Autophagy Modulation for Alzheimer’s Disease Therapy

Xi-Chen Zhu & Jin-Tai Yu & Teng Jiang & Lan Tan

Received: 25 February 2013 /Accepted: 9 April 2013# Springer Science+Business Media New York 2013

Abstract Autophagy is an essential and conserved lyso-somal degradation pathway that controls the quality of cy-toplasm by eliminating the intracellular aggregated proteinsand damaged organelles. Autophagy works in mammaliantarget of rapamycin (mTOR)-dependent pathway or mTOR-independent pathway to keep the neuronal homeostasis.Mounting evidence has implicated the importance of defec-tive autophagy in the pathogenesis of aging and neurode-generative diseases, especially in Alzheimer’s disease (AD).It has also demonstrated a neuroprotective role of autophagyin mediating the degradation of amyloid beta and tau whichare major factors of AD. Amounts of molecules function ineither mTOR-dependent pathway or mTOR-independentpathway to induce autophagy, which maybe a potentialtreatment for AD. In this review, we summarize the lateststudies concerning the role of autophagy in AD and exploreautophagy modulation as a potential therapeutic strategy forAD. However, to date, little of the researches on autophagyhave been performed to investigate the modulation in AD;more investigations need to be confirmed in the future.

Keywords Autophagy . Alzheimer’s disease . Mechanism .

Therapy

Introduction

The word “autophagy” (“self-eating”) is the Greek term thatwas coined about half a century ago by Christian Deduve [1].Autophagy functions in the homeostatic process and works asa survival tactic employed by cells to combat against hostilesituations [2]. Autophagy is utilized to clear cytoplasmicconstituents including misfolded or aggregated proteins anddamaged organelles such as endoplasmic reticulum (ER),mitochondria, peroxisomes, and intracellular pathogens.Autophagy includes macroautophagy, chaperone-mediatedautophagy (CMA) and microautophagy. Neurodegenerativedisorders are widely regarded as protein conformational dis-orders which may be regulated by autophagy. An emergingconsensus is that autophagy is a neuroprotective response anddefective autophagy or inadequate autophagy promotes neu-ronal cell death in most neurodegenerative disorders [3].Some studies indicate that autophagy may increase the forma-tion of autophagosome in Alzheimer’s disease (AD) patientsand the deficient autophagy induces the pathogenesis of AD,particularly at the late stage of AD, so it may be a therapy toAD by enhancing autophagy. In this review, we summarizecurrent understanding of autophagy mechanisms in AD anddiscuss how to use current knowledge to find potential thera-peutic approaches.

The Machinery of Autophagy

Autophagy begins with the formation of preautophagosomalstructure that includes part of the cytoplasm and turns into adouble-membrane-limited autophagosome [4]. Autophagy-related genes (ATGs) are indentified, Atg1∼Atg35, whichinvolved in autophagosome formation, and their functionsare considered to be conserved from yeast to humans[5]. The term “autophagic flux” is used to represent the

X.-C. Zhu : J.-T. Yu : T. Jiang : L. TanDepartment of Neurology, Qingdao Municipal Hospital,Nanjing Medical University, Nanjing, China

J.-T. Yu (*) : L. Tan (*)Department of Neurology, Qingdao Municipal Hospital,School of Medicine, Qingdao University, Qingdao, Chinae-mail: yu-jintai@163.come-mail: dr.tanlan@163.com

J.-T. Yu : L. TanCollege of Medicine and Pharmaceutics,Ocean University of China, Qingdao, China

Mol NeurobiolDOI 10.1007/s12035-013-8457-z

dynamic process of autophagy. Autophgaic flux influencesautophagosome formation, maturation, fusion with lyso-somes, subsequent breakdown, and the release of macromol-ecules into the cytosol [6]. The deficiency of autophagy flux inautophagy induction and autophagosome formation or thedegradation of autophagic vesicles (AVs) in the lysosomesall will lead to appearance of AD [6]. At the stage of initiation,the autophagic membrane forms phagophore and then elon-gates. Neurons are especially rich in acid phosphatase-positivelysosomes to be a preferred model in the initial investigationsof autophagy [7]. Postmitotic neurons are more susceptible tothe effects of dysfunctional autophagy because the specificcell compartments (axon, synapse, and dendrite) that are char-acterized by high-energy demand and protein turnover, whichare regulated in autophagic pathway [8]. Phagophore forma-tion is thought as autophagosome precursor or pre-autophagosomal structure which needs the class IIIphosphoinositide3-kinase (PI3K) Vps34, which functionswith Beclin-1, Atg14, and Vps15 in a large macromolecularcomplex. Atg5, Atg12, Atg16, focal adhesion kinase, family-interacting protein of 200 kDa which interacts with Atg1 andthe mammalian ortholog of Atg13 are other proteins in-volved in the early stages of autophagy [9]. Quantities ofcellular organelles have been regarded as sources of theelongating autophagosome membranes [10]. Recently, someevidences suggest that soluble N-ethylmaleimide-sensitivefactor attachment protein receptor (SNARE) proteins medi-ate homotypic fusion of phagophore precursors and enlargethe size of structures. The process makes contributions toelongation of the autophagosome precursor membranes andrecruitment of proteins that enable maturation intophagophores [11] (Fig. 1). Two ubiquitination-like reactionsare critical for autophagosome formation in the elongationof membranes. One is Atg12 conjugated to Atg5 by Atg7(which is similar to an E1 ubiquitin-activating enzyme) andAtg10 (which is similar to an E2 ubiquitin-conjugatingenzyme). The complex of Atg5–Atg12 conjugatesnoncovalently with Atg16L1 and associates with phagophoresbut dissociates from completed autophagosomes. Anotherreaction is that microtubule-associated protein 1 light chain3 (LC3)-I is conjugated to the lipid phosphatidylethanolamineby Atg7 (E1-like) and Atg3 (E2-like) to form LC3-II [9].Autophagosome formation can appear in the cytoplasm atrandom sites. Autophagosomes are transported along micro-tubules towards the microtubule-organizing center where ly-sosomes are abundant to increase the chances for lysosomalfusion [12]. Numerous of SNARE proteins including vesicle-associated membrane protein 8 and Vti1B are believed to berelated to regulating heterotypic fusion betweenautophagosomes and the lysosomal compartment [10, 13].Because of the quickly fusing with lysosome makes the pro-tein turnover processes go smoothly in autophagy-lysosomepathway, so it is rare to see AVs in the normal brain [14].

The Autophagic Pathway

There are three basic forms of autophagy: macroautophagy,microautophagy, and CMA. They are primarily different in theway of transporting cytosolic components to lysosomes [1, 15].

Compared with macroautophagy, little is currentlyknown about microautophagy and CMA. The most familiarkind of autophagy is macroautophagy. Lysosomal hydro-lases the limited membrane of autophagosome, then thefusion of lysosomes accesses to the sequestered cargo tomake its complete degradation. The limited membranes aregiven rise by coordinated recruitment of lipid-modifyingenzymes and proteins at the autophagosome formation[16]. There are two pathways in macroautophagy to induceautophagy: mammalian target of rapamycin (mTOR)-depen-dent and mTOR-independent signaling pathways.

mTOR-Dependent Signaling Pathways

The primordial negative regulator of autophagy is mTOR.mTOR links to starvation-induced autophagy via activation ofmTOR target Atg13, UNC51-like kinase (ULK)1 and ULK2[17]. Of the mTOR complex (mTORC)1 and mTORC2, onlymTORC1 inhibits autophagy directly with stimulating proteinsynthesis and mitosis [18]. Nutrient starvation inhibits mTOR,thus leading to enhanced autophagosome biogenesis. RagGTPases are required components in mTORC1. Mammalshave four Rag proteins, Rag A to Rag D. Rag A and Rag Bform a component, and Rag C and Rag D make up anothercomponent. Amino acids induce Rag A/B loading with GTPand Rag C/D loading with GDP, and finally promote theactivity of mTORC1 [19]. The tumor suppressor protein tuber-ous sclerosis complex (TSC)2 inhibits mTOR [20]. TSC1/2 iscenter to many pathways, such as growth factor signaling.Growth factor stimulates PI3K which inhibits TSC1/2. WhenTSC1/2 is inhibited, it will enable the ability of mTORC1.Several immunological relevant accessory molecules inducethe activation of mTOR. Tumor necrosis factor-α (TNF-α), aproinflammatory cytokine, is the upregulators in mTORC.Proinflammatory cytokines induce the inhibitor of κB kinaseβ (IKKβ) which phosphorylates TSC1 leading to TSC1/2inhibition, and IKKβ also downregulates nuclear factor κBleading to the resistance to inflammation stress [21]. It isnotable that CD28 is a potent activator of PI3K to upregulatemTOR1 in T cells, and interleukin (IL)-2 and IL-4 are alsopotent activators of mTORC1 activity via PI3K activation [22,23]. IL-12 and interferon-γ activate mTOR in CD8+ T cells[24]. Protein kinase B (Akt) signals is a TSC1/2-independentpathway by phosphorylating and causing the dissociation fromraptor of the proline-rich Akt substrate of 40 kDa, a mTORC1inhibitor. TSC1/2 is a GTPase-activating protein. Tumor sup-pressor, p53, is one intracellular protein that regulates mTORactivity. Transcription of autophagy-inducing genes, sestrin2,

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and dram are activated by nuclear p53 under condition ofcellular stress [25]. Auophagy is inhibited by cytosplic p53through direct inhibition of AMP-activated protein kinase(AMPK) [26]. The canonical Wnt pathway also provokes theactivation of mTORC1 by modulating TSC1/2. The Wnt path-way inhibits glacogen synthase kinase (GSK)-3β, and finallyphosphorylates and supports TSC2 activity (Fig. 2). TheULK1/2–Atg13 complex is inhibited by mTORC1 throughphosphorylation. Dephosphorylation of ULK1/2 activates itskinase activity: when mTORC1 is inactive and ULK1/2,Atg13, and the fungal immunodulatory protein 200 will bephosphorylated [27]. Active ULK1/2–Atg13 complex locatesat the isolation membrane [28]. The isolation membrane is anunusual double layered phospholipid bilayer which originsfrom mitochondrial [29] or endoplasmic reticular [30]. WhenmTORC1 is triggered in this pathway, it will increase formationof autophagosomes which decomposes the aggregated, patho-genic or hyperphosphorylated proteins, so as to release AD.

mTOR-Independent Signaling Pathways

Autophagy can be induced by compounds which decreaseinositol or inositol 1,4,5-trisphosphate (IP3) levels in thispathway. When this IP3 receptor is inhibited, the IP3 leveldecreases, then autophagy will reduce, so it could be a targetto treat AD to activate IP3 receptors to induce autophagydecreasing the level of Aβ and tau [31]. Another mTOR-independent signaling pathway is regulated by the L-typeCa2+ channel modulators which affect intracytosolic Ca2+

levels in inducing cyclic AMP (cAMP), so as to regulatephospholipase C-ε activity through the Epac pathway.Epac is mainly the target of cAMP, protein kinase A(PKA), and a guanine nucleotide exchange factor. WhenEpac pathway is triggered, a small G-protein can beactivated to induce IP3, then upregulates autophagy, itwould be another pathway to treat AD [32]. mTOR-independent signaling pathway can directly influencecAMP–Epac–PLC-ε–IP3 pathway.

Autophagy in Maintaining Neuronal Homeostasis

Autophagy plays a vital role in remaining cellular metabolicbalance, controlling cellular quality, remodeling cellular,influencing in cellular death and survival and guardinggenome [1, 33–36]. Different from other cell types, neuronsare post-mitotic and highly dependent in active signaling inthe axons and dendrites in the endo-lysosomal pathway.When failed to remove toxic components, it will requireeffective protein degradation to control the cell survival.Abnormal proteins accumulate in Atg5−/− cells and formaggregates and inclusions. This suggests that preventingthe accumulation of abnormal proteins mainly depend onthe continuous clearance of diffusing cytosolic proteins inbasal autophagy, and it can ruin neural function and finallycause neurodegeneration. It has also been reported thatneurodegeneration can be caused by the loss of Atg7 (agene essential for autophagy) [37].

Fig. 1 The machinery of autophagy and its role in AD. At initiation,many cellular organelles are sources of the elongatin autophagosomemembranes. Soluble N-ethylmaleimide-sensitive factor attachmentprotein receptor (SNARE) proteins mediate homotypic fusion ofphagophore precursors and enlarge the size of structures. The initiationstage includes conjugation of autophagy-related genes (ATGs), and in

the elongation stage involves conjugation of the LC3 protein.Autophagic vesicles (AVs) are increased when under the stress ofmutant amyloid beta (Aβ) precursor protein (APP) and injured organ-elles. When the maturation and degradation of autophagosomes areblocked, it will cause inhibiting autophagic pathway and finally lead tothe increase of intracellular level of Aβ and tau leading to AD

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Autophagy and Aging

Aging is a vital factor in AD. Neuronal autophagic lysosomalsystem (ALS) may turn into the pathological and deleteriousstate from the functional and protective state during brainaging [38]. The aging-associated defect is changed inmacroautophagy and CMA when loop-mediated isothermalamplification 2a reinstates to normal levels, and it also con-tributes to reducing oxidized proteins, polyubiquitinated pro-teins, and apoptotic cells in liver [39]; it indicates thatinsufficient autophagy affects aging.

The theory of increased autophagy delays aging, extendslongevity, and improves AD origins from the seminal ob-servation that inhibiting the insulin-like growth factor path-way leads to autophagy in Caenorhabditis elegans andinhibiting autophagy by mutation of essential ATGs wouldshorten longevity [40]. Autophagy abolishes the life-span-extending effects of rapamycin in all investigated species forknockout or knockdown of ATGs [41]. When inhibitingTOR, it will pharmacologically or genetically extend lifespan in yeast; it indicates that autophagy affects aging. S6kinases (S6K) deletion induces the activation of AMPK anddecreases longevity in AMPK-deficient nematodes [42].Although AMPK is a potent activator of autophagy to be a

potential target to treat AD [9], it remains unknown whetherS6K deletion leads to autophagy and whether the life-spanextension of S6K-deficient animals is caused by autophagy[17]. Since many related researchers have shown amounts ofcompounds regulating autophagy both in aging and AD, soit is promising to find the optimal strategy to improvehealthy aging and AD.

Autophagy in Alzheimer’s Disease

AD is the late-onset, progressive, neurodegenerative disorder,and it is the most familiar cause of dementia in aging people.Aging is a complex process of mitochondrial and macromo-lecular damage. Aging may appear when the damage exceedsthe continual repair and turnover [43]. Defective lysosomalclearance of autophagic substrates and impaired autophagyinitiation are the two independent evidences to implicatedautophagic failure in AD [44]. The pathogenic events associ-ated with AD and related neurodegenerative diseases may beunderscored by the correlation between perturbed autophagyand aging. In the early stage of AD, AVs are increased whenunder the stress of APP and injured organelles. Conversely, inthe late stage of AD, the maturation and degradation of

Fig. 2 The regulation of mammalian target of rapamycin (mTOR).mTORC1 can be regulated by amino acids, growth factor, inflamma-tion mediators, hypoxia, DNA damage, and the Wnt pathway, and then

mTORC1 inhibits the formation of autophagy which works in decreas-ing the level of Aβ and tau

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autophagosomes are blocked by the microtubule disruptioncaused by tau hyperphosphorylation. Meanwhile, the lyso-some enzyme dysfunction also inhibits autophagosome–lyso-some fusion in AD. All these indicate that autophagic pathwaycontribute to the increase of Aβ and tau [45] (Fig. 1).

Aβ-Dependent Roles for Autophagy in AD

The key protein in AD pathology is amyloid beta (Aβ)precursor protein (APP). APP, one family of conserved type1 membrane proteins has APP-like protein (APLP)1 andAPLP2 [46]. Recent studies indicate that APLP decreaseswith stress, just as proteasome inhibition in neuronal cells[47]. The glycosylated form of APP in the trans-Golginetwork (TGN) after synthesizing APP is transported intothe plasma membrane [48]. In tumor necrosis factor αconverting enzyme or a disintegrin and metalloproteinase10 (ADAM10), the aspartyl protease at the cell surface isregarded to work in α-secretase, then induces α-cleavage ofAPP in luminal/extracellular to generate a large solubleterminal fragment from cells and the remaining membraneattaches to the C-terminal fragment (CTF) [48, 49]. Withinearly endosomes, inner cell surface, APP cleavages at amore distal site controlled by β-site-APPcleaving enzyme(BACE and β-secretase) to generate a soluble APP fragmentand β-secretase-cleaved carboxyl-terminal fragment(βCTF) with all Aβ peptide along the luminal/extracellular.At low pH, transmembrane aspartyl protease has optimalactivity that distributes predominantly to endosomes, atwhich βCTF is generated and additional BACE can bediscovered in the TGN [50–52]. An intramembrane γ-cleavage generates Aβ from βCTF to produce predominant-ly a 40-mer peptide (Aβ40) and small amounts of a 42-merpeptide (Aβ42). The γ-secretase complex combined withproteins presenilin, nicastrin, anterior pharynx defective-1and presenilin enhancer 2 induces the γ-cleavage of APP[48, 53]. The locations of the complex have been confirmedat the membrane, in early and late endosomes, AVs andlysosomes [54, 55]. In endosomal–lysosomal compart-ments, the soluble Aβ in intracellular pool rises substantial-ly before Aβ is deposited extracellularly and even in theheavily plaque-laden AD [56, 57]. The accumulation ofextracellular Aβ will progressively lead to the extracellularsenile plaques which are compromised with a dense core ofAβ surrounded with dystrophic neuritis, and all these indi-cate that Aβ is a causal factor in AD [58].

Many studies have explored the relationship between relat-ed molecules in autophagy and the clearance and generationof Aβ, and they indicated that autophagy plays a vital role inmodulating Aβ. The marker of macroautophagy, Atg5,Atg12, and LC3, are found to be associated with plaque andtangle pathologies in AD [59]. Aβ could be an autophagicsubstrate and be subjected to autophagy-mediated clearance,

since the morphological evidence reveals that APP and Aβpeptides can co-localized with LC3-positive autophagosomesin an APP-overexpresing cell line and ADmousemodels [60].Cells depleted of Beclin-1, an initiator of autophagy, showsignificant accumulation of Aβ, APP, and CTFwith inhibitionof autophagosome turnover [61]. It has been confirmed bydownregulation of Beclin-1 in an APP transgenic mousemodel which shows the accumulation of both intraneuronaland extracellular Aβ deposition and combined with markedneurodegeneration [62]. All these indicate that autophagy isnecessary for the removal of detrimental Aβ peptides andaggregates. Autophagy can enhance the translocation of γ-secretase complexes from an endosome/ER pool toautophagosomes [54]. Presenilin 1 (PS1), the γ-secretase coresubunits, functions as an ER chaperone to assist with the N-glycosylation of the v-ATPase V0a1 subunit [63]. The de-crease of PS1 would lead to the accumulation of immatureunglycosylated v-ATPase which is needed in the acidificationof autolysosomes/lysosomes, the abnormal accumulation oflate-stage autophagosomes with undigested contents can bediscovered in PS1-null cells, just like the ultrastructures pres-ent in AD neurons [64]. The autophagy-mediated clearance oftelencephalin and α-synuclein (α-syn) is impaired in neuronsfrom PS1−/− mice [65, 66]. All these indicate thatdownregulation of the autophagic system may induce ADpathogenesis and neuronal deficits.

Tau-Dependent Roles for Autophagy in AD

Compared with tau in normal brain, AD tau lies in bothsomatodendritic and axonal compartments in oxidized, C-terminally proteolyzed and hyperphosphorylated forms [67].There are at least 30 phosphorylation sites in tau isoforms,and in normal condition most of them are in the de-phosphorylated form, and some of them are usually phos-phorylated in AD [67]. Among several identified tau kinasesprotein, phosphatase-2A (PP2A) seems to be the major tauphosphatase [67]. In AD pathogenesis the microtubule-associated protein tau affects the accumulation of neurofi-brillary tangles (NFT). Abnormal hyperphosphorlation oftau can be caused by the dysfunction of PP2A, and it willinhibit the ability of microtubule assembly and promote self-aggregation, and finally lead to the pathogenesis of AD [68].In AD brain, the functions of tau depend on the degree ofphosphorylation, and there are four- to eightfold of abnor-mal hyperphosphorylated tau in deficient brain [68]. Thedecrease in its turnover resulted from hyperphosphorylationcaused by the increasing level of tau in AD brain [69]. Thetypical hallmark in AD is NFT. NFT polymerized by tau areinert, and it neither combines with tubulin nor promotesmicrotubules assembling [70]; 40 % of the abnormalhyperphosphorylated tau is in the cytosol without polymer-izing into paired helical filaments/NFT in AD brain [68].

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The AD cytosolic abnormally hyperphosphorylated tau in-hibits assembling and disrupting microtubules [71].

There are two pathways to complete clearance of tau, theubiquitin-proteasome system (UPS) and the ALS. The short-lived nuclear and cytosolic proteins are selectivelydecomposed by the UPS, and the long-lived proteins andorganelles are cleared by the ALS [72]. The present studyindicates that mild oxidative stress requires UPS activity toregulate APP metabolism with a simultaneous reduction inthe autophagy/lysosome system [73]. The aggregated neu-rotoxicity downstream of tau is essential in AD, the Aβ-elicited neurodegeneration [74]. Full-length tau is degradedby macroautophagy, while TauRDΔK280, a truncated taucontaining pro-aggregated repeat domains and functions as aseed for initiating tau aggregate formation, is translocated tolysosomes through CMA machinery. Caspase-cleaved tauwhich is prone to aggregate and induce neurotoxicity thannature tau in AD brain, and it is degraded through autophagyin a faster turnover rate than full-length tau. These suggest thatdifferent tau fragments might be degraded in differentautophagic pathways and autophagy-mediated degradationof tau may be important to keep the homeostasis of intracel-lular tau [75–77].

The therapeutic strategies which affect tau neurotoxicitymay be a potential approach to develop next-generation anti-AD drugs, and regulating mTOR may be a valid therapeuticapproach to AD [78]. Autophagic pathway may degrade theoverexpressedmutant tau and Tu aggregates in N2a cell model,and the activated autophagy inhibits tau aggregation and de-creases cytotoxicity [72]. The intraneuronal NFT are caused bytau hyperphosphorylation and neuronal apoptosis. The defi-cient ALS leads to the over expression of formation of Aβ,tau oligomers, and insoluble aggregates. The mTOR pathwayinducing ALS functions in keeping protein homeostasis, so themTOR-related agents in ALS may be a potential therapeutictarget for AD [79]. It is suggested that autophagy can partiallymediate the intracellular clearance of soluble tau and aggregat-ed NFTs. Tau-enriched granules are detected in lysosomes ofneurons from human brains [80]. Rapamycin, an autophagyinducer, can reduce aggregation of tau and tau-induced neuro-toxicity to treat tau-overexpressingDrosophila [81].Methyleneblue (MB) can decrease tau in autophagic pathway by alteringthe amounts of LC3-II, cathepsin D, Beclin-1, and p62, andMB can be used as a therapeutic agent in neurodegenerativediseases [82]. Phospholipase D1 (PLD1), the downregulator ofVps34, induces autophagosomes maturation, and blockade ofPLD1 activity leads to higher levels of tau and p62 aggregatingin the brain; all these indicate that autophagy plays a vital rolein the degradation of tau [83].

Even though many studies have indicated the function ofautophagy in ameliorating the pathology of AD, some re-searchers suggest that abnormal regulation of autophagy isan unsuccessful rescue mechanism instead of being protective

to AD pathogenesis [84]. Emerging data supports that theabnormal regulation of autophagy may function in the patho-genesis of neurodegenerative disorders [44, 85]. Autophagymay be regarded as a double-edged sword in AD: it canfunction as either a gatekeeper of cell survival or a culprit ofneurotoxicity [85]. Motivated autophagy may induce overaccumulation of autophagosome in the brain neurons and itmay be a major source of intracellular Aβ in AD [54]. Itremains a significant issue that if cell homeostasis can bedisturbed by abnormal activation of autophagy–lysosomefunction, over-activating autophagy may lead to the death ofautophagic cell [45]

Autophagy Modulation as a Potential TherapeuticTarget for AD

One of the most rapidly growing fields is autophagy inbiomedical studies. The latest studies suggested that thelevel of autophagy are regulated and autophagic processfunctions in some processes such as development, aging,and several major neurodegenerative diseases, particularlyAD [5].

Targeting the mTOR-Dependent Pathway

Amounts of studies show that deficient autophagy inducesthe pathogenesis of AD, particularly at the late stage of ADto alter the autophagy–lysosomal pathway and it may be atherapy to AD. A potential treatment to neurodegernativediseases is stimulating autophagy [86]. The most familiardrug used to treat AD in mTOR-dependent pathway israpamycin. Rapamycin and the immunophilin FK506-binding protein 12 form a complex to stabilize the raptor-mTOR association and decrease the activity of its kinase toinduce autophagy, so as to reduce the production of Aβ [32,54]. Latest study suggests that rapamycin decreases tauphosphorylation at Ser214 (pS214) via regulation PKA,and the decrease in pS214 may inhibit phosphorylation oftau by other kinases [87]. For instance, rapamycin and itsanalogue temsirolimus increase autophagy to protect againstneurodegeneration in AD models in Drosophila and mice,rapamycin improves the ability of learning and memory anddecreases Aβ and tau pathology [88, 89].

There are many substances functioning in mTOR-dependent pathway, some of them have been researched inother neurodegenerative diseases, but the exact influence onAD still needs to be confirmed. Small molecular enhancersof rapamycin 28, may induce autophagy and reduce APP-CTF (apparent EC 50 of −20 μM) and Aβ peptide (apparentEC 50 of −10 μM), which may be a potential target to treatAD and other proteinopathies [90]. The autophagosome–lysosome fusion could be inhibited at the late stage of AD

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[54]. TauDeltaC and FL-tau are significant in degradationsystems to clear pathological proteins, and it may be devel-opment therapeutic strategies [75]. Beclin-1 is involved inthe initiation of autophagy, some study showed that theactivation of Beclin-1-dependent autophagy can stop neuro-nal cell death, while inhibition of Beclin-1-dependentautophagy can hastened cell death, all these suggest thatincreasing Beclin-1-depent autophagy may be preventive inAD [91]. Cystain C (CysC) plays an important role in theinhibition of Aβ oligomerization and fibril formation bybinding both Aβ40 and Aβ42. CysC also induces autophagyin mTOR pathway; recent study indicates that regulation ofCysc may be an alternative therapeutic strategy in dementiawith amyloid deposits, such as AD [91]. Parkin is an E3ubiquitin ligase involved in degradation of proteins viaautophagy pathway, its insolubility co-localization with p-tau and Aβ42 and the ability of compromising the cellautophagic clearance indicate the promising function ofparkin in autophagic clearance in AD [92]. Sirtuin 1(Sirt1) is a class III histone deacetylase that plays role inprotecting neurons via regulating autophagy in neurodegen-erative disorders, such as AD, so the Sirt1 activation may bea potential target for AD [93].

Targeting the mTOR-Independent Pathway

Autophagy is increased by the mood stabilizer lithium inmTOR-independent pathway through inhibiting inositolmonophosphatase (IMPase) and lowering inositol and IP3levels [10]. Decreasing this enzyme inhibits inositol recyclingwhich causes depleting cellular inositol and decreasing thephosphoinositol cycle [10]. The autophagy-inducing ability oflithium was due to inhibition of IMPase, leading to depletionof cellular inositol and inhibition of the phosphoinositol cycle.Lithium increases the clearance of aggregate-prone proteins,for instance α-syn and huntingtin (htt) [31]. Scyllo-inositol(SI), an endogenous inositol stereoisomer, inhibits Aβ fibrilformation. SI can reduce the amount and size of AVs, andautophagic deficiencies in AD may be reduced by SI–Aβinteractions [94]. Trimethyltin (TMT), a triorganotin com-pound, damages the limbic system, and at least lithium canmodify TMT neurotoxicity in vitro [95].

There are some compounds functioning in mTOR-independent pathway to induce autophagy, and they have beenidentified to be useful to modulate other neurodegenerativediseases in mTOR-independent pathway, so they may be usedas the promising targets to decrease the level of Aβ and tau totreat AD, but more studies should be performed to investigate it.L-690,330, carbamazepine and sodium valproate can induceautophay by inhibiting IMPase and decrease inositol and IP3,and it can induce clearance of the accumulation and toxic effectsof aggregation-prone mutant proteins in cell models and protectagainst neurodegeneration in vivo [32]. Fourteen compounds

identified by FDA-approved medications can increaseautophagy in an mTOR-independent pathway [96]. These com-pounds play roles in Ca2+–calpain–Gsα and cAMP–Epac–PLC-ε–IP3 mTOR-independent pathways [10]. Nivadipine,clonidine, and rilmenidine are indentified active agents and theyare used to treat hypertension patients [10]. These drugs playroles in imidazoline II receptors which are widely in brain, α2-adrenoreceptors decrease G protein signaling pathways to re-duce adenyl cyclase and link to reduce cAMP to induceautophagy [10]. When under abnormal conditions, cytosolicphospholipasesA (2) (cPLA2) may protect neuronal functionsby attaching to ionotropic glutamate receptors, then Ca2+ com-binedwith calpain and possible redox-based S-nitrosylation, andthen works in Ca2+–calpain–Gsα and cAMP–Epac–PLC-ε–IP3mTOR-independent pathways and finally induces autophagy, socPLA2 could be a potential therapeutic manner to decrease thedetrimental effects of AD [97]. The Ca2+ channel blockers(verapamil, loperamide, amiodarone, and nimodipine),calpastain, and calpeptin inhibit calpain. 2′5′-dideoxyadenosineinhibit adenylyl cyclase inhibitor to reduce cAMP, NF449 in-hibits Gsα inhibitor, they will finally lead to the induction ofautophagy (Fig. 3). Trehalose, a disaccharide present in manynon-mammalian species, is an activator of mTOR-independentautophagy and enhances the clearance of the autophagy sub-strates mutant α-syn and htt [98].

Combination Therapy

It may be a potential treatment strategy to use mTOR-dependentpathways and mTOR-independent pathways at the same time.Compared with either treatment alone using two kinds of drugsmay lead to induction of autophagy and may decrease either thedose of treatment or the likely adverse effects.

Using both rapamycin and lithium are the most familiarcombination therapy to induce autophagy. Lithium can in-duce autophagy in mTOR-independent pathway, but it hasopposite influences on autophagy in mTOR-dependent path-way. Lithium inhibits GSK-3-dependent phosphorylation ofTSC protein TSC2 which can activate mTOR signaling [99].In order to neutralize the lithium influences on GSK inhibi-tion, rapamycin is used to induce autophagy by inhibitingmTOR and downregulating IP3 levels. Combination therapywith lithium and rapamycin can be used against cell deathand clear more mutant htt compared with using one kind oftherapy [32]. However, the combination therapy is challeng-ing and remains investigated.

Pharmacological Modulation of Autophagy by UnknownMechanisms

There are many other molecules improving AD; however, thedetailed mechanisms remain to be explored. Nicotinamide is ahistone deacetilasa inhibitor to upregulate Atg12, so as to

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improve AD by activating autophagy [5]. There are severalmolecules possibly downregulating autophagy-related genes toinhibit autophagy, galanthamine (agonist of the nicotinic), hy-drochloride (acetylcholine receptor), and ghrelin (natural ligandfor the growth hormone secretagogue receptor) [100]. In theearly AD, the downregulation of autophagy may reduce theimpaired energy metabolism, and hesperetin or hesperidin maytreat AD [101]. Autophagy is necessary to neuroprotectioninduced by mild ER stress, and ER preconditioning may be apotential therapeutic manner in neurodegenerative diseases[102]. Isorhynchophylline, a tetracyclic oxindole alkaloid fromChinese herbal medicine Uncaria rhynchophylla, Jacks(Gouteng in Chinese) and in East Asia it has been utilized totreat neurological diseases, and IsoRhy-induced autophagy ismTOR-independent pathway but rely on Beclin-1, so oxindolealkaloid derivatives may induce autophagy in neuronal cells andmay protect neurodegenerative diseases [103]. Orally adminis-tered glucosamine could prevent neurodegenerative diseasesand promote antiaging effects. Glucosamine (GlcN) induceLC3 and LC3-positive AVs, degradation of polyglutamine,and ubiquitinated proteins in cytosol. GlcN functions inmTOR-independent pathway for it has not changed the level

of S6 and 4E-BP1 proteins. But the comprehensive mechanismremains unclear [104]. A recent study suggest that utilizinglongitudinal measures of cholesterol measures (total cholesterol,27-OHC, and 24-OHC) and longitudinal measures of cognitivefunction and assessment of white matter integrity with MRI andamyloid deposition using PET ligands is greatly needed, thenew tools may indicate that if AD is a “brain lipid disorder”, thatcould possibly be prevented or treated by modifying brain lipidmetabolism and production of amyloidogenic peptides [105].Some researchers observed that latrepridine (an anti-histamine)promote the removal of α-syn protein aggregates in vivo, fur-thermore, latrepridine reduced GFP-Aβ42 in wild-type com-pared with the Atg8Δ mutant, while latrepirdine treatmentattenuated Aβ42-induced toxicity in wild-type cells but not inthe Atg8Δ mutant; all these indicate a novel mechanism ofaction for laterpirdine in inducing autophagy and reducingintracellular levels of GFP-Aβ42 [106]. S-adenosyl methionine(SAM) functions in multiple pathways to keep neuronal homeo-stasis, several are in age-related neurodegeneration and AD.Dietary supplementation of transgenic mice with SAM canmaintain cognitive performance, acetylcholine levels, oxidativebuffering capacity, and phosphatase activity and reduce calcium

Fig. 3 Targeting the mTOR-independent pathway. Autophagy can beinduced in mTOR-independent pathway via decreasing IP3 levels,inhibiting IMPase activity or reduce inositol (INs). Another mTOR-independent signaling pathway is regulated by the L-type Ca2+ channelmodulators which affects intracytosolic Ca2+ levels to induce cyclicAMP (cAMP) to regulate phospholipase C-ε (PLC-ε) activity throughthe Epac pathway. When Epac pathway is triggered, a small G-protein(Rap2B) can be activated to induce IP3. mTOR-independent signalingpathway can directly influence the cAMP–Epac–PLC-ε–IP3 pathway.

Lithium and SI functions via the first pathway, and Nivadipine, cloni-dine, rilmenidine, and cPLA2 are regulated in Ca2+–calpain–Gsα andcAMP–Epac–PLC-ε–IP3 mTOR-independent pathways. L-690,330,sodium valproate, carbamzepine, verapamil, loperamide, amiodarone,nimodipine, and 2′5′-dideoxyadenosine also function in mTOR-inde-pendent pathway, but the clinical effects need to be confirmed by moreresearches. When autophagy is activated by these substances, the levelof AD hallmarks, Aβ and tau, will decrease

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influx, endogenous PS1 expression, gamma-secretase activity,and levels of Aβ and phospho-tau. All these indicate that SAMcan modulate the time course of AD neuropathology [107].Some researchers examined the changes in amyloid pathologyin the hippocampus and neocortex following three anti-inflammatory treatments aimed at reducing the amyloid burdento investigate the relation between amyloid neuropathology andinflammation, they concluded that changing the activity of glialcells can lead to both a decrease of the amyloid burden anddetrimental changes, maybe caused by the interplay between theactivation levels of astrocytes and microglial cells; these showthe possibility of potential anti-AD treatments on inflammation[108]. Some study indicates the benefits of aerobic physicalexercise on synapse, redox homeostasis, and general brain func-tion demonstrated in the 3xTg-AD mouse, further supports thevalue of this healthy life-style against neurodegeneration [109].Synapses are ultrastructural sites for memory storage in brain,and synaptic damage is the best pathologic correlate of cognitivedecline in AD, thus the mitochondrial NH-2-derived tau peptidedistribution may exacerbate the synapse degeneration occurringin AD and support the in vivo NH-2 tau cleavage inhibitors asan alternative drug discovery strategy for AD therapy [110].Mitochondrial functions as various aspects in AD, such assustain homeostasis, and mitochondrial could potentially servesas AD therapeutic targets [111].

To date, many studies have recovered the potential ther-apy of autophagy to many neurodegenerative diseases, suchas hunting disease. However, limited attention of autophagyhas been paid to AD, even though autophagy plays a vitalrole in removing defective organelles and potentially toxicproteins. These studies in other neurodegenerative diseaseshave indicated the influence on relieving the pathology inautophagic pathway, and it indicates the promising future inAD, while more studies needs to be performed to verifica-tion the hypothesis.

Since AD is a disease caused by various factors, treatingAD by a multi-factor therapy may be a potential method.The combination of therapy targeted in other aspects of ADpathogenesis and autophagy-medicated therapy may be mo-re effective to relive the pathology of AD, while moreresearches need to be done to indicate the hypothesis.

Concluding Remarks and Future Perspectives

Autophagy is necessary in mammalian health, as it keepsneuronal homeostasis and the balance of nutrient and energyhomeostasis which is necessary in delaying aging and ADprocessing. Many molecular related to autophagy in mTOR-dependent pathway or mTOR-independent pathway caninfluence the clearance of Aβ and tau, and this indicatesthat autophagy plays a vital role in keeping homeostasis inAD neurons. Autophagy regulates AD in mTOR-dependent

pathway or mTOR-independent pathway, and many relat-ed compounds work in the two ways to affect autophagy.These compounds may be potential targets to release AD.However, we have no complete knowledge of the precisemolecular mechanisms of autophagy abnormality. It re-mains an issue about the precise role of autophagy indiseases, and it is still a challenge in neurodegenerativediseases to indentify the cellular events. The ALS couldbe the foundation to develop therapeutic strategiesagainst AD and some other neurodegenerative disease.Understanding the precise mechanisms of autophagy andthe specific role in different stages of neurodegenerativediseases will be an important step in developing thera-peutic approaches.

Above all, we sincerely hope these researches of autophagymay help to open up avenues to find the ideal manner inautophagy to treat AD and related neurodegenerative diseaseswhich have better efficacy and less side effects.

Acknowledgments This work was supported in part by grants from theNational Natural Science Foundation of China (81000544, 81171209),the Shandong Provincial Natural Science Foundation, China(ZR2010HQ004, ZR2011HZ001), and the Shandong Provincial Out-standing Medical Academic Professional Program.

Conflicts of interest The authors declare no conflicts of interest.

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