The Roles of Autophagy in Parkinson's disease

Review Article

Clinical Pharmacology and Translational Medicine © All rights are reserved by R.A. González-Polo and J.M. Fuentes, et al.

AutophagyDefinition

To maintain intercellular homeostasis, cells must constantly adapt their metabolism in response to different stimuli (e.g., cellular energy status, stress, growth, and death). Therefore, almost all cell types possess 2 types of degradative pathways: autophagy and the ubiquitin-proteasome system (UPS). The term “autophagy” comes from the Greek words “auto” (self) and “phagy” (eating) and was mentioned in ca. 1860 by M. Auselmier [1]. However, de Duve discovered cellular compartments that contained cytoplasmic mate-rials and that were able to fuse with degradative vesicles, termed lysosomes, during his observations of autophagy-related structures by electron microscopy in the 1950s [2]. This catabolic pathway is the major process for degrading and recycling unwanted and potentially toxic cellular components, such as misfolded proteins and dysfuncti-onal organelles, as well as pathogens and other materials.

ISSN-2572-7656

*Address for Correspondence: R.A. González-Polo and J.M. Fuentes, Center for Biomedical Research in Network on Neurodegenerative Diseases (CIBERNED), Department of Biochemistry and Molecular Molecular Biology and Genetics. Faculty of Nursing and Occupational Therapy. University of Extremadura, Avda, De la Universidad S / N, C.P. 10003 Cáceres (Cáceres); Tel: +34 927257450; E-Mail: jfuentes@unex.es; rosapolo@unex.es

Received: March 23, 2017; Accepted: July 8, 2017; Published: July 10, 2017

S.M.S. Yakhine-Diop1, 2, R. Gómez-Sánchez3, J.M. Bravo-San Pedro4, 5, 6, 7, 8, R.A. González-Polo1, 2*, J.M.Fuentes 1, 2*.1Center for Biomedical Research in Network in Neurodegenerative Diseases (CIBERNED).2Department of Biochemistry and Molecular Biology and Genetics. Faculty of Nursing and Occupational Therapy. University ofExtremadura. Avda. De la Universidad s / n, C.P. 10003 Cáceres (Cáceres).3Department of Cell Biology, University of Groningen, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, Netherlands.4Equipe 11 labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers, 75006 Paris, France.5INSERM U1138, 75006 Paris, France.6Université Paris Descartes / Paris V, Sorbonne Paris Cité, 75006 Paris, France.7 Université Pierre et Marie Curie / Paris VI, 75006 Paris, France.8Gustave Roussy Comprehensive Cancer Institute, 94805 Villejuif, France.

Abstract Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. This process was initially described as an adaptive mechanism in response to a lack of nutrients. However, its role in the origin of different pathologies, including neurodegenerative diseases, is now recognized. The results of genetic, cellular and toxicological studies show that many of the etiological factors associated with Parkinson's disease alter the cellular process of autophagy in different models and systems. These data support the hypothesis that autophagy dysregulation may play a fundamental role in the etiopathogenesis of Parkinson's disease

Keywords: Autophagy; Parkinson’s disease; Modulation; PARK genes; mTOR; LRRK2.

In the 1990s, the proteins involved in this pathway, termed autophagy-related (ATG) proteins, were discovered and characterized in the budding yeast Saccharomyces cerevisiae [3-5]. Interestingly, this mechanism is highly conserved throughout evolution, and the core machinery has mammalian orthologues [6].

The relevance of autophagy in human pathologies has emerged with the discovery that impairment or a defect in autophagy can cause several diseases, such as muscular dystrophies or neurodegenerative disorders [7]. Crucially, modulation of autophagy has been shown to be effective as a therapy to delay the onset of specific diseases, including cancer [8].

Types of autophagy The term autophagy encompasses all of the pathways that are

involved in the delivery of materials within lysosomes for further degradation and recycling. There are three types of autophagy: macro-autophagy, microautophagy and Chaperone-Mediated Autophagy (CMA).

Macroautophagy Macroautophagy is the major form of autophagy and has been

the most broadly analyzed. During macroautophagy, a portion of the cytoplasm is enwrapped by a cup-shaped double-membrane cistern, termed a phagophore (or isolation membrane). The phagophore expands and eventually is sealed, forming a double-membrane vesicle, the autophagosome. Lastly, the autophagosome fuses with a

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ATG101); the class III phosphatidylinositol 3-kinase (PI3K) complex (vacuolar protein sorting 34 (VPS34), VPS15/p150, (BECN1) and ATG14L); the ATG12 conjugation system (consists of ATG5, ATG7, ATG10, ATG12 and ATG16L); the ATG8/microtubule-associated protein 1 light chain 3 (LC3) conjugation system (ATG3, ATG4, ATG7 and LC3); the ATG2-ATG18/WD-repeat domain phospho-inositide-interacting (WIPI) complex (ATG2 and WIPIs); and ATG9 vesicles.

Role of autophagy machinery during autophagosome biogenesisInduction/initiation

Autophagy is highly regulable, dependent on cellular energy status

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

Figure 1: Steps of macroautophagy Once autophagy is induced, different types of materials are encompassed in a double-membrane cistern termed a phagophore (or isolation membrane). The expansion of the phagophore leads to its final closure, generating an autophagosome. Then, the autophagosome is transported to lysosomes, and fusion of the external autophagosomal membrane occurs. The internal membrane as well as the autophagosome cargo is degraded by the action of lysosomal hydrolases. ER: endoplasmic reticulum; LD: lipid droplets; Mitoc. mitochondria; Pathog. pathogens; Perox. peroxisomes; Prot. protein aggregates; Ribos. ribosomes.

Chaperone-Mediated Autophagy (CMA) Chaperone-mediated autophagy is only involved in the selective

degradation of specific cytosolic proteins (not organelles), which are imported into the lysosomal membrane for degradation [10].

The regulation of autophagySteps of autophagy pathway and the involved protein complexes

In mammals, macroautophagy (hereafter referred to as auto-phagy) can be broken down into different steps: induction/initiation, phagophore nucleation, elongation, maturation into an autophago-some, docking and fusion with the lysosome, degradation, and efflux [Figure 1]. This pathway requires the hierarchical and coordinated function of the following several protein complexes [Table 1]: the uncoordinated-51 like kinase 1 (ULK1) complex (ULK1/2, ATG13, FAK family kinase-interacting protein of 200 k Da (FIP200) and

lysosome (termed autolysosome), and both the inner autophagoso-mal membrane and the corresponding cargo are degraded by lysoso-mal hydrolases. Depending on the substrates to be removed, the process is referred to be different terminology, as follows: mitophagy (mitochondria), reticulophagy/endoplasmic reticulum (ER)-phagy, pexophagy (peroxisomes), ribophagy (ribosomes), lipophagy (lipid droplets), aggrephagy (protein aggregates), xenophagy (pathogens), etc. [Figure 1].

Microautophagy

Similarly to the macroautophagy, small cytoplasmic cargoes are delivered into the lysosomes during microautophagy; however, these vesicles are formed via invaginations of the lysosomal membrane [9].

(i.e., growth factors, nutrients, energy, etc.). The primary regulator is the mammalian/mechanistic target of rapamycin complex 1 (mTORC1) [11]. In nutrient-rich contexts, mTORC1 is active and phosphorylates two components of the ULK1 complex, ULK1 and ATG13, suppressing the autophagy pathway [12-14]. However, the deprivation of nutrients (especially amino acids) induces mTORC1 inhibition, leading to ULK1 complex activation and the promotion of autophagy induction. A second regulator is AMP-activated protein kinase (AMPK), which senses the energy status of the cell, specifically the AMP: ATP ratio. In response to energy depletion, AMPK not only negatively regulates mTORC1 [15] but also phosphorylates ULK1, promoting autophagy induction [16, 17] [Figure 2].

Phagophore nucleation

In mammalian cells, various reports suggest that certain ER microdomains are the membrane source and/or platform for phagophore nucleation (and therefore the origin of the autophagoso-

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Citation: Swerdlow RH, Lyons KE, Khosla SK, Nashatizadeh M, Pahwa R. A Pilot Study of Oxaloacetate 100 mg Capsules in Parkinson ’sdisease Patients. J Parkinsons Dis Alzheimer Dis. 2016;3(2): 4.

*Address for Correspondence:Leandro Bueno Bergantin,Rua Pedro de Toledo, 669 – Vila Clementino, São Paulo– SP, Brazil, CEP: 04039-032. Fax: 1-913-588-0681;E-mail: rleanbio39@yahoo.com.br

Complex Protein Yeast

homolog Function References

ULK1 kinase

complex

ULK1/2 Atg1 Ser/Thr kinase; phosphorylated by

mTORC1 [110]

ATG13 Atg13 Phosphorylated by mTORC1 [111]

FIP200,

RB1CC1 Atg17 Scaffold protein for ULK1/2 and ATG13 [112]

ATG101 - Interacts with ULK1 and ATG13 [113, 114]

Class III PI3K

complex

VPS15,

p150,

PIK3R4

Vps15 Ser/Thr kinase; also involved in

endocytic trafficking [115]

VPS34,

PIK3C3

Vps34 PI3-kinase (catalyzes synthesis of PI3P

from PI); also involved in endocytic

trafficking

[115]

BECLIN1 Atg6/

Vps30

Presents BH3 domain (interacts with

BCL2; regulates autophagy/apoptosis); also

involved in endocytic trafficking [116]

ATG14L,

BARKOR Atg14

Autophagy-specific subunit of class III

PI3K complex [117-119]

ATG12

conjugation

system

ATG5 Atg5

E3-like enzyme during ATG8–PE

conjugation; conjugates to ATG12 [120]

ATG7 Atg7 E1-like enzyme; activates ATG12 and

ATG8 proteins [120, 121]

ATG10 Atg10 E2-like enzyme; conjugates ATG12 to

ATG5 [120]

ATG12 Atg12 Ubiquitin-like protein; conjugates to

ATG5 [120]

ATG16L1 Atg16

Forms a homodimer; interacts with

ATG5–ATG12 and guides LC3

conjugation at the phagophore [122]

ATG8/LC3

conjugation

system

ATG3 Atg3 E2-like enzyme; conjugates ATG8

family proteins to PE [123]

ATG4A,B,C,

D Atg4

ATG8 C-terminal hydrolase (Cys

protease); activates ATG8 proteins for PE

conjugation and releases ATG8-PE from

autophagosomal outer membrane

[124]

ATG7 Atg7 E1-like enzyme [120,121]

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

Table 1: Protein complexes (and their composition) that regulate the mammalian autophagy pathway. The core protein machinery involved in autophagy, their functions and yeast homologs are noted.

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Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

LC3A,B,C;

GABARAPL

1,2,3

Atg8 Ubiquitin-like proteins; conjugated to PE

[124, 125]

ATG2-

ATG18/WIPI

complex

ATG2A,B Atg2 Required for recruitment of ATG9 in

growing autophagosomes [126]

WIPI1,2,3,4 Atg18,

Atg21

PI3P-binding proteins; recruited to

omegasome [24, 127]

ATG9 vesicles ATG9A,B Atg9

Transmembrane protein; assists during

autophagosome formation; primarily

localized in the trans-Golgi network [22]

Figure 2: Autophagy machinery during autophagosome formation

During stress cell situations (nutrient deprivation, low energy levels, etc.), several protein kinases (mTORC1, AMPK) modulate the ULK1 complex via phosphorylation, leading its mobilization to specific microdomains of the ER. These regions are termed omegasomes, where the class III PI3K complex generates high local concentration of PI3P, a phospholipid required for the recruitment of different proteins, such as DFCP1 and WIPIs. The incipient phagophore then begins to grow, with its elongation depending on the recruitment of two conjugations systems (ATG12 and LC3). These systems are responsible for the lipidation and binding of LC3 to the autophagosomal membranes until this structure is sealed.

somal membrane), being physically connected to both membranes [18-20]. In this regard, ATG9-positive vesicles are thought to deliver membranes for autophagosomal biogenesis [21] in an ULK1-depe-ndent manner [22]; however, little is known regarding its function. However, contributions from mitochondria, Golgi, endosomes and the plasma membrane cannot be excluded as secondary sources. Specifically, ER reorganizes during starvation, forming Ω-shaped structures termed omegasomes. Once the ULK1 complex activates, it translocates to these ER-subdomains [23]. Here, the class III PI3K complex increases the local concentration of phosphatidylinositol 3-phosphate (PI3P) via VPS34 kinase activity. PI3P is an essential phospholipid for recruiting downstream effectors, such as double FYVE domain containing protein 1 (DFCP1) and WIPIs [23-25]. Although autophagosomes can bind PI3P, there is no evidence regarding the functional role of this interaction during autophagoso-me formation.

Elongation and autophagosome maturation The phagophore expands during the steps of elongation and

maturation, which conclude when the pore closes at its extremities, forming the autophagosome. Autophagosomal elongation requires two ubiquitin-like conjugation systems, the ATG12 and LC3 conjugation systems. Both complexes act coordinately in the conjugation of the cytosolic form of LC3 (LC3-I) to phosphatidyle-thanolamine (PE), allowing the anchoring of this protein (LC3-II form) with the nascent autophagosomal membrane. PI3P may also have a role in this step, as it was recently shown that ATG16L1 directly interacts with WIPI2b, being responsible for the recruitment of the ATG12 conjugation system to the omegasome [26]. In addition, ATG8 family proteins recruit the ULK1 complex [27, 28], supporting their role as scaffold proteins and contributing to the stabilization of the autophagy machinery until autophagosome closure.

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Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

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lysosomal-autophagic degradation [48]. Another PD-related protein that is involved in controlling the autophagic process is leucine-rich repeat kinase 2 (LRRK2). LRRK2 mutations lead to the accumulation of autophagic structures [49] and the deregulation of this degradative process [50-52].

In contrast to decreased autophagy, the role of excessive auto-phagy in PD and whether it plays a role in PD pathology has been poorly studied. This issue is particularly important given that excessive activation of autophagy is associated with neuronal loss [53]. In fact, in contrast to the rare detection of autophagosomes in the normal brain due to their rapid elimination in the central nervous system, the abnormal presence of autophagic vacuoles is evident in the brains of patients with PD [54, 55]. The presence of autophagosomes in the PD brains could represent defective activation of macroautophagy. An example of this phenomenon is the deregulation of autophagy mecha-nisms and changes in sensitivity observed in cells with or without the LRRK2 G2019S mutation [Figure 3]. Alterations in basal autophagy flux levels, either genetically (i.e., via the LRRK2 G2019S mutation [51]) or by inhibiting autophagy flux, augment the sensitivity of cells to MPP+. However, the elimination of autophagy flux differences between 3-methyladenine-treated and untreated cells eliminates differences of sensitivity [52]. These studies indicate that while the activation of macroautophagy may be protective, excessive activation can result in neuronal death and hence contribute to cell loss in PD.

Role of PARK genes in autophagy response One of the main hallmarks of PD is the presence of LB, in which

are aggregated several proteins. LB accumulation is due to dysfunctional cell degradation processes, autophagy or UPS. For this reason, the role of PARK genes on autophagy regulation has been studied. Some of these genes affect autophagosome maturation or the lysosome biogenesis. Below, we review the most-studied PARK genes in autophagy dysregulation and their relationship with PD.

α-synuclein

There is a good deal of evidence that α-synuclein inclusions or insoluble oligomers are due to impaired autophagy and that the induction of autophagy may be beneficial. Interestingly, due to autophagosome maturation defects, α-synuclein aggregates can, in turn, prevent autophagic degradation in neurons and other cells [56]. In contrast, a recent work by Koch and coworkers does not support the autophagy flux inhibition by α-synuclein (WT or A53T) in neurons [57]. Colasanti et al. were the first to show that systemic lupus erythematosus patient T lymphocytes, which are characterized by a high level of α-synuclein, are resistant to autophagy induction [58]. It was previously found that α-synuclein overexpression, both in vivo and in vitro, inhibits autophagy by perturbing autophagosome formation. In fact, α-synuclein overexpression induces Golgi fragmentation by hindering the activity of Rab1a, a protein that is crucial for both autophagy and secretion pathways. Rab1a regulates ATG9 roles in omegasome formation and subsequent autophagosome synthesis [59]. It is evident that α-synuclein overexpression inhibits autophagy at an early stage; both WT and mutant (A30P or A53T) α-synuclein can reduce the levels of ATG7 and BECN1 protein as well as LC3-II [60]. α-synuclein binds to HMGB1 (high-mobility group box 1), a protein that translocate from the nucleus to the cytosol and which is both essential for autophagy induction and affects BECN1 function. During autophagy activation, HMGB1 translocates to the cytosol and interacts with BECN1. However, α-synuclein overexpression prevents this interaction and favors the BECN1-BCL2 complex in the cytosol [61].

Autophagy in Parkinson’s disease One of the common characteristics of all neurodegenerative

diseases is the accumulation of misfolded or modified proteins, such as β-amyloid protein plaques in Alzheimer's disease [29], huntingtin in Huntington's disease (HD) [30] or α-synuclein in Lewy bodies (LB) in Parkinson's disease (PD) [31]. Although the role of these protein aggregates in the development of these pathologies is not clear, it can be hypothesized that the clearing of these aggregates by the activation of autophagy could reduce their toxic effects, allevia-ting disease progression. Various data suggest that the modification of basal levels of autophagy may be very important for the elimina-tion of protein inclusions, resulting in a protective effect in the case of neurodegenerative diseases. Specifically, the activation of macro-autophagy limits neuronal damage in the case of HD [32], and the knock-down of key genes that are required for macroautophagy results in an increase in the number of intracellular inclusions with ubiquitinated proteins [33].

Some of the earliest evidence linking autophagy to PD was the observation that WT α-synuclein was degraded via both macroauto-phagy and CMA [34, 35], whereas mutant forms (A53T or A30P) of α-synuclein block CMA by interacting with lysosome-associated membrane protein type 2A (LAMP2A) receptor. This situation results in the formation of high-molecular weight and detergent-insoluble species of α-synuclein [35]. Therefore, in healthy neurons, CMA-mediated clearance of α-synuclein is crucial for limiting α-synuclein oligomerization. CMA inhibition produces a compensatory activation of macroautophagy, although the functional relevance of this mechanism is not well defined. As α-synuclein is a fundamental component of LB, which are pathological hallmarks of both sporadic and familial PD, understanding the fundamental role of autophagy in the formation of etiopathogenic protein aggregates is extremely relevant.

In addition to the degradation of α-synuclein, the autophagic pathway is also involved in mitochondrial turnover. Mitochondrial dysfunction is one of the most characteristic cellular events in PD, and deficits in mitochondrial complex I are observed in patients with this disease [36]. Moreover, 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP), a neurotoxin that generates PD symptoms in humans via the selective degeneration of dopaminergic neurons in the Substantia nigra [37], is a complex I inhibitor following its transformation in MPP+ [38, 39]. Mitochondria are also the target of other neurotoxins related to PD, such as paraquat or rotenone [40, 41]. Two proteins involved in the control of mitochondrial morpho-logy and function, Parkin and PTEN-induced putative kinase 1 (PINK1), have been found to be associated with familial PD [42, 43] and to be involved in the selective mitochondrial elimination process known as mitophagy [44-46]. Specifically, Parkin and PINK1 are recruited to altered mitochondria with a low membrane potential, promoting the clearance of these organelles; it is therefore accepted that altered mitophagy activation mechanisms participate decisively in the etiopathogenesis of PD.

Parkin and PINK1 are not the only proteins derived from the so-termed PARK genes, which are involved in both PD and autophagy control, confirming the close relationship between PD and this cellular process. For example, silencing DJ-1 reduces autophagic events triggered after exposure to paraquat [47]. Furthermore, loss-of-function mutations in DJ-1 lead to mitochondrial dysfunction, elevated reactive oxygen species (ROS) levels, and decreased

While BECN1 or HMGB1 overexpression promotes the clearance of aggregated α-synuclein [60], rapamycin (or serum starvation)-induced autophagy inhibits clearance [56]. Therefore, depending on the α-synuclein species, overexpression has distinct effects on autophagy. The mechanisms by which α-synuclein inhibits autophagy and further prevents starvation-induced autophagy will be interesting topics of further study. It will also be important to assess whether α-synuclein inclusions are a cause or effect of autophagy impairment. It is likely that α-synuclein overexpression activates autophagy at early steps but fails to maintain this flux over the long term.

LRRK2 LRRK2 is shown to regulate autophagy because of its recruitment to caveolae [62], its participation in various signaling pathways and its multiple targets. The activation of endogenous LRRK2 (Lipopolysa-ccharides-stimulated monocytes) promotes its recruitment to membranes; such translocation is more efficient with rapamycin treatment, and LRRK2 is thereby directed to autophagosome membranes to colocalize with LC3-II [63]. The ability of LRRK2 to translocate to membranes after activation may be due to its GTPase and kinase domains. Thus, endogenous expression of the LRRK2 G2019S mutant leads to an increase in kinase activity and excessive autophagy-mediated degradation [51]. Additionally, LRRK2 G2019S elicits mitophagy by interacting and phosphorylating BCL2 (Thr56), a negative regulator of autophagy [64].

However, other studies associate autophagy inhibition with LRRK2. Under proteasome inhibition, LRRK2 G2019S overexpression

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

Figure 3: The effects of autophagy levels on cell sensitivityAlterations in basal autophagy flux levels due to the LRRK2 G2019S mutation induce a higher MPP+ sensitivity, leading to cell death.

disrupts autophagy clearance in vivo (transgenic mice) and in vitro (differentiated neuronal cells). It was reported that elevated p62 levels co-localized with high concentrations of MG132-ubiquitinated protei-ns in these models. Notably, MG132 induces aggresome formation, which generally precedes autophagic clearance. The overexpression of WT or LRRK2 G2019S appears to disrupt the perinuclear distribution of aggresomes in neurons but not in non-neuronal cells [65]. Although LRRK2 protein does not induce aggresome formation, LRRK2 R1441C may affect the maturation of autophagic vacuoles [62]. We hypothesize that if LRRK2 can be degraded by UPS, CMA [66] and autophagy [67], its overexpression and, especially, mutations may perturb these processes, as it has been demonstrated [68]. Such a disruption would slow protein clearance when a maximum degree of degradation is reached. Interestingly, the LRRK2 inhibitor IN1 induces BECN1-dependent autophagy in H4 cells [69], while rapamycin activates LRRK2 in monocytes [63]. Given its numerous interactions, LRRK2 can be thought as a crossroad protein that regulates unexpected signaling pathways depending on cellular stimuli. In the context of autophagy, it would be interesting to examine the cellular mechanisms by which LRRK2 functions, as well as the effects of its forced overex-pression given that this manipulation can have different effects than endogenous expression. There is no doubt that LRRK2 is a constituent of the autophagosome, but it remains to be determined whether it functions to regulate autophagy or is only present at the autophago-some to be degraded. PINK1/PARKIN

PINK1 and Parkin are proteins that are crucial for the clearance of

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damaged mitochondria, a process known as mitophagy. Parkin is an ubiquitin ligase that is selectively recruited to depolarized mitocho-ndria that are slated for autophagic degradation [44]. Parkin appears to negatively regulate autophagy and positively regulate mitophagy. Under normal conditions or in the context of starvation-induced autophagy, Parkin remains in the cytosol and mono-ubiquitinates BCL2 for its stabilization, promoting the formation of the BECN1-BCL2 complex [70]. BECN1-BCL2 forms a ternary complex with LRPPRC (leucine rich pentatricopeptide repeat containing); the latter can also interact with Parkin at depolarized mitochondria. For these reasons, LRPPRC is thought to regulate autophagy/mitophagy in HEK293T and HeLa cells. In fact, LRPPRC stabilizes Parkin and diminishes mitochondria clearance. Therefore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 6-Hydroxydopamine (6-OHDA)-damaged mitochondria, as well as depletion of LRPPRC, lead to mitophagy and autophagy through a reduction in BCL2 protein levels. Of note, Parkin and LRPPRC proteins are eliminated during mitophagy [71]. The translocation of Parkin to depolarized mitoc-hondria is mediated by PINK1, which is present of the outer mitochondrial membrane. In healthy mitochondria, PINK1 is degraded by Lon proteases; therefore, independently of mitochondrial depolarization, PINK1 may also accumulate due to inactivation of these proteases [72]. PINK1 recruits Parkin, which ubiquitinates itself as well as specific mitochondrial proteins in fragmented mitocho-ndria. The PINK1-Parkin interaction induces mitophagy in response to palmitic acid-/CCCP-induced cell stress, among other stimuli. Nevertheless, the downregulation or overexpression of PINK1 or Parkin reduces the mitochondrial membrane potential in basal cellular state, suggesting that both proteins act as quality control regulators of mitochondria [73]. Consistent with this role, PINK1-deficient MEFs increase mitophagy and autophagy in an mTOR-independent manner [46]. Given the compensatory role of Parkin, the loss of PINK1 does not prevent mitophagy induction [74]. Conversely to PINK1, Parkin deficiency eases mitochondrial accumulation. Overall, the PINK1-Parkin pathway preserves mitochondrial integri-ty; while PINK1 serves as an alert to the presence of impaired mito-chondria, Parkin promotes mitochondrial degradation. Moreover, mitophagy induction can be independent of or dependent on mitochondrial depolarization.

ATP13A2 ATP13A2 is a transmembrane lysosomal P5-type ATPase, and

mutations in this enzyme are associated with the Kufor-Rakeb syndrome, an autosomal recessive form of PD [75]. The loss of ATP13A2 function leads to lysosomal alterations, which are characte-rized by increased lysosomal pH, reduced proteolytic activity and, consequently, impaired macroautophagy and CMA-mediated degra-dation [76]. Despite this lysosomal dysfunction, the levels of lysosomal markers, including LAMP1/2 and cathepsins B/D, are increased in fibroblasts from PD patients who carry ATP13A2 mutati-ons (L3292, L6025, 1550C>T) as well as in ATP13A2-deficient dopa-minergic cells [76, 77]. Thus, cells lacking ATP13A2 function display an accumulation of lysosomes that are dysfunctional in degradation, leading to decreased autophagic vacuoles clearance. Moreover, ATP13A2 deficiency preferentially fosters α-synuclein accumulation and induces cell death. Together, the loss of ATP13A2 function and α-synuclein accumulation is responsible for cell toxicity [77]. Indeed, ATP13A2 regulates the lysosomal biogenesis through the mTOR pathway. The down-regulation of ATP13A2 affects Transcription factor EB (TFEB) expression, which is phosphorylated by mTORC1, and inhibits autophagy [78]. It is unclear how ATP13A2 depletion

leads to α-synuclein accumulation. It may be the case that α-synuclein aggregates are primarily degraded through the autophagy-lysosome pathway; either ATP13A2 overexpression or α-synuclein knock-down are cytoprotective.

Pharmacological modulation of autophagy

In recent years, new drugs have been investigated to modulate autophagy in mTOR-dependent or -independent manners. The bala-nce of this regulation is very delicate, given the complexity of intera-ctions between different signaling pathways that lead to autophagy activation.

Pharmacological regulation of autophagy by mTOR-depe-ndent pathways

The modulation of autophagy through mTOR represents a possi-ble mechanism to treat PD and prevent the progression of neurode-generative disorders.

mTOR protein kinase is one of the most important molecules in regulating the cellular response to a decrease or absence of nutrients. Various signals, such as amino acids, growth factors, energy status and stress factors, can activate mTORC1, which negatively regulates autophagy. Autophagy is stimulated by the inhibition of mTORC1 during starvation or pharmacologically with rapamycin (or its analogs), metformin or resveratrol.

The pharmacological agent most widely used to stimulate autophagy is rapamycin. This drug activates autophagy by inhibiting the mTORC1 pathway by binding to the FKBP12 receptor. Both rapamycin and its analogs (including CCI-779, RAD001 and AP23573) have been tested in different models of neurodegeneration, such as SH-SY5Y cells overexpressing α-synuclein. In this context, rapamycin reduces the levels of α-synuclein serine 129 phosphoryla-tion. This effect has also been attributed to metformin, in the same model, through the activation of protein phosphatase 2A (PP2A) [79]. In this regard, it has been reported that metformin mitigates dopami-nergic dysfunction and mitochondrial abnormalities in LRRK2-mutant Drosophila melanogaster through the AMPK pathway by simultaneously inhibiting mTOR and activating ULK1 [80].

In addition, rapamycin and its analogues have been shown to reduce cell death in several studies making use of the following in vitro and in vivo PD models: rotenone-exposed SH-SY5Y cells [81-83]; MPP+ and/or 6-OHDA-treated PC2 cells [84]; α-synuclein transgenic mice and rats [85, 86]; MPTP-treated mice [84, 87]; and Drosophila melanogaster PINK1 and Parkin mutants [88]. In animal models, it has been shown that these agents improve motor function and reduce synaptic injury [89], levodopa-induced dyskinesia [90, 91] and mitochondrial dysfunction [88].

Resveratrol, a dietary polyphenol, is another indirect mTORC1-dependent activator of autophagy that has been demonstrated to increase α-synuclein clearance in α-synuclein-overexpressing PC12 cells (92), to reduce rotenone-exposed SH-SY5Y cell death [92, 93] and to improve mitochondrial function in cultured PARK2-mutant fibroblasts [94].

Pharmacological regulation of autophagy by mTOR-indepen-dent pathways

Multiple drug targets acting at distinct stages of these mTOR-independent signaling pathways induce autophagy. In this sense, lithium, (an inositol monophosphatase inhibitor), and other mood-stabilizing agents (e.g., valproic acid or carbamazepine [two inositol-

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

Clin Pharmacol Transl Med, 2017 Volume 1(3): 68 - 72

lowering agents]) have been used in rotenone-exposed SH-SY5Y cells to reduce apoptosis and mitochondrial dysfunction [82, 83]. In addition, the use of both lithium and valproic acid in a model of PD, such as mice treated with MPTP, has shown a significant potential to improve motor function, increase dopaminergic cell viability and decrease the loss of, 4-Dihydroxyphenylacetic acid (DOPAC) [95]. The most commonly used of these three stabilizing agents in PD models has been lithium, which has also been shown to increase the clearance of the A53T and A30P α-synuclein mutant proteins in PC12 cells that overexpress these forms [96]. The use of other mTOR-independent autophagy enhancers with unknown mechanisms of action (termed small-molecule enhancers of rapamycin (SMERs), and including SMERs 10, 18 and 28) has similarly shown an increase in the clearance of A53T α-synuclein from PC12 cells [97].

Other mTOR-independent autophagy enhancers, including trehalose, have been shown to have multiple beneficial effects in different models of PD. Trehalose is a disaccharide that inhibits the GLUT family of glucose transporters. The use of this drug has been shown to reduce the death of rotenone-exposed PC12 cells [98]; motor deficits in MPTP-treated mice, in which it also reduces the neuroinflammation [99] and A53T α-synuclein rats, in which α-synuclein clearance is also increased [100]. In addition, trehalose increases α-synuclein clearance in others in vitro PD models, such as PC12 cells that overexpress WT and A53T α-synuclein [101, 102], NB69 human neuroblastoma cells that show protein accumulation following proteasome inhibition [103] and rotenone-treated PC12 cells [98].

Other drugs that activate autophagy independently of mTOR have unclear mechanisms of action but exhibit beneficial effects in different PD models. Among these drugs, we note latrepirdine, an antihistamine that increases the clearance of α-synuclein, both in Saccharomyces cerevisae, (where it also decreases cell death) and in SH-SY5Y cells expressing α-synuclein [104]. Spermidine has been tested in models of Drosophila melanogaster and Caenorhabditis elegans that express α-synuclein. In both models, spermidine reduced motor dysfunction and neuronal loss, increasing lifespan [105]. Nilo-tinib is another mTOR-independent autophagy inducer with an unknown mechanism of action. This drug has been shown to increase α-synuclein clearance and to improve motor function in mice expres-sing A53T α-synuclein [106], as well as to reduce cell death in mouse primary cortical neurons [107].

Curcumin and kaempferol are two polyphenols that induce autophagy through an unclear mechanism. Curcumin has been repo-rted to reduce α-synuclein accumulation in SH-SY5Y cells that overexpress WT and A53T forms of this protein [108]. In addition, it has been shown that incubation with kaempherol in PD models of rotenone-mediated acute toxicity reduces ROS production, apoptosis and mitochondrial dysfunction [109].

Conclusion From our perspective, regulation of the role of autophagy in

eliminating protein aggregates and its involvement with mitochondr-ial homeostasis are two phenomena that are of great importance for the pathogenesis of PD. A better understanding of the mechanisms that control the basal activation of autophagy or its deregulation, as well as the regulatory systems involved in autophagy and mitocho-ndrial function, may contribute to the development of therapeutic strategies that prevent or delay the development of PD. A reduction in basal autophagy leads to an accumulation of altered proteins or organelles as their hyperactivation can cause damage or even cell death. Therefore, it is necessary to focus on future studies determini-

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

1. Karanasios E and Ktistakis NT. Autophagy at the Cell, Tissue and Organismal Level: Springer International Publishing. 2016; 103. [Crossref]

2. De Duve C. The lysosome. Scientific American. 1963; 208:64-72. [Crossref]

3. Harding TM, Morano KA, Scott SV and Klionsky DJ. Isolation and characterizationof yeast mutants in the cytoplasm to vacuole protein targeting pathway. J Cell Mol Biol. 1995; 131:591-602. [Crossref]

4. Tsukada M and Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS letters. 1993; 333:169-174. [Crossref]

5. Thumm M, Egner R, Koch B, Schlumpberger M, Straub M, Veenhuis M, et al. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS letters. 1994; 349:275-80. [Crossref]

6. Lamb CA, Yoshimori T and Tooze SA. The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol. 2013 Dec; 14:759-774. [Crossref]

7. Choi AM, Ryter SW and Levine B. Autophagy in human health and disease. N Engl J Med. 2013; 368:651-662. [Crossref]

8. Thorburn A, Thamm DH and Gustafson DL. Autophagy and cancer therapy. Mol Pharmacol. 2014; 85:830-838. [Crossref]

ng the precise mechanisms for optimizing autophagic activity, with the aim of favoring neuronal protection to treat PD.Disclosure Statement

R. G.-S. was supported by a Marie Sklodowska-Curie Individual Fellowship (IF-EF) from the European Commission. J.M. B.-SP. was funded by La Ligue Contre le Cancer. J. M. F. received research support from the Instituto de Salud Carlos III, CIBERNED (CB06/ 05/004) and from Instituto de Salud Carlos III, FIS, (PI15/00034). R. A. G.-P. was supported by a "Contrato destinado a la retención y atracción del talento investigador, TA13009" from Junta de Extre-madura, as well as research support from the Instituto de Salud Carlos III, FIS, (PI14/00170). This work was also supported by “Fondo Europeo de Desarrollo Regional” (FEDER), from European Union.

AbbreviationsAMPK: AMP-activated protein kinase; Atg: autophagy-related; ATP13A2: ATPase 13A2; BECN1: Beclin-1; CCCP: carbonyl cyanide m-chlorophenylhydrazone; BARKOR: Beclin 1-associated autophagy related key regulator; CMA: Chaperone-Mediated Autophagy; DFCP1: double FYVE-containing protein 1; ER: endoplasmic reticulum; FKBP12: FK506-binding protein 12; GABARAPL: γ-aminobutyric acid type A receptor-associated protein-like; GLUT: Glucose transporter; HEK293T: Human embryonic kidney cells 293 T; HMGB1: High Mobility Group Box 1; LAMP1/-2: Lysosomal associated membrane protein 1/2; LB: Lewy bodies; LRRK2: Leucine-Rich Repeat Kinase 2; LRPPRC: Leucine-rich pentatricopeptide repeat-containing; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; mTOR: mammalian/mechanistic target of rapamycin; mTORC1: mammalian/mechanistic target of rapamycin complex 1; 6-OHDA: 6-Hydroxydopamine; PARK2: Parkin RBR E3 Ubiquitin Protein Ligase; PC12: pheochro-mocytoma 12 cells; PD: Parkinson’s disease; PE: phosphati-dylethano-lamine; PI: phosphatidylinositol; PI3P: phosphatidylinositol 3-phosp-hate; PI3K: phosphatidylinositol 3-kinase; PIK3C3: phosphatidyli-nositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide 3-kinase regulatory subunit 4; PINK1: PTEN-induced putative kinase 1; ROS: Reactive oxygen species; SMERs: Small-molecule enhancers of rapamycin; SQSTM1/p62: Sequesto-some 1; TFEB: Transcription factor EB; ULK: uncoordinated-51 like kinase; UPS: ubiquitin-proteasome system; VPS: vacuolar protein sorting; WIPI: WD-repeat domain phosphoinosidei-nteracting; WT: Wild Type.

References

Clin Pharmacol Transl Med, 2017 Volume 1(3): 69 - 72

9. Ahlberg J and Glaumann H. Uptake--microautophagy--and degradation ofexogenous proteins by isolated rat liver lysosomes. Effects of pH, ATP, andinhibitors of proteolysis. Exp Mol Pathol. 1985; 42:78-88. [Crossref]

10. Kaushik S, Cuervo AM. Chaperone-mediated autophagy: a unique way to enter thelysosome world. Trends cell Biol. 2012; 22:407-417. [Crossref]

11. Laplante M and Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012; 149:274-793. [Crossref]

12. Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, Cao J, et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell.2009; 20:1992-2003. [Crossref]

13. Ganley IG, Lam du H, Wang J, Ding X, Chen S and Jiang X. ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. J Biol Chem. 2009; 284:12297-123305. [Crossref]

14. Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, et al. Nutrient- dependent mTORC1 association with the ULK1-Atg13-FIP200 complex requiredfor autophagy. Mol Biol Cell. 2009; 20:1981-1991. [Crossref]

15. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol cell.2008; 30:214-226. [Crossref]

16. Kim J, Kundu M, Viollet B and Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat cell Biol. 2011; 13:132-141. [Crossref]

17. Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011; 331:456-461. [Crossref]

18. Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmicreticulum.J Cell Biol. 2008; 182:685-701. [Crossref]

19. Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T and YamamotoA. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat cell biol. 2009; 11:1433-1437. [Crossref]

20. Yla-Anttila P, Vihinen H, Jokitalo E and Eskelinen EL. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy. 2009; 5:1180-1185. [Crossref]

21. Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, et al. Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol boil cell. 2012;23:1860- 1873. [Crossref]

22. Young AR, Chan EY, Hu XW, Kochl R, Crawshaw SG, High S, et al. Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes. J cell sci. 2006; 119:3888-3900. [Crossref]

23. Karanasios E, Stapleton E, Manifava M, Kaizuka T, Mizushima N, Walker SA, et al. Dynamic association of the ULK1 complex with omegasomes during autophagyinduction. J cell sci. 2013; 126:5224-5238. [Crossref]

24. Polson HE, de Lartigue J, Rigden DJ, Reedijk M, Urbe S, Clague MJ, et al. Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores andpositively regulates LC3 lipidation. Autophagy. 2010; 6:506-522. [Crossref]

25. Koyama-Honda I, Itakura E, Fujiwara TK, Mizushima N. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site.Autophagy. 2013 ;9:1491-1499. [Crossref]

26. Dooley HC, Razi M, Polson HE, Girardin SE, Wilson MI and Tooze SA. WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1. Mol Cell. 2014; 55:238-252. [Crossref]

27. Alemu EA, Lamark T, Torgersen KM, Birgisdottir AB, Larsen KB, Jain A, et al. ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequencerequirements for LC3-interacting region (LIR) motifs. J Biol Chem. 2012;287:39275-39290. [Crossref]

28. Kraft C, Kijanska M, Kalie E, Siergiejuk E, Lee SS, Semplicio G, et al. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. EMBO J. 2012 ; 31:3691-3703. [Crossref]

29. Herring A, Munster Y, Metzdorf J, Bolczek B, Krussel S, Krieter D, et al. Late running is not too late against Alzheimer's pathology. Neurobiol Dis. 2016; 94:44- 54. [Crossref]

30. Nopoulos PC. Huntington disease: a single-gene degenerative disorder of the striatum. Dialogues Clin Neurosci. 2016; 18:91-98. [Crossref]

31. Surmeier DJ, Obeso JA and Halliday GM. Selective neuronal vulnerability in Parkinson disease. Nat Rev Neurosci. 2017; 18:101-113. [Crossref]

32. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. Inhibition ofmTOR induces autophagy and reduces toxicity of polyglutamine expansions infly and mouse models of Huntington disease. Nat Genet. 2004; 36:585-595. [Crossref]

33. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nat. 2006; 441:885-889. [Crossref]

34. Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT and Sulzer D. Impaireddegradation of mutant alpha-synuclein by chaperone-mediated autophagy.Science. 2004; 305:1292-1295. [Crossref]

35. Vogiatzi T, Xilouri M, Vekrellis K and Stefanis L. Wild type alpha-synuclein isdegraded by chaperone-mediated autophagy and macroautophagy in neuronal cells.J Biol Chem. 2008; 283:23542-23556. [Crossref]

36. Truban D, Hou X, Caulfield TR, Fiesel FC and Springer W. PINK1, Parkin, and Mitochondrial Quality Control: What can we Learn about Parkinson'sDisease Pathobiology? J Parkinsons Dis. 2017; 7:13-29. [Crossref]

37. Langston JW, Ballard P, Tetrud JW and Irwin I. Chronic Parkinsonism in humansdue to a product of meperidine-analog synthesis. Science. 1983; 219:979-980. [Crossref]

38. Gonzalez-Polo RA, Soler G, Alonso JC, Rodriguez-Martin A and Fuentes JM. MPP(+) causes inhibition of cellular energy supply in cerebellar granulecells. Neurotoxicology. 2003; 24:219-225. [Crossref]

39. Gonzalez-Polo RA, Soler G and Fuentes JM. MPP+: mechanism for its toxicity in cerebellar granule cells. Molecular neurobiology. 2004; 30:253-264. [Crossref]

40. Niso-Santano M, Gonzalez-Polo RA, Bravo-San Pedro JM, Gomez-Sanchez R,Lastres-Becker I, Ortiz-Ortiz MA, et al. Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: modulation by the Nrf2/Trxaxis. Free Radic Biol Med. 2010; 48:1370-1381. [Crossref]

41. Niso-Santano M, Moran JM, Garcia-Rubio L, Gomez-Martin A, Gonzalez-Polo RA,Soler G, et al. Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways: role of c-Jun N-terminal kinase in paraquat-induced cell death. Toxicol Sci. 2006; 92:507-515. [Crossref]

42. Darios F, Corti O, Lucking CB, Hampe C, Muriel MP, Abbas N, et al. Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Hum Mol Genet. 2003; 12:517-526. [Crossref]

43. Silvestri L, Caputo V, Bellacchio E, Atorino L, Dallapiccola B, Valente EM, et al.Mitochondrial import and enzymatic activity of PINK1 mutants associatedto recessive parkinsonism. Hum Mol Genet. 2005; 14:3477-3492. [Crossref]

44. Narendra D, Tanaka A, Suen DF and Youle RJ. Parkin is recruited selectively toimpaired mitochondria and promotes their autophagy. J Cell Biol. 2008; 183:795-803. [Crossref]

45. Gomez-Sanchez R, Gegg ME, Bravo-San Pedro JM, Niso-Santano M, Alvarez-Erviti L, Pizarro-Estrella E, et al. Mitochondrial impairment increases FL-PINK1 levels bycalcium-dependent gene expression. Neurobiol Dis. 2014; 62:426-440. [Crossref]

46. Gomez-Sanchez R, Yakhine-Diop SM, Bravo-San Pedro JM, Pizarro-Estrella E,Rodriguez-Arribas M, Climent V, et al. PINK1 deficiency enhances autophagy andmitophagy induction. Mol Cell Oncol. 2015; 3:e1046579. [Crossref]

47. Gonzalez-Polo R, Niso-Santano M, Moran JM, Ortiz-Ortiz MA, Bravo-San Pedro JM, Soler G, et al. Silencing DJ-1 reveals its contribution in paraquat-induced autophagy. J Neurochem. 2009; 109:889-898. [Crossref]

48. Krebiehl G, Ruckerbauer S, Burbulla LF, Kieper N, Maurer B, Waak J, etal. Reduced basal autophagy and impaired mitochondrial dynamics due toloss of Parkinson's disease-associated protein DJ-1. PloS one. 2010; 5:e9367. [Crossref]

49. Plowey ED, Cherra SJ, 3rd, Liu YJ and Chu CT. Role of autophagy in G2019S- LRRK2-associated neurite shortening in differentiated SH-SY5Y cells. J Neurochem. 2008; 105:1048-1056. [Crossref]

50. Bravo-San Pedro JM, Gomez-Sanchez R, Niso-Santano M, Pizarro-Estrella E, Aiastui- Pujana A, Gorostidi A, et al. The MAPK1/3 pathway is essential for the deregulation of autophagy observed in G2019S LRRK2 mutantfibroblasts. Autophagy. 2012; 8:1537- 1539. [Crossref]

51. Bravo-San Pedro JM, Niso-Santano M, Gomez-Sanchez R, Pizarro-Estrella E, Aiastui- Pujana A, Gorostidi A, et al. The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway. Cell Mol Life Sci. 2013; 70:121-136. [Crossref]

52. Yakhine-Diop SM, Bravo-San Pedro JM, Gomez-Sanchez R, Pizarro-Estrella E,Rodriguez-Arribas M, Climent V, et al. G2019S LRRK2 mutant fibroblasts fromParkinson's disease patients show increased sensitivity to neurotoxin 1-methyl-4- phenylpyridinium dependent of autophagy. Toxicology. 2014; 324:1-9. [Crossref]

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

Clin Pharmacol Transl Med, 2017 Volume 1(3): 70 - 72

53. Bredesen DE, Rao RV and Mehlen P. Cell death in the nervous system. Nat. 2006;443:796-802. [Crossref]

54. Zhu JH, Guo F, Shelburne J, Watkins S and Chu CT. Localization of phosphorylated ERK/MAP kinases to mitochondria and autophagosomes in Lewy body diseases. Brain Pathol. 2003; 13:473-481. [Crossref]

55. Anglade P, Vyas S, Javoy-Agid F, Herrero MT, Michel PP, Marquez J, et al. Apoptosis and autophagy in nigral neurons of patients with Parkinson's disease. Histol Histopathol. 1997; 12:25-31. [Crossref]

56. Tanik SA, Schultheiss CE, Volpicelli-Daley LA, Brunden KR and Lee VM. Lewy body-like alpha synuclein aggregates resist degradation and impair macroautophagy. J Biol Chem. 2013; 288:15194-15210. [Crossref]

57. Koch JC, Bitow F, Haack J, d'Hedouville Z, Zhang JN, Tonges L, et al. Alpha- Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons. Cell Death Dis. 2015; 6:e1811. [Crossref]

58. Colasanti T, Vomero M, Alessandri C, Barbati C, Maselli A, Camperio C, et al. Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes. Cell Death Dis. 2014; 5:e1265. [Crossref]

59. Winslow AR, Chen CW, Corrochano S, Acevedo-Arozena A, Gordon DE, Peden AA, et al. alpha-Synuclein impairs macroautophagy: implications for Parkinson's disease. J Cell Biol. 2010; 190:1023-1037. [Crossref]

60. Wang K, Huang J, Xie W, Huang L, Zhong C and Chen Z. Beclin1 and HMGB1 ameliorate the alpha-synuclein-mediated autophagy inhibition in PC12 cells. Diagn Pathol. 2016; 11:15. [Crossref]

61. Song JX, Lu JH, Liu LF, Chen LL, Durairajan SS, Yue Z, et al. HMGB1 is involved in autophagy inhibition caused by SNCA/alpha-synuclein overexpression: a process modulated by the natural autophagy inducer corynoxine B. Autophagy. 2014; 10:144-154. [Crossref]

62. Alegre-Abarrategui J, Christian H, Lufino MM, Mutihac R, Venda LL, Ansorge O, et al. LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model. Hum Mol Genet. 2009; 18:4022-4034. [Crossref]

63. Schapansky J, Nardozzi JD, Felizia F and LaVoie MJ. Membrane recruitment of endogenous LRRK2 precedes its potent regulation of autophagy. Hum Mol Genet. 2014; 23:4201-4214. [Crossref]

64. Su YC, Guo X and Qi X. Threonine 56 phosphorylation of Bcl-2 is required for LRRK2 G2019S-induced mitochondrial depolarization and autophagy. Biochim Biophys Acta. 2015; 1852:12-21. [Crossref]

65. Bang Y, Kim KS, Seol W and Choi HJ. LRRK2 interferes with aggresomeformation for autophagic clearance. Mol Cell Neurosci. 2016; 75:71-80. [Crossref]

66. Orenstein SJ, Kuo SH, Tasset I, Arias E, Koga H, Fernandez-Carasa I, et al. Interplay of LRRK2 with chaperone-mediated autophagy. Nat Neurosci. 2013; 16:394-406. [Crossref]

67. Park S, Han S, Choi I, Kim B, Park SP, Joe EH, et al. Interplay between Leucine- Rich Repeat Kinase 2 (LRRK2) and p62/SQSTM-1 in Selective Autophagy. PloS one. 2016; 11:e0163029. [Crossref]

68. Lichtenberg M, Mansilla A, Zecchini VR, Fleming A and Rubinsztein DC. TheParkinson's disease protein LRRK2 impairs proteasome substrate clearance without affecting proteasome catalytic activity. Cell Death Dis. 2011; 2:e196. [Crossref]

69. Manzoni C, Mamais A, Roosen DA, Dihanich S, Soutar MP, Plun-Favreau H, et al. mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1. Sci Rep. 2016; 6:35106. [Crossref]

70. Chen D, Gao F, Li B, Wang H, Xu Y, Zhu C, et al. Parkin mono-ubiquitinates Bcl- 2 and regulates autophagy. J Biol Chem. 2010; 285:38214-38223. [Crossref]

71. Zou J, Yue F, Li W, Song K, Jiang X, Yi J, et al. Autophagy inhibitor LRPPRC suppresses mitophagy through interaction with mitophagy initiator Parkin. PloS one. 2014; 9:e94903. [Crossref]

72. Thomas RE, Andrews LA, Burman JL, Lin WY and Pallanck LJ. PINK1-Parkin pathway activity is regulated by degradation of PINK1 in the mitochondrial matrix. PLoS Genet. 2014; 10:e1004279. [Crossref]

73. Wu W, Xu H, Wang Z, Mao Y, Yuan L, Luo W, et al. PINK1-Parkin-MediatedMitophagy Protects Mitochondrial Integrity and Prevents Metabolic Stress-InducedEndothelial Injury. PloS one. 2015; 10:e0132499. [Crossref]

74. Cherra SJ, 3rd, Dagda RK, Tandon A and Chu CT. Mitochondrial autophagy as a compensatory response to PINK1 deficiency. Autophagy. 2009; 5:1213-1214. [Crossref]

75. Williams DR, Hadeed A, al-Din AS, Wreikat AL and Lees AJ. Kufor Rakeb disease: autosomal recessive, levodopa-responsive parkinsonism with pyramidal degeneration, supranuclear gaze palsy, and dementia. Mov Disord. 2005; 20:1264- 1271. [Crossref]

76. Dehay B, Ramirez A, Martinez-Vicente M, Perier C, Canron MH, Doudnikoff E, etal. Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration. Pro Natl Acad Sci USA 2012; 109: 9611-9616. [Crossref]

77. Usenovic M, Tresse E, Mazzulli JR, Taylor JP and Krainc D. Deficiency ofATP13A2 leads to lysosomal dysfunction, alpha-synuclein accumulation, andneurotoxicity. The Journal of neuroscience : the official journal of the Society forNeuroscience. 2012; 32: 4240-4246. [Crossref]

78. Bento CF, Ashkenazi A, Jimenez-Sanchez M and Rubinsztein DC. The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway. Nat Commun. 2016; 7: 11803. [Crossref]

79. Perez-Revuelta BI, Hettich MM, Ciociaro A, Rotermund C, Kahle PJ, Krauss S, et al. Metformin lowers Ser-129 phosphorylated alpha-synuclein levels via mTOR- dependent protein phosphatase 2A activation. Cell Death Dis. 2014; 5: e1209.[Crossref]

80. Ng CH, Guan MS, Koh C, Ouyang X, Yu F, Tan EK, et al. AMP kinase activation mitigates dopaminergic dysfunction and mitochondrial abnormalities in Drosophila models of Parkinson's disease. The Journal of neuroscience : the official journal ofthe Society for Neuroscience. 2012; 32: 14311-14317. [Crossref]

81. Pan T, Rawal P, Wu Y, Xie W, Jankovic J, Le W, et al. Rapamycin protects against rotenone-induced apoptosis through autophagy induction. Neuroscience. 2009; 164: 541-551. [Crossref]

82. Hou L, Xiong N, Liu L, Huang J, Han C, Zhang G, et al. Lithium protectsdopaminergic cells from rotenone toxicity via autophagy enhancement. BMCNeurosci. 2015; 16:82. [Crossref]

83. Xiong N, Jia M, Chen C, Xiong J, Zhang Z, Huang J, et al. Potential autophagy enhancers attenuate rotenone-induced toxicity in SH-SY5Y. Neuroscience. 2011;199: 292-302. [Crossref]

84. Malagelada C, Jin ZH, Jackson-Lewis V, Przedborski S and Greene LA. Rapamycin protects against neuron death in in vitro and in vivo models of Parkinson's disease. J Neurosci : the official journal of the Society for Neuroscience. 2010; 30: 1166-1175. [Crossref]

85. Crews L, Spencer B, Desplats P, Patrick C, Paulino A, Rockenstein E, et al. Selective molecular alterations in the autophagy pathway in patients with Lewy body diseaseand in models of alpha-synucleinopathy. PloS one. 2010; 5: e9313. [Crossref]

86. Decressac M, Mattsson B, Weikop P, Lundblad M, Jakobsson J, Bjorklund A, et al. TFEB-mediated autophagy rescues midbrain dopamine neurons from alpha-synuclein toxicity. Pro Natl Acad Sci USA 2013; 110:E1817-1826. [Crossref]

87. Dehay B, Bove J, Rodriguez-Muela N, Perier C, Recasens A, Boya P, et al.Pathogenic lysosomal depletion in Parkinson's disease. J Neurosci : the officialjournal of the Society for Neuroscience. 2010; 30: 12535-12544. [Crossref]

88. Tain LS, Mortiboys H, Tao RN, Ziviani E, Bandmann O, Whitworth AJ et al.Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss.Nature neuroscience. 2009; 12: 1129-1135. [Crossref]

89. Bai X, Wey MC, Fernandez E, Hart MJ, Gelfond J, Bokov AF, et al. Rapamycin improves motor function, reduces 4-hydroxynonenal adducted protein in brain, and attenuates synaptic injury in a mouse model of synucleinopathy. Patho boil AgingAge Relat Dis. 2015; 5: 28743. [Crossref]

90. Santini E, Heiman M, Greengard P, Valjent E and Fisone G. Inhibition of mTOR signaling in Parkinson's disease prevents L-DOPA-induced dyskinesia. Sci signal. 2009; 2:ra36. [Crossref]

91. Decressac M and Bjorklund A. mTOR inhibition alleviates L-DOPA-induced dyskinesia in parkinsonian rats. J Parkinsons Dis. 2013; 3:13-17. [Crossref]

92. Wu Y, Li X, Zhu JX, Xie W, Le W, Fan Z, et al. Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson's disease. NeuroSignals. 2011; 19:163-174. [Crossref]

93. Lin TK, Chen SD, Chuang YC, Lin HY, Huang CR, Chuang JH, et al. Resveratrol partially prevents rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through induction of heme oxygenase-1 dependent autophagy. Int Mol Sci. 2014; 15: 1625-1646. [Crossref]

94. Ferretta A, Gaballo A, Tanzarella P, Piccoli C, Capitanio N, Nico B, et al. Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease. Biochimica et biophysica acta. 2014; 1842: 902-915. [Crossref]

95. Li XZ, Chen XP, Zhao K, Bai LM, Zhang H, Zhou XP et al. Therapeutic effects of valproate combined with lithium carbonate on MPTP-induced parkinsonism in mice: possible mediation through enhanced autophagy. Int J Neurosci. 2013; 123: 73-79.[Crossref]

96. Sarkar S, Floto RA, Berger Z, Imarisio S, Cordenier A, Pasco M, et al. Lithiuminduces autophagy by inhibiting inositol monophosphatase. J Cell Biol. 2005; 170: 1101-11011 [Crossref]

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

Clin Pharmacol Transl Med, 2017 Volume 1(3): 71 - 72

97. Sarkar S, Perlstein EO, Imarisio S, Pineau S, Cordenier A, Maglathlin RL, et al.Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol. 2007; 3: 331-338. [Crossref]

98. Wu F, Xu HD, Guan JJ, Hou YS, Gu JH, Zhen XC, et al. Rotenone impairsautophagic flux and lysosomal functions in Parkinson's disease. Neuroscience.2015; 284: 900-11. [Crossref]

99. Sarkar S, Chigurupati S, Raymick J, Mann D, Bowyer JF, Schmitt T, et al.Neuroprotective effect of the chemical chaperone, trehalose in a chronic MPTP- induced Parkinson's disease mouse model. Neurotoxicology. 2014; 44:250-262. [Crossref]

100. He Q, Koprich JB, Wang Y, Yu WB, Xiao BG, Brotchie JM, et al. Treatment withTrehalose Prevents Behavioral and Neurochemical Deficits Produced in an AAValpha-Synuclein Rat Model of Parkinson's Disease. Mol Neurobiol. 2016; 53:2258- 2268. [Crossref]

101. Sarkar S, Davies JE, Huang Z, Tunnacliffe A and Rubinsztein DC. Trehalose, anovel mTOR-independent autophagy enhancer, accelerates the clearance of mutanthuntingtin and alpha-synuclein. J Biol Chem. 2007; 282:5641-5652. [Crossref]

102. Lan DM, Liu FT, Zhao J, Chen Y, Wu JJ, Ding ZT, et al. Effect of trehalose onPC12 cells overexpressing wild-type or A53T mutant alpha-synuclein. Neurochemical research. 2012; 37: 2025-2032. [Crossref]

103. Casarejos MJ, Solano RM, Gomez A, Perucho J, de Yebenes JG, Mena MA et al. The accumulation of neurotoxic proteins, induced by proteasome inhibition, is reverted by trehalose, an enhancer of autophagy, in human neuroblastoma cells.Neurochem Int. 2011; 58:512-520. [Crossref]

104. Steele JW, Lachenmayer ML, Ju S, Stock A, Liken J, Kim SH, et al. Latrepirdineimproves cognition and arrests progression of neuropathology in an Alzheimer'smouse model. Mol Psychiatry. 2013; 18:889-897. [Crossref]

105. Buttner S, Broeskamp F, Sommer C, Markaki M, Habernig L, Alavian-Ghavanini A,et al. Spermidine protects against alpha-synuclein neurotoxicity. Cell cycle. 2014;13: 3903-3908. [Crossref]

106. Hebron ML, Lonskaya I and Moussa CE. Nilotinib reverses loss of dopamineneurons and improves motor behavior via autophagic degradation of alpha-synucleinin Parkinson's disease models. Hum Mol Genet. 2013; 22:3315-3328. [Crossref]

107. Mahul-Mellier AL, Fauvet B, Gysbers A, Dikiy I, Oueslati A, Georgeon S, et al. c- Abl phosphorylates alpha-synuclein and regulates its degradation: implication for alpha-synuclein clearance and contribution to the pathogenesis of Parkinson'sdisease. Hum Mol Genet. 2014; 23: 2858-2879. [Crossref]

108. Jiang TF, Zhang YJ, Zhou HY, Wang HM, Tian LP, Liu J, et al. Curcumin ameliorates the neurodegenerative pathology in A53T alpha-synuclein cell model of Parkinson's disease through the downregulation of mTOR/p70S6K signaling and the recovery of macroautophagy. J Neuroimmune Pharmacol : the official journal of the Society on NeuroImmune Pharmacology. 2013; 8:356-369. [Crossref]

109. Filomeni G, Graziani I, De Zio D, Dini L, Centonze D, Rotilio G, et al.Neuroprotection of kaempferol by autophagy in models of rotenone-mediated acutetoxicity: possible implications for Parkinson's disease. Neurobiol Aging. 2012; 33:767-785. [Crossref]

110. Chan EY, Kir S and Tooze SA. siRNA screening of the kinome identifies ULK1 asa multidomain modulator of autophagy. J Biol Chem. 2007; 282: 25464-25474. [Crossref]

111. Chan EY, Longatti A, McKnight NC, Tooze SA. Kinase-inactivated ULK proteinsinhibit autophagy via their conserved C-terminal domains using an Atg13- independent mechanism. Mol Cell Biol. 2009; 29: 157-171. [Crossref]

112. Hara T, Takamura A, Kishi C, Iemura S, Natsume T, Guan JL, et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. JCell Biol. 2008; 181: 497-51. [Crossref]

113. Hosokawa N, Sasaki T, Iemura S, Natsume T, Hara T, Mizushima N, et al. Atg101,a novel mammalian autophagy protein interacting with Atg13. Autophagy. 2009;5:973-979. [Crossref]

114. Mercer CA, Kaliappan A and Dennis PB. A novel, human Atg13 binding protein,Atg101, interacts with ULK1 and is essential for macroautophagy. Autophagy.2009; 5: 649-662. [Crossref]

115. Volinia S, Dhand R, Vanhaesebroeck B, MacDougall LK, Stein R, Zvelebil MJ, et al. A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p- Vps15p protein sorting system. EMBO J. 1995; 14: 3339-3348. [Crossref]

116. H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature.1999; 402: 672-676. [Crossref]

117. Sun Q, Fan W, Chen K, Ding X, Chen S, Zhong Q et al. Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol3- kinase. Proc Natl Acad Sci of the U S A. 2008; 105: 19211-19216. [Crossref]

118. Matsunaga K, Saitoh T, Tabata K, Omori H, Satoh T, Kurotori N, et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at differentstages. Nat Cell Biol. 2009; 11: 385-396. [Crossref]

119. Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT, et al. Distinct regulation ofautophagic activity by Atg14L and Rubicon associated with Beclin 1- phosphatidylinositol-3-kinase complex. Nat Cell Biol. 2009; 11:468-476. [Crossref]

120. Mizushima N, Sugita H, Yoshimori T and Ohsumi Y. A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essentialfor autophagy. J Biol Chem. 1998; 273: 33889-33892. [Crossref]

121. Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol. 2005; 169: 425-434. [Crossref]

122. Mizushima N, Kuma A, Kobayashi Y, Yamamoto A, Matsubae M, Takao T, et al. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate. J Cell Sci. 2003;116: 1679-1688. [Crossref]

123. Tanida I, Tanida-Miyake E, Komatsu M, Ueno T and Kominami E. Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p tohApg5p. J Biol Chem. 2002; 277: 13739-13744. [Crossref]

124. Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T et al. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci. 2004; 117:2805-2812. [Crossref]

125. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. LC3, amammalian homologue of yeast Apg8p, is localized in autophagosome membranesafter processing. The EMBO journal. 2000; 19:5720-5728. [Crossref]

126. Velikkakath AK, Nishimura T, Oita E, Ishihara N and Mizushima N. Mammalian Atg2 proteins are essential for autophagosome formation and important forregulation of size and distribution of lipid droplets. Molecular biology of the cell.2012; 23: 896-909. [Crossref]

127. Proikas-Cezanne T, Waddell S, Gaugel A, Frickey T, Lupas A, Nordheim A, et al.WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene. 2004; 23: 9314-9325. [Crossref]

Acknowledgements The authors also thank FUNDESALUD for helpful assistance.

Yakhine-Diop SMS, Gómez-Sánchez R, Bravo-San Pedro JM, González-Polo RA, Fuentes JM. The Roles of Autophagy in Parkinson's disease. Clin Pharmacol Transl Med. 2017; 1(3): 62-72.

Clin Pharmacol Transl Med, 2017 Volume 1(3): 72 - 72