The Implications of Autophagy in Alzheimer's Disease

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REVIEW ARTICLE

The Implications of Autophagy in Alzheimer’s Disease Tadanori Hamano1,2,3,*, Kouji Hayashi1, Norimichi Shirafuji1 and Yasunari Nakamoto1 1

Second Department of Internal Medicine, 2LifeScience Innovation Center, 3Department of Aging and Dementia (DAD), Faculty of Medical Sciences, University of Fukui, Fukui, Japan

ARTICLE HISTORY Received: March 18, 2018 Revised: August 10, 2018 Accepted: October 01, 2018 DOI: 10.2174/1567205015666181004143432

Abstract: The pathogenic mechanisms of Alzheimer’s Disease (AD) involve the deposition of abnormally misfolded proteins, amyloid β protein (Aβ) and tau protein. Aβ comprises senile plaques, and tau aggregates form Neurofibrillary Tangles (NFTs), both of which are hallmarks of AD. Autophagy is the main conserved pathway for the degeneration of aggregated proteins, Aβ, tau and dysfunctional organelles in the cell. Many animal model studies have demonstrated that autophagy normally functions as the protective factor against AD progression associated with intracytoplasmic toxic Aβ and tau aggregates. The upregulation of autophagy can also be favorable in AD treatment. An improved understanding of the signaling pathways that regulate autophagy is critical to developing AD treatments. The cellular and molecular machineries of autophagy, their function in the pathogenesis of AD, and current drug discovery strategies will be discussed in this review.

Keywords: Alzheimer’s disease, autophagy, tau, amyloid β protein, autophagosome, therapy. 1. INTRODUCTION Alzheimer’s Disease (AD) is one of the most common diseases causing progressive dementia. Pathological hallmarks of AD are Extracellular Senile Plaques (SPs) and intra-neuronal Neurofibrillary Tangles (NFTs). The SPs mainly consist of amyloid β protein (Aβ), which is proteolytically generated by the transmembrane protein Amyloid Precursor Protein (APP). APP is cleaved by β- and γsecretase, and produces the Aβ fragment. NFTs consist of microtubule associated protein tau [1] and ubiquitin [2]. The aggregated hyper-phosphorylated tau by tau kinases, such as glycogen synthase kinase (GSK)3β, cyclin dependent kinase (cdk)5, and Jun N-terminal kinase (JNK), is the major factor enhancing the intracellular accumulation of NFTs [3-5]. Tau cleavage by caspase at the C terminus also makes tau prone to forming aggregates [3-5] (Fig. 1). Autophagy, i.e. self-eating, is a lysosomal degradation mechanism that enables cells to capture cytoplasmic proteins, lipids, and organelles. Then, they are transported to the lysosomal compartment by vesicles with double membranes termed autophagosomes [6]. Autophagy is a dynamic recycling machinery that supplies new energy and building blocks. Therefore, autophagy is important for homeostasis, and maintains it via the synthesis, degradation, and turnover of cytoplasmic materials under stress. Three types of autophagy have been found; macroautophagy, Chaperon *Address correspondence to this author at the Second Department of Internal Medicine, LifeScinece Innovation Center, Department of Aging and Dementia (DAD), Faculty of Medical Sciences, University of Fukui, Fukui, Japan, 23-3 Matsuokashimoaizuki, Eiheiiji-cho, Yoshida-gun, Fukui 9101193, Japan; Tel: +81 776618351; Fax: +81 776618110; E-mail: [email protected] 1567-2050/18 $58.00+.00

Mediated Autophagy (CMA) [7], and microautophagy (Fig. 2). Macroautophagy is considered to be the main type of autophagy, and it has been explored more extensively than the others. Recent researches have found that autophagic vacuoles (AVs), the commonly used term for autophagyrelated vesicle formation, are abundantly observed in neurons in AD [3] and other neurodegenerative diseases, including Parkinson’s disease (PD) [8], amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), which manifest due to the deposition of pathogenic proteins [9]. Moreover, in neurodegenerative disorders, the induction of autophagy has neuroprotective effects. This is why abnormal autophagy induces neuronal death in most neurodegenerative diseases. Therefore, autophagy is a target of therapy for AD and other neurodegenerative diseases, including PD, ALS, and HD. 2. THE MACHINERY OF AUTOPHAGY Phagophore formation likely begins in the lipid bilayer of the membrane [10] (Fig. 3). The Endoplasmic Reticulum (ER) is one of the possible origins of the membrane for preautophagosomes. Indeed, isolated membranes are found in an ER subdomain and connected with it [11]. Furthermore, Atg14 and Atg5 (encoded by autophagy related genes)positive isolated membranes are observed in the immediate vicinity of the ER subdomain in contact with mitochondria during nutrient depletion [12]. Post-Golgi tubulovesicular compartments undergoing remodeling and homotypic fusion and the ER-Golgi intermediate compartment (ERGIC) have been thought to be the origin for pre-autophagosomal membrane, too [13]. In addition, the plasma membrane and the endocytic compartments are regarded as membrane origins for the early autophagosomal precursor structure. In this process, clathrin-dependent endocytosis is involved by de© 2018 Bentham Science Publishers

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Fig. (1). Pathological hallmarks of Alzheimer’s disease are senile plaques (SPs) and neurofibrillary tangles (NFTs). Sps consist of amyloid β protein (Aβ), which is proteolytically cleaved by β secretase and γ secretase. Aβ forms oligomers that cause neuronal death. NFTs are composed of tau protein. Tau is a microtubule-associated protein that stabilizes microtubules. However, once tau is phosphorylated by tau kinases, including GSk3β, Cdk5, or Jun N-terminal kinase (JNK), tau no longer binds microtubules and aggregates. C-terminal truncation of tau by caspase3 also stimulates tau aggregation.

Fig. (2). Scheme of the three types of autophagy: Macroautophagy: A part of the cytoplasm, including organelles, is engulfed by an isolated membrane, the phagophore, to form an autophagosome. The outer membrane of the autophagosomes fuse with the lysosomes, and the internal material is digested in the autolysosome. Microautophagy: Small pieces of the cytoplasm are immediately enclosed by invaginated lysosomal or late endosomal membranes. Chaperon-mediated autophagy (CMA): Substrate proteins harboring a KFERQ-like polypeptide sequence are first identified by cytosolic Hsc70 and cochaperones. They are then translocated to the lysosomal lumen after connecting with the lysosomal receptor Lamp2A. All 3 autophagy types have similar purposes, including the synthesis of protein, energy, and gluconeogenesis.

The Implications of Autophagy in Alzheimer’s Disease

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Fig. (3). Scheme of the autophagy flux. Depletion of nutrients, growth factors, and energy is well-characterized to induce autophagy by eventually inhibiting mTORC1 and activating AMPK. mTORC1 downregulation and AMPK upregulation positively regulate the ULK1 complex via a phosphorylation cascade. ULK1 complex induction leads to activation of the Vps34 complex, which leads to PI3P synthesis in preautophagosomal structures. The membranes of these structures are of varying origin, including the ER, Golgi, trans-Golgi network, plasma membrane, endosome, and mitochondria. PI3P discriminates the LC3 lipidation sites for autophagosomal precursors by helping in the mobilization of the Atg12-Atg5-Atg16L1 complex. This complex is required for the conjugation of LC31 to PE in membranes, which supports the elongation of membranes to engulf substances, including aggregated protein, infective agents, and altered mitochondria that are finally degraded in lysosomes after fusion with autophagosomes. Inactivation of mTORC1 causes translocation of TFEB to the nucleus, where TFEB becomes activated, and many genes of autophagy and lysosome are translated. Key components in autophagy are produced, and effective autophagy-dependent digestion occurs. Abbreviations: AMPK, AMP-dependent protein kinase; mTORC1, mammalian target of rapamycin complex 1; PE, phosphatidylethanolamine; PI3P, phosphatidylinositol3-phosphate; TFEB, transcription factor EB; ULK, unc51 like kinase.

livering Atg16L1 and mAtg9, a core Atg protein and a multipass transmembrane protein, to recycle endosomes through different pathways via vesicle associated membrane protein3 (VAMP3)-dependent fusion events of membranes, which induces the formation of the early autophagosome and mature autophagosomes [14]. Overexpression of the recycling endosome proteins TBC1D14 (a Rab 11 effector) or pxcontaining SNX 18 was reported to induce mAtg9 and Atg16L1 accumulation, respectively, and other autophagic proteins, including unc51 like kinase (ULK)1 and microtubule associated protein 1 light chain (LC3), in their compartment [15]. These observations suggested that the recycling endosome is important for the origin of phagophore membrane. Mitochondria are also considered to be a possible source of membrane. In the formation of autophagosomes, ER-mitochondria contact loci are important for assembling the phagophore [12]. Phagophore expands to engulf the cytoplasmic region or selected organelles, then isolating the cargo in an autophagosome with double membrane. In the cytoplasm, autophagosome formation happens at random sites. The autophagosomes are delivered to the mi-

crotubule organizing center along microtubules. At the microtubule organizing center, lysosomes are enriched to enhance lysosomal fusion [16]. Proteins like dynein and the LC3-II are required for the transport along microtubules. During transport, fusion of the autophagosome with a late endosome may occur, generating an amphisome, which eventually fuses with a lysosome and digests its cargo, or the autophagosome may immediately fuse with a lysosome to produce an autolysosome (Fig. 2) [17]. The content of the autolysosome is then digested by lysosomal proteases (Figs. 2, 3). The amino acids and other by-products of digestion are used for metabolic recycling and for formation of macromolecules. Atg1-35 organizes into functional complexes that act as mediators of the following steps in the process of autophagy; initiation, elongation, maturation, fusion, and degradation (Fig. 3). One essential molecule for the maturation autophagosomes or endosomes is Rab7. Rab7, a small GTPase, is associated with late endosomes/lysosomes. Rab7 is necessary for autophagosome formation, transportation of the cargo along microtubules, and the fusion with lysosomes [18]. Furthermore, it was reported that Synthaxin 17 is also


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recruited to the outer membrane of autophagosomes to mediate fusion via its interactions with ubisnaps (SNAP29) and vesicle-associated membrane protein 7(VAMP7) in drosophila melanogaster [19]. During the proteolytic digestion through lysosomes, transcription factor EB (TFEB) controls autophagy by modulating the transcription of lysosomal enzymes. Furthermore, it has been demonstrated that TFEB overexpression results in the new production of lysosomes and increases autophagosomes in several types of cells [20] (Fig. 3).

ULK complex activation is needed for recruiting the class III PI3K Vps34 to the phagophore start sites where, Vps34 produces phosphatidylinositol 3-phosphate (PI3P) while in a complex with Vps15, Atg14, and beclin 1(Atg6). Although the precise roles of PI3P in autophagy are not clear, it likely aids in the recruitment of WIPI (tryptophan aspartic acid (WD) repeat domain phosphoinositideinteracting) proteins to the phagophore membrane. WIPI2 recruits Atg16L1 (Atg16- like 1) to phagophore formation sites [34].

Autophagy was first qualified as a bulk, non-selective, digestion flux caused by the depletion of nutrients (Fig. 3). Recent studies clarified that in non-starved cells, autophagy also contributes to the homeostasis of the cytoplasm by degrading materials such as aggregate prone proteins, including Aβ and tau (aggrephagy), impaired mitochondria (mitophagy) [21, 22], excess peroxisomes (pexophagy), and invading pathogens (xenophagy) [23]. Autophagy can be initiated by varying stimuli, including reactive oxygen species (ROS) [24] and hypoxia [25]. For example, abnormally folded aggregated proteins are tagged with ubiquitin chains and identified by ubiquitin biding domain with receptors, including p62 (Sequestosome-1 (SQSTM1)) [26], neighbor of BRCA1 gene1 [27], optineurin (OPTN) [28], Tax-1 binding protein 1 (TAX1BP1), nuclear dot protein 52 (NDP52), Toll-interacting protein (TOLLIP) [29], and 26S proteasome regulatory subunit RPN10 [30]. In their sequences, these receptors harbor LC3-interacting region (LIR) motifs that can identify LC3, an important autophagosome-associated protein, as described above (Fig. 3). Thus, these receptors bridge between autophagosomes and ubiquitinated cargo, and improve the inclusion of cargo into autophagosomes for lysosomal digestion [26].

The Atg12 and Atg8/LC3 ubiquitin-like conjugation systems are necessary to maintain phagophore expansion. Initially, Atg12 is conjugated to Atg5 in a reaction that involves Atg7 and Atg10 (E2 like enzyme), and this complex binds to Atg16L1 non-covalently. Atg12-Atg5-Atg16L1 associates with pre-autophagosomal membranes, allowing the elongation by helping in the recruitment of LC3. However, before this can occur, the C terminus of early phase LC3 is processed by Atg4 and becomes LC3-I. The C-terminal end of LC3-I contains a glycine residue. This cleavage is required for LC3-1 conjugation to phosphatidyl ethanolamine (PE), leading to LC3-II via a mechanism dependent on Atg7, Atg3, and Atg12-Atg5-Atg16L1 (E1-like, E2 like, and E3 like enzymes, respectively). LC3-II is strongly linked to autophagosomal membranes (Fig. 3). Then, to form mature autophagosomes, this cascade reactions enables phagophore edge elongation and closure [10].

3. KEY AUTOPHAGIC REGULATORS Autophagy activation induced by the primary stimuli of starvation or low energy status in the cell is controlled by signaling networks based on ULK1 and ULK2 (Fig. 3). ULK1/2 forms complexes with Atg13, Atg101, and focal adhesion kinase (FAK) family interacting protein of 200 kDa (FIP200). The availability of nutrition and growth factors, and the amount of adenosine monophosphate (AMP)/adenosine triphoshophate (ATP) reflect the cellular energy levels and are detected by mechanistic target of rapamycin (mTOR) complex1 (mTORC1) and AMP dependent protein kinase (AMPK), respectively. These control the ULK1/2 complexes via a series of phosphorylation cascades. For example, AMPK upregulation by allosteric binding of AMP and Thr172 phosphorylation induces autophagy by direct ULK1 activation via Ser317 and Ser77 phosphorylation under glucose depletion [31] or Ser555 phosphorylation under amino acid depletion and mitophagy [32]. Conversely, in medium containing amino acids (detected by the Regulator-Rag complex), and growth factors (signal by tyrosine kinase) and the phosphatidylinositol-3 kinase (PI3K)/AKT pathway, mTORC1 is upregulated and downregulates autophagy by connecting the ULK1 complex through RaptorULK1 interaction, and by Atg13 and ULK1 (Ser757) phosphorylation. Consequently, ULK1 kinase is suppressed, and the interaction of ULK1 with AMPK is prevented [33].

Phagophore extension is also assisted by mAtg9. mAtg9 localizes to the trans-Golgi network and the endocytic compartment such as early, late, and recycling endosomes. Moreover, it is suspected to aid in the supply of lipid bilayers to the initial phagophore, allowing further elongation before closing the fully formed autophagosomes [10]. Why the late stage, and the inner and outer membranes of the preautophagosomal structure are separate entities is not fully explored. However, Atg2 in combination with WIPI1 may control closure of autophagosomes [35], a process that functions in membrane fission /scission-type events similar to the production of multivesicular bodies by endosomal sorting complexes required for transport (ESCRT)-mediated membrane sprouting [36]. 4. TRANSCRIPTIONAL REGULATOR OF AUTOPHAGY Besides phosphorylation, autophagy is controlled at the translational level. mTORC1, besides regulating the ULK1/2 complex, binds the lysosome nutrient-sensing (LYNS) mechanisms to the transcriptional regulation of autophagy genes through TFEB [37] (Fig. 3). At rest, TFEB phosphorylation via activated mTORC1 causes TFEB to bind to 14-3-3 proteins, thereby causing cytosomal retention of TFEB [38]. On the other hand, under nutrient depletion and subsequent mTORC1 downregulation, TFEB is not phosphorylated and translocates to the nucleus. In the nucleus, TFEB binds to interconnected lysosomal expression and regulation (CLEAR) consensus sequences in promoters of target genes and initiates transcription. Many of these genes, including lysosomal hydrolases, vacuolar type H+-ATPase (VATPase) subunits, and Atg proteins are related to lysosomes and autophagy directly. V-ATPase, a lysosome membrane


multimeric channel protein, mainly functions as an ATPase and H+ channel, but it also acts as a sensor for the lysosomal amino acid concentration. Therefore, TFEB coordinates the transcription of many of the key genes needed for autophagic-lysosomal pathway [20]. Zinc-finger protein with KRAB and Scan domain 3 (ZKSCAN3) in a transcriptional repressor of autophagy that likely opposes TFEB. ZKSCAN3 silencing augments autophagosome and lysosomal synthesis, and mTORC1 inhibition, which causes it to be deposited in the cytoplasm [39]. Currently, quite a few (>20) transcription factors have been linked to transcriptional modulation of autophagy after a variety of stimuli [40]. For example, MITF9 (microphthalmia-associated transcription factor9; belonging to the same protein family as TFEB) [41], p53 [42], and forkhead box 03 (FOX03) [43] have been reported to trans-activate Atg genes. 5. THE AUTOPHAGIC PATHWAY 5.1. mTORC1-dependent Signaling Pathways mTORC1 is a protein complex that works as a nutrient, energy, and redox sensor, and controls the synthesis of proteins. mTORC1 is a fundamentally inhibiting signal that functions in the first step of signal transduction upstream of Atg proteins. Rapamycin sensitive mTORC1 directly controls homeostasis of cell through autophagy downregulation [44]. If growth factors, including insulin-like growth factor (ILGF), bind to ILGF1 receptors (ILGF1R), the class 1 PI3K flux, which catalyzes the change of phosphatidylinositol 4,5bisposphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), recruits Akt and PDK1 to the membrane, allowing PDK1 to phosphorylate Akt (Fig. 4) [22, 45]. The activation of Akt inactivates the TSC1 and /or TSC2 complex, whose activity inhibits general mTORC1 signaling (Fig. 4) [46]. On the other hand, intracellular signals, such as nutrient depletion, low energy status (increased AMP and/or ATP ratio), DNA damage, and hypoxia, inactivate mTORC1 (Fig. 3). P53, which is frequently mutated in human cancers, also controls mTORC1 activity. Under oncogenic or genotoxic stress, nuclear p53 promotes translation of the autophagy-induced gene sestrin1/2. Sestrin 1/2 stimulates autophagy by activating AMPK [47]. Furthermore, mutation of the p53 orthologue CEP-1 extends the life span of Caenorhabditis elegans by increasing baseline autophagic activity [48]. Furthermore, p53 controls the expression of damage regulated autophagy modulater1 (DRAM1), thereby inducing autophagy. Recent studies suggest that DRAM1 controls autophagosome elimination by promoting lysosomal acidification and activating enzymes in lysosomes [49]. Moreover, TFEB was found to be regulated by lysosomes through the mTORC1 pathway. Distribution of TFEB is directly controlled by the mTORC1 signaling pathway as follows: The Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to mediate nutrient depletion- and stress-induced translocation of TFEB to the nucleus [37]. 5.2. mTORC1 Independent Signaling Pathways In addition to the standard mTORC1-dependent signaling pathway, mTORC1-independent pathways are important for autophagy regulation. Autophagy can be directly initiated by


AMPK, leading to the phosphorylation of ULK1 and beclin1 (Fig. 3) [32]. Although ULK1 activation by energy depletion happens via a different manner than that mediated by amino acid depletion, the coordinated phosphorylation of ULK1 by mTORC1 and AMPK may regulate the autophagic pathway response to metabolite needs. Furthermore, the LKB1/AMPK-dependent phosphorylation of P27 stabilizes this cdk inhibitor, enabling cells to survive under nutrient derivation and oxidative stress via autophagy activation [50] (Fig. 4). Beclin-1, an important factor for autophagy, binds to Bcl-2 [5], which is an anti-apoptotic protein under nutrient-rich conditions. On the other hand, under nutrient-poor conditions, Bcl-2 is phosphorylated by JNK1 [51] and dissociates from beclin-1 [52]. This promotes the formation of the autophagy-stimulatory beclin-1-Vps34 complex. mTORC1 is also inhibited by nutritional depletion, but active JNK1 does not influence the activity of mTORC1. Furthermore, rapamycin does not influence JNK1 or Bcl-2 phosphorylation. In TSC2-deficient cells, these phosphor-proteins are not affected even when mTOCR1 is active [53]. These results imply that autophagy is controlled by JNK1/beclin-1/PI3K and mTORC1 independently. A cyclical pathway involving inositol and myoinositol 1, 4, 5 triphosphate (IP3), both of which downregulate autophagy, has also been reported [54]. Cytosolic cyclic AMP (cAMP) increases the IP3 production. IP3 then binds to ER membrane receptors, and releases Ca2+ from the ER stores, which upregulates calpain and inhibits autophagy. Sirtuin 1 (SIRT1) is a physiogenetically preserved NADdependent protein deacetylase. SIRT1 is one of the essential regulators of autophagy causing aging and age-related disorders. SIRT1 is activated by 3 mechanisms, including overexpression, pharmacological activation with resveratrol, and nicotine amide starvation, in which its negative regulator prolongs the life span via stimulation of basal autophagy rates in yeast, flies, and C. elegans [55]. SITR1 was suggested to form complexes with several important autophagic molecules, including Atg5, Atg7, and Atg8. (Fig. 4). As SIRT1 can also deacetylate autophagy-related proteins [55], it likely induces autophagy in this manner [56]. 6. AUTOPHAGY IN NEURONS Regarding the maintenance of homeostasis, the role of autophagy is extremely important for postmitotic neurons because the levels of disturbed proteins and disturbed organelles cannot be diluted by cell division. Furthermore, the neuron has specific intracellular structures for interneuronal communication. Thus, there is a heavy reliance on autophagy in the brain based on the observation that the most seriously damaged organ is the brain in the primary lysosomal network by autophagy and endosomal pathways. Therefore, disturbed autophagy and endosomal pathways are frequently linked to neurodegenerative diseases. As neurons normally have large dendrites and axonal cytoplasm, it is difficult to prevent damaged organelles and cell waste from accumulating with no aid from cell division. Young neurons can clean autophagic substrates very effectively. However, many AVs produced in axons have to be transported a long distance to lysosomes, which mainly accumulate close to the cell body. As such neurons are extremely fragile to disturbed prote-


olytic clearance of autolysosomal substrates [57], ubiquitinated protein aggregates and degenerated substrates are accumulated in neurons without autophagy [58, 59]. Therefore, postmitotic neurons with specific cellular structures have different mechanisms to regulate autophagy compared with autophagic regulation systems in non-neuronal cells. In primary neuronal cultures, blockage of autophagosome clearance by cathepsin inhibition induces rapid AV accumulation with no hindrance by autophagic induction [60]. This indicates that in neurons, autophagy is constitutively active. High clearance of autophagosomes rather than low levels of autophagic production maintains a low number of autophagosomes [17]. Higher specialized neuronal structures are present in high-energy demand regions, and a high protein turnover is needed for quality control for neuronal survival. Imbalance of abnormal protein quantity and capacity of the quality control system can cause abnormal protein and organelle accumulation. Indeed, autophagy is more effective in younger neurons [60] because autophagy-related proteins, including beclin-1, Atg5, and Atg7, decrease with aging [61, 62]. With aging, Atg1, Atg8a, and Atg18 are also downregulated in drosophila melanogaster [63], which may be the cause of several late onset neurodegenerative disorders, including AD [64]. This ultimately leads to neuronal degeneration and death. The roles of basal autophagy in maintaining neuronal homeostasis were demonstrated by experiments using knockout mice for Atg5 or Atg7, which exhibited neurobehavioral abnormalities [65]. Moreover, in these autophagy-deficient neurons, inclusion bodies consisting of polyubiquitinated proteins increased with aging in both size and number. These observations imply that constitutive clearance of uncommon proteins via autophagy markedly impacts neuronal homeostasis. Many pathological proteins can be digested through either the ubiquitin proteasomal system (Ups) [66, 67] or CMA only when they are soluble [68]. Selective autophagy, CMA, is distinct from other autophagy systems due to the uniqueness in which its substrate proteins are selectively targeted to the lysosomal surface and subsequently taken into the digestive organelles (Fig. 2). 7. AUTOPHAGY MULFUNCTION IN ALZHEIMER’S DISEASE Neurodegenerative disorders, including AD, present dynamic variety in the stage of failure, susceptible regions, and pathological mechanisms. Almost all hereditary and sporadic neurodegenerative disorders, including AD, are age dependent and present as progressive neuronal function loss. Many studies have reported that dysfunctional autophagy occurs in the AD animal model and AD patients. In 1967, Suzuki discovered that in AD brains, many abnormally aggregated tau and subcellular vesicles are deposited in the neurites with dystrophy or swelling [69], but these vesicles were not identified at that time. In 2005, Nixon et al. observed immature AVs deposited in dystrophic neuritis in a brain with AD by immunoelectron microscopy [70]. This was the direct, first evidence that autophagy is disturbed in AD. Similar findings were obtained using presenilin 1(PS1)/APP double transgenic animals [71] in which AVs were increased in the dendrites and cell body of neurons even at the early phase when Aβ plaques were not observed compared with age-matched controls. The expression level of lysosomal protease was

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increased in the early stage of AD patients [64]. As noted previously in Drosophila melanogaster, Atg1, Atg8a, and Atg18 expression levels gradually decrease with aging, subsequently reducing the autophagic activity and increasing Aβ production. This was thought to correlate with the lateonset dysfunctional neurons and AD phenotype [63]. Thus, a decrease in autophagy-related gene expression with aging is associated with late onset AD. In hippocampal neurons of AD mice, abnormally accumulated immature AVs in axons were found preceding the loss of synapses and neurons [71]. However, whether the disturbance of autophagy is the cause or result of AD remains controversial [72, 73]. Universal characteristics of neurodegenerative disorders in their pathogenesis are the presence of pathogenic proteins. However, once the proteins form irreversible oligomers or multimers, they cannot be completely unfolded. Therefore, proteins can be cleaved though distinct methodmacroautophagy (Fig. 2) [74]. The mechanisms to initiate macroautophagy under these conditions are unclear. Failure of the Ups or CMA system to digest the aggregated proteins, including tau and Aβ may be involved (Fig. 3). Moreover, this hypothesis was confirmed by an experiment blocking Ups- or CMA-upregulated macroautophagy [75]. Macroautophagy does not only function at the compensatory stage, it may also play a main role in the elimination of pathologic proteins. Consequently, once macroautophagy is dysfunctional by further inactivation of Ups and CMA, it can not perform quality control. Consequently, toxicity due to aberrant AVs frequently leads to abnormal neurons and neuron death. In the AD brain, dystrophic and degenerated neurites have abundant autophagosomes and other types of AVs, which become the main reservoir of toxic peptides in the cell [70]. Combined factors cause this immature AV accumulation in aged neurons such as increased initiation of autophagy, retrograde transport of Avs, and maturation deficits in autophagosomes [17]. Local deposition of autophagosomes in dystrophic neurites leads to Aβ production, which occurs due to both enhanced turnover of APP and enrichment of the γ-secretase complex. Furthermore, studies using APP/PS1 double transgenic model mice also reported that AV generation was mediated via the activation of AMPK [76]. 7.1. Amyloid β Protein and Autophagy Autophagy plays important roles in Aβ metabolism. First, similar with Aβ degrading enzymes [77], autophagy is considered to be another main Aβ eliminating pathway. Second, the autophagy-lysosome system is necessary for degrading Aβ under normal conditions. However, it is presented as a new method for Aβ production under pathological conditions or throughout the course of aging [78]. Although Aβ is considered to be generated in lysosomes, ER, and the Golgi apparatus, Aβ has been found in AVs after autophagy activation [79]. Thus, the accumulated immature AVs observed in AD patients and APP/PS1 transgenic mice may be origins of Aβ production. Immunohistochemical studies have revealed Aβ42 in AEL (autophagy-endosomal-lysosomal) vesicles in neurons of Drosophila [80]. Another study found that APP and its cleaving enzyme BACE1 are separated into different vesicles under normal conditions in neurons. How-


ever, they became highly co-localized and co-trafficked into autophagy-lysosome pathways with the GABAA antagonist Picrotoxin (PTX) or glycine/NMDA-receptor agonist/K+ stimulation [81]. This implies that under pathological situations, the convergence of APP and BACE1 in autophagosomes acts as a new area for Aβ production. Third, autophagy itself may cause Aβ secretion. It was confirmed that autophagy plays an essential role in the extracellular release of Aβ. In autophagy-deficient mice, Aβ production was decreased by 90%. However, recovery of autophagy reversed Aβ secretion to normal levels [82]. Another study demonstrated that Aβ secretion was markedly decreased with high intracellular Aβ deposition in Atg7 knockout mice [83]. As Aβ can also regulate autophagy, vascular Aβ40 may initiate autophagy in endothelial cells and disturb neurovascular regeneration [84]. Membranes of autophagosomes are rich in PS1, whose mutation results in early onset autosomal dominantly inherited AD. PS1 has numerous biological functions, including the outgrowth of neurites, cell adhesion, plasticity calcium homeostasis, and apoptosis of synapses. One of the most important roles of PS1 is being a constituent of the γ secretase complex for APP cleavage. PS1 has γ secretaseindependent functions for lysosomal proteases during autophagy, too. Reduction in PS1 function induces immature unglycosylated V-ATPase accumulation, which is needed for autolysosome and/or lysosome acidification. Abnormal deposition of immature late stage autophagosomes with undigested components is observed in PS-deficient cells, which is similar with the ultrastructure present in AD neurons [85]. Furthermore, it was reported that fibroblasts from AD patients with PS1 mutation lacked macroautophagy. This was due to the disturbed maturation of the V0a1 subunit of the bimodular V-type H+-ATPase proton pump that supports acidification of autolysosomes, resulting in Aβ accumulation [86]. Hyper-activation of GSK-3β, which is also an important risk factor for AD [4, 5], can disturb lysosome acidification in a similar manner as PS1 [87]. Moreover, many studies have reported that the beclin1 expression level was downregulated in the early phase of AD [88]. The autophagy-inducing protein beclin-1 was demonstrated to lead to AD progression [89] because in moderate to severe AD, the beclin-1 protein level was reduced in microglia in the gray matter of the superior and middle frontal cortex. In APP transgenic mice, decreased beclin-1 expression reduced neuronal autophagy, which interrupted lysosomes and enhanced intracellular Aβ accumulation, causing neurodegeneration [89]. However, the opposite pattern was observed in mice with increased beclin-1 expression [90]. Furthermore, beclin1 is involved in retromer trafficking and receptor mediated phagocytosis. Indeed, beclin 1 and retromers are decreased in microglia obtained from postmortem AD human brains [91]. In addition, in APP double transgenic mice (TgCRND8), genetic amelioration of cystatin B, an endogenous lysosomal cysteine protease inhibitor, markedly decreased Aβ levels, and prevented memory and learning deficits, which supports that autophagy clearance is involved in Aβ metabolism regulation [92]. Allelic variation in the apolipoprotein E (APOE) gene (ApoE4) is an important risk factor for sporadic AD. In the brain, ApoE is the primary cholesterol carrier. Overexpression of ApoE4 increases Aβ42


levels in lysosomes, and consequently causes neuronal death in the hippocampus [93]. Furthermore, ApoE4 potentiates lysosomal leakage and apoptosis induced by Aβ peptides in Neuro-2a cells [94]. In conclusion, these studies demonstrated that ApoE4 and Aβ may function with each other to increase the susceptibility to lysosomal membrane disruption, lysosomal enzyme release, and degeneration of neurons [94]. Moreover, ApoE variants and Aβ oligomers enhance lysosomal membrane permeabilization (LMP) [95]. It was also found that ApoE enhanced Rab7 recycling, which is a small GTPase responsible for recruiting the motor complex to late endosomes/lysosomes [96]. Therefore, ApoE4 may be one cause of dysfunction of autophagy. 7.2. Tau Protein and Autophagy The 20S proteasome (core particle) and trypsin-like activity in human AD brains is markedly low [97]. It has been reported that Ups may be the primary mechanism to degrade endogenous tau [98]. However, PHF-tau isolated from postmortem brains did not undergo degradation by proteasomes in the soluble state. Instead, it functioned as a proteasomal inhibitor [99]. Moreover, tau was found to be degraded by proteasomes in vitro; however, in primary neurons or neuroblastoma cells, tau was not degraded by proteasomes [98]. Autophagy is involved, in at least in part, in the clearance of soluble and aggregated tau, and NFTs in the cell. Several autophagy inhibitors, including chloroquine, NH4Cl, 3methyladenine (3MA), and cathepsin inhibitors, delay the degradation of tau and increase the high molecular weight tau [3, 100]. In cultured hippocampal slices, the lysosomal protease cathepsin D can degrade tau [101]. Multivesicular body (late endosome) formation was also observed after lysosomal inhibition by chloroquine [3]. Furthermore, phospholipase D1 (PLD1), which is primarily associated with the endosomal system, partially relocalized to the outer membrane of autophagosome-like structures in nutrient-poor conditions. PLD1 localization and starvation-induced PLD activation were altered by wortmannin, a PI3K inhibitor, suggesting that PLD1 acts downstream of Vps34. This increases tau and p62 aggregates in the brain [102]. The C terminal truncation of tau is known to be important for tau aggregation [4, 5, 67, 103] (Fig. 1). Moreover, autophagy functions in the degeneration of caspase-cleaved tau levels [104]. It was reported that caspase-cleaved tau (TauD421) (Fig. 1) was colocalized with the immunophilin FKBP52 in the autophagy endolysosomal system in AD neurons [105]. FKBP52, a member of the immunophilin protein family, is required for immunoregulation and basic cellular processes, including folding and trafficking of proteins. FKBP52 binds to the immunosuppressant FK506 and rapamycin. Furthermore, the formation of oligomeric tau and insoluble aggregates disturbs the autophagy-lysosomal system. Increased tau phosphorylation reduces its binding to microtubules, and consequently, the number of tau particles capable of movement increases. Decreased autophagosome formation and enhanced phosphorylated tau levels are highly associated in down syndrome patients during AD development [106]. Therefore, these findings all highlight autophagy in the pathogenesis of AD, especially for tau metabolism.


Multiple studies, mainly on AD, clarified that mTORC1 signaling activation enhances tau pathology. On the other hand, the inhibition of mTORC1 signaling decreases the progression of tau pathology [107]. Two possible mechanisms for how hyperactive mTORC1 signaling leads to tau hyperphosphorylation are proposed: direct tau phosphorylation regulation and autophagic downregulation. 7.2.1 Alzheimer’s Disease and CMA CMA is a selective and essential autophagy system that mediates the lysosomal degradation of a large number of proteins with the consensus pentapeptide motif KFERQ in their sequence. Although tau has the KVEF motif, only a small amount of tau is degraded by CMA. Truncated tau is recognized by Hsc70 at its C terminus and is delivered to CMA receptors [9]. The colocalization of tau in autophagic vacuoles was also observed. CMA is vulnerable in neurodegenerative disorders because altered tau protein, including tau in frontotemporal dementia (FTD), strongly binds Lamp2A, blocking their translocation across the lysosomal membrane (Fig. 2). Another report noted that APP has the KFERQ motif identified by hsc70. Deleting this sequence protects APP from lysosomes and increases its cleavage products by secretase, Aβ, without affecting its binding ability to hsc70 [108]. 7.3. Mitophagy and Alzheimer’s Disease Neurons affected in AD exhibit disturbed mitochondria and bioenergetic dysfunction that occurs early stage AD. These factors enhance Aβ and tau pathologies. It was recently suggested that the lysosome pathway that clears disturbed mitochondria (mitophagy) is affected in AD [109] (Fig. 3). This causes damaged mitochondria to accumulate in the AD brain. It was previously demonstrated that the recruitment of p62, LC3, and histone deacetylase (HDAC) initiates mitophagy. Results from animal models and cell culture models of AD, and from sporadic, late onset AD patients revealed that disturbed mitophagy causes dysfunctional synapses and cognition by triggering the deposition of Aβ and tau due to increased oxidative stress and deficient cellular energy [110]. 7.4. Axonal Transport and Autophagy in Alzheimer’s Disease Axonal transport is a necessary process for maintaining neuronal homeostasis. Newly built autophagosomes travel along microtubules in mammalian cells [111]. During this process, autophagosomes engulf aged and misfolded proteins or impaired organelles, including mitochondria. Then, they are degraded after fusing with lysosomes. Impaired axonal transport in both early- and late-stage AD was observed [112]. Phosphorylated tau impairs axonal transport and degradation [113], which supports the observation that unusual aggregated proteins disturb axons. Autophagosomes were not transported to the cytoplasm and were unable to fuse with lysosomes. On the other hand, other studies have proposed opposite hypotheses such as that lysosomal protease abnormalities induce axonal degeneration [114].

Hamano et al.

7.5 Aging or Disease Progression of Alzheimer’s Disease and Autophagy As stated above, autophagy-related proteins, including beclin-1, Atg5, Atg7, [61, 62], Atg1, Atg8a, and Atg18, are downregulated with aging in Drosophila melanogaster [63], which may be the cause of late onset neurodegenerative diseases, including AD [64]. Mutated APP and Aβ were reported to induce disturbed autophagy. They also cause mitochondrial stress, and functional change and synaptic damage to hippocampal neurons in AD. These observations imply that hippocampal deposition of mutant APP and Aβ is due to altered mitophagy [109]. The activating molecule in beclin 1 regulated-autophagy Ambra1 has a high molecular weight (130 kDa) and is involved in neural development. Ambra1 is abundantly found in the central and peripheral nervous system in mouse embryos. In the hippocampus, Ambra1 expression decreased with aging. In the WT mouse, Ambra1 was significantly reduced at 12 months, and in the mutant tau (Tg2576) mouse, Ambra1 levels were markedly low at 6 months, unchanged at 12 months, and became the lowest at 18 months [115]. 8. AUTOPHAGY AS THE POTENTIAL THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE 8.1. Autophagy Induction and Amyloid β Protein The recovery of normal autophagy function may be a groundbreaking therapy for AD. As such, small molecules that upregulate autophagy are desired. Rapamycin can decrease Aβ and tau pathologies in AD animal models by inhibiting mTORC1. Many studies on APP mutant (V717F) transgenic mice demonstrated that mTORC1 inhibition by rapamycin for long periods rescued AD-like memory and learning impairments, and decreased Aβ42 levels [116]. In rapamycin-treated mice, autophagy was actively initiated in the hippocampus. In addition, SMER28 was found to enhance the clearance of Aβ and Aβ CTFs in cultured cells by initiating autophagy in an mTORC1-independent manner via the Atg5 -dependent autophagy pathway, which may be one potential target for AD treatment [117]. It was noted that patients on long-term lithium therapy have a markedly lower rate of AD due to the induction of autophagy in an mTORC1 independent manner [22, 118] (Fig. 4, Table 1). Furthermore, βCa2+ channel blockers, including nilvadipine, were also noted as potential therapeutics for AD in a clinical trial. Nilvadipine can induce autophagy via Ca2+-calpain Gsα and mTORC1-independent pathways [119]. Other compounds may also activate autophagy in an mTORC1independent manner. Resveratrol and its analogues RSVA314 and RSVA405 induce autophagy and promote transcellular elimination of Aβ in vitro [120]. Nicotinamide prevents the pathology and cognitive deficits in AD model mice by affecting enhanced β-nicotinamide adenosine dinucleotide biosynthesis, PI3K signaling, and the autophagy systems [121]. Another study found that the anti-convulsive drug carbamazepine has strong autophagy-inducing effects and anti-AD effects in APP (swe)/PS1(deltaE9) transgenic mice [122].



Fig. (4). mTORC1-dependent and mTORC1-independent pathways of autophagy, and autophagy activating drugs. The autophagic flux and potential drug targets. Two signaling pathways are involved in the regulation of autophagy: mTORC1-dependent signaling pathway and mTORC1-independent signaling pathway. Abbreviations: mTORC1, mammalian target of rapamycin 1; IRS-1, insulin receptor substrate 1; PI3K, phosphoinositide 3-kinase; SIRT1: sirtuin 1.

It was previously reported that latrepirdine significantly attenuated the toxicity induced by Aβ42 in WT mouse compared with in autophagy deficient mice (Atg8delta). This suggests that autophagy induced by lateropirdine decreases intraneuronal Aβ42 [123]. However, rapamycin was found to induce Aβ production [78], whereas 3MA and inhibitors of autophagy prevented the rapamycin-induced deposition of Aβ in vitro. Disturbed autophagosome and/or lysosome fusion and reduced lysosomal activity may function in the late stage of AD [57, 86]. Autophagy stimulation is involved in the deposition of autophagosomes in neurons, resulting in significant intraneuronal Aβ accumulation in AD. On the other hand, augmenting lysosomal activity or lysosomal fusion may be therapeutic. As such treatments that simply enhance autophagy initiation are not always beneficial to AD patients, we should consider the entire autophagy process in AD and tightly regulate autophagic activity for AD treatment. 8.2. Autophagy Induction and Tau Protein Autophagy inducers, such as rapamycin, enhance insoluble tau degradation and protect against toxicity of tau in Drosophila [124] and tau transgenic mouse models [125]. In transgenic mice harboring mutant human tau (P301L) that were administered lithium chloride (LiCl) orally for 4 months starting at the age of 5 months, LiCl-exposed mice exhibited higher scores for sensory motor tasks. Insoluble and soluble phosphorylated tau was decreased. Lithium therapy also induced autophagosome-like puncta with LC3 and decreased p62 (substrate of autophagy) in the spinal cord

[22, 126] (Fig. 4). Trehalose, a natural disaccharide and activator of mTORC1-independent autophagy [127], enhanced neuronal survival and decreased aggregated tau in the brains of a human tauopathy mouse model [128]. Similarly, reduced tau aggregation and tau-induced neurotoxicity was observed in a mouse model of tauopathy with parkinsonism (PK(-/-)/Tau(VLW) treated by trehalose [129]. This effect was considered to be mediated by the upregulation of autophagy. The A152T mutation of MAPT was reported as a risk factor for FTD-spectrum and AD. Recently, using a zebrafish model of A152T mutation, autophagy upregulation therapy by clonidine, rilmenidine, and rapamycin was found to ameliorate the pathology [130]. The FDA-approved agent, bexarotene, was found to augment autophagy in the ischemic brain by a reducing the affected area, and ameliorating behavioral deficiencies in aged transgenic mice harboring human P301L tau. Bexarotoene also restored mitochondrial respiration in P301L-tau neurons [131]. The anti-diabetic drug metformin is a biguanide-type drug that activates AMPK by increasing AMP. Metformin decreases glucose, cholesterol, and triglyceride levels in the blood, but it has both AMPK-dependent (Fig. 4) and – independent effects. Metformin activated PP2A and subsequently reduced tau phosphorylation at sites dependent on PP2A dephosphorylation in both mouse brains and cultured neurons [132]. It is known that okadaic acid, a PP2A inhibitor, markedly augments tau hyper-phosphorylation and NFT formation. However, Son et al. reported that metformin al-


Table 1.

Hamano et al.

Small molecules inducing autophagy that are potentially effective for the treatment of Alzheimer’s disease.

Drugs Possibly Effective for Alzheimer’s disease by Activating Autophagy Autophagy Target

Action

References

Drugs mTORC1 dependent autophagy inducer Rapamycin Rapamycin analogues (CCI-779, RAD001, AP23573) Selective ATP-competitive small molecule (PP242, Torin1, WYE-354, ku-0063794) PI-103, NVP-BEZ235 Metformin, Phenphormin Resveratrol Latrepirdine mTORC1-independent autophagy inducers Lithium Carbamazepine, Valproate Clonidine, rilmenidine 2’5’-dideoxyadenosine Nilvadipine, Verapamil, loperamide, amiodarone Calpastatin, calpeptin

[107, 116, 124, 125]

mTORC1 inhibitor

mTORC1 and 2 inhibitor

AMPK activator

[132]

Inhibits AMPK target mTORC1 Antihistaminic compound (Enhance mTORC1- and Atg5- dependent autophagy)

[120] [123]

IMPase inhibitor (Reduces inositol and IP3) MIP synthase inhibitor Imidazoline receptor agonists (Reduces cAMP) Adenylate cyclase inhibitor (Reduces cAMP) Ca+ channel blocker (Decreases Ca+) Inhibit calpain activation

[118, 126] [122] [130]

[127-129]

Bexarotene

Chemical chaperone Sirtuin-1 activator Unknowns Up-regulates Akt and mTORC1-independent autophagy Enhances lysosome/autolysosome acidification reduce autophagosome accumulation Unknown

Gene therapy AAV/Aβ vaccine lentivirus encoding mouse beclin 1 MicorRNA

increases the clearance of Aβ from the brain Beclin 1 induction, significantly reduce intracellular Aβ Increase the clearance of tau in AD mice

Other autophagy-modulating drugs Trehalose Resveratrol, (analogue RSVA314, RSVA405) SMER 10, 18, 28 An N(10)-substituted phenoxazine Nicotinamide

[119]

[117, 127] [121] [131] [134] [89] [135]

Abbreviations: AMPK: adenosine monophosphate-activated protein kinase; cAMP, cyclic adenosine monophosphate; IMPase, inositol monophosphatase; MIPS: myo-inositol-1- phosphate synthase.

tered Aβ production by activating AMPK via mTORC1 suppression. mTOCR1 suppression upregulated autophagy. Previously, highly augmented γ-secretase activation was observed in autophagic vacuoles, which are new sites of Aβ production [133]. 8.3. Autophagy Regulation by Gene Therapy In addition to pharmacological treatments, gene therapy is another selective method to alter autophagy activity. As it functions in a tissue-directed manner, oral administration of the recombinant AAV/Aβ vaccine effectively removes brain Aβ. Cognition in AD animal models was improved via autophagy enhancement [134]. Beclin-1 is a molecular platform that brings together components regulating the initiation of autophagosome formation. Beclin-1 activation also activates autophagy. In beclin1-/- transgenic mice, significant reduction of beclin 1 reduced neuronal autophagy and subsequently led to neu-

rodegeneration [89]. Lentivirus administration of mouse beclin 1 to the hippocampus and frontal cortex in 6-month-old APP transgenic mice for 8 weeks enhanced Beclin 1 expression and significantly decreased intracytoplasmic Aβ [89]. MicroRNAs (miRNAs) are plentiful, endogenous, noncoding, short essential RNA molecules that negatively regulate target genes posttranscriptionally. Several biological processes are dependent on miRNAs, changes in the profiles of these miRNAs may be biomarkers of neurodegenerative disorders, including AD. Misfolded Aβ or tau protein is degraded via the autophagy pathway to decrease chronic ER stress and enhance cell survival. It was suggested that specific miRNA targets control the autophagy mechanisms. miRNA-mediated alteration of specific proteins involved in autophagy can be a significant therapeutic method. Indeed, miR-132/212 knockout mice exhibited altered autophagy activity, including reduced Atg 5-12, Atg 9a, and increased p62 expression, which increases the size of AVs. Conse-


quently, the total amount of tau, including phosphorylated tau, was also increased. Conversely, treatment with MiR132/212 partially reversed memory deficits and decreased phosphorylated tau [135]. As such, miRNA may be a target for regulating autophagy in AD.

Current Alzheimer Research, 2018, Vol. 15, No. 14 1293 [5] [6] [7]

CONCLUSION Neuronal autophagy is known as an important keystone for delaying aging and many neurodegenerative disorders. In order to use the beneficial aspects of autophagy, the precise roles of autophagy in AD must be clarified. However, our knowledge of the precise pathways of autophagy impairment in AD is lacking. Recent studies targeting autophagy in the nervous system have advanced the development of new treatment strategies. An understanding of the complexities of autophagy and its significant pathways is essential to minimize potential adverse effects. Another important key factor is the timing and duration that activators can be effectively maintained. The reason for failure of autophagy with aging must be elucidated.

[8]

Taken together, autophagy is undoubtedly involved in aging and neurodegenerative disorders, including AD. Autophagy is a double-edged sword because its activation can be cell protective or cell destructive depending on the type and length of exposure. A greater understanding of neuronal autophagy will allow for the development of future therapies for AD, especially for preclinical AD or prodromal AD (ADmild cognitive impairment (MCI)).

[12]

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CONSENT FOR PUBLICATION Not applicable.

[16]

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDEGEMENTS The authors thank Mrs. Aiko Ishida, Akiko Kitade, and Junko Nakane for excellent technical help. We are grateful to Prof Koji Aoki, Department of Pharmacology, University of Fukui, for his helpful advice. A part of this study was supported by JSPS KAKEN Grant Number JP (25460893, 15K08904, 16K09235), JST (AS242Z03676Q), and a research grant from University of Fukui. REFERENCES [1] [2] [3]

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