes the release of pro-apoptotic factors and apoptosis and dysregulates the autophagy-lysosome system, which is a critical pathway for eliminating damaged mitochondria. Thus, the imbalance between apoptosis and autophagy contributes to PD pathogenesis, and restoring this balance is a potential therapeutic strategy. Figure 5. Roles of BCL-2 phosphorylation in apoptosis and autophagy. The phosphorylation of BCL-2, which is mainly mediated by JNK1 signaling, is critical for the regulation of apoptosis and autophagy. BCL-2 phosphorylation at Ser70 promotes BAX and BAD binding and is essential for its anti-apoptotic activity, whereas phosphorylation at Thr69, Ser70, and Ser87 cause its dissociation from BECN1 and induction of autophagy.


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Balancing Apoptosis and Autophagy for Parkinson's Disease Therapy

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