Neurobiology of Aging 35 (2014) 941e957
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Review
Autophagy in aging and neurodegenerative diseases: implications for pathogenesis and therapy Chen-Chen Tan a, Jin-Tai Yu a, b, c, **, Meng-Shan Tan b, Teng Jiang c, Xi-Chen Zhu c, Lan Tan a, b, c, * a b c
Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, Qingdao, China Department of Neurology, Qingdao Municipal Hospital, College of Medicine and Pharmaceutics, Ocean University of China, Qingdao, China Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Qingdao, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 July 2013 Received in revised form 17 November 2013 Accepted 19 November 2013 Available online 28 November 2013
Neurodegenerative diseases, such as Alzheimer’s disease Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis, share a common cellular and molecular pathogenetic mechanism involving aberrant misfolded protein or peptide aggregation and deposition. Autophagy represents a major route for degradation of aggregated cellular proteins and dysfunctional organelles. Emerging studies have demonstrated that up-regulation of autophagy can lead to decreased levels of these toxic aggregateprone proteins, and is beneficial in the context of aging and various models of neurodegenerative diseases. Understanding the signaling pathways involved in the regulation of autophagy is crucial to the development of strategies for therapy. This review will discuss the cellular and molecular mechanisms of autophagy and its important role in the pathogenesis of aging and neurodegenerative diseases, and the ongoing drug discovery strategies for therapeutic modulation. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Autophagy Aging Alzheimer’s disease Parkinson’s disease Huntington’s disease Amyotrophic lateral sclerosis Lysosomal storage disorders Therapy
1. Introduction Autophagy (which means “self-eating”) is a lysosome degradation pathway by which cells capture intracellular proteins, lipids and organelles, and deliver them to the lysosomal compartment where they are degraded (Levine and Klionsky, 2004; Levine and Kroemer, 2008). It plays an important homeostatic role in cells and keeps the metabolic balance between synthesis, degradation, and subsequent turnover of cytoplasmic materials in stressful environment. Autophagy is classified into 3 types: macroautophagy, chaperone-mediated autophagy (CMA), and macroautophagy (Cuervo, 2004). Increasing studies have observed that autophagic vacuoles (AVs), the general term for autophagy-related vesicular structures, are abundant in neurons in an increasing * Corresponding author at: Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No. 5 Donghai Middle Road, Qingdao, Shandong Province 266071, China. Tel.: þ86 532 8890 5659; fax: þ86 532 8890 5659. ** Alternate corresponding author at: Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No. 5 Donghai Middle Road, Qingdao, Shandong Province 266071, China. Tel.: þ86 532 8890 5659; fax: þ86 532 8890 5659. E-mail addresses: [email protected] (J.-T. Yu), [email protected] (L. Tan). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.019
number of neurodegenerative disorders that are manifested by the presence of pathogenic proteins. Moreover, in the context of neurodegenerative disorders, the view that induction of autophagy is a neuroprotective response and that aberrant autophagy promotes neuronal cell death in most of these disorders has been widely accepted. Given these observations, autophagy as a potential therapeutic target in aging and neurodegenerative diseases is currently receiving more and more attention. In this review, we will focus on aging and neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and lysosomal storage disorders (LSDs), after briefly reviewing the mechanisms and function of autophagy. Furthermore, we also discuss how current knowledge about autophagy could be harnessed to open exciting new therapeutic perspectives for aging and neurodegenerative disorders. 2. The machinery of autophagy The proteins that are encoded by autophagy-related-genes (ATGs), ATG1-ATG35, organize into functional complexes that mediate the following steps in the autophagic processes: initiation,
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elongation, maturation and fusion, and degradation (Table 1) (Fig. 1). The process begins with the formation of a preautophagosomal structure or phagophore, which is probably derived from the lipid bilayer of the plasma membrane, including endoplasmic reticulum, golgi apparatus, plasma membrane, or mitochondria (Axe et al., 2008; Hailey et al., 2010; Ravikumar et al., 2010). Furthermore, recent studies suggest that the endoplasmic reticulum (ER)-mitochondria contact sites are involved in phagophore assembly (Hamasaki et al., 2013). This phagophore expands to engulf a region of cytoplasm or selected organelles, thereby sequestering the cargo in a double-membrane autophagosome. Several hetero-oligomeric protein complexes play an important role in the initiation stages. Nutrient depletion inhibits mammalian target of rapamycin complex 1 (mTORC1), which leads to dephosphorylation and activation of the ULK1-ATG13-FIP200 complexda crucial initiating step in autophagy (Ganley et al., 2009). Following closely, activation of this complex activates the beclin-1 interacting complex, which consists of beclin-1, BCL-2 family proteins, class III phosphatidylinositol 3-kinase (VPS34), and ATG14L (Li et al., 2012). Vps34 has a PI3K activity and produces phosphatidylinositol3phosphate, which promotes autophagosomal membrane nucleation (Juhasz et al., 2008). In addition, there are 2 specialized ubiquitin-like conjugation systems that control the elongation stages, the ATG5eATG12 conjugation system and the microtubuleassociated protein light chain 3 (LC3eATG8)- conjugation system. The first conjugation is facilitated by ATG7, ATG10, and ATG12 while the second one is facilitated by ATG3 and ATG4 (Hanada et al., 2007). LC3 (that originates from microtubularassociated protein-light chain) is cleaved at its C terminus by ATG4 to form LC3I, which then combined with lipid phosphatidyletanolamine to form Atg8-phosphatidyletanolamine or lapidated LC3-II, which localizes to the autophagosome membrane (Nakatogawa et al., 2007). As the autophagosome formation occurs at random sites in the cytoplasm, the autophagosome are transported along the microtubule toward the microtubule organizing-center, where lysosomes are plentiful to improve the opportunity for lysosomal fusion (Marambio et al., 2010). The protein, such as dynein and LC3-II, has the significant effect on the microtubular transport. During the transport, the autophagosome may fuse with a late endosome to generate an amphisome, which finally fuses with lysosome to
digest its cargo, or it may fuse immediately with lysosome to generate an autolysosome (Nixon and Yang, 2011). Then autophagosomal contents are degraded by lysosomal acid proteases and the amino acids and other by-products of degradation are released for metabolic recycling and for building of macromolecules. One of the most substantial molecules in the maturation of autophagosomes and/or endosomes is Rab7. It designates the maturation of autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes (Jager et al., 2004). In addition, recent data shows that Syntaxin17 is also recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster (Takats et al., 2013). During the proteolytic clearance via lysosomes, transcription factor EB (TFEB) modulates lysosomal biogenesis and autophagy by controlling the expression of lysosomal enzymes. Moreover, it has been shown that overexpression of TFEB leads to the generation of new lysosomes and increased numbers of autophagosomes in a variety of cell types (Sardiello et al., 2009; Settembre and Ballabio, 2011; Settembre et al., 2011).
3. The autophagic pathway 3.1. mTOR-dependent signaling pathways The mTOR is a primordial inhibitory signal, which participates in the initial process of signal transduction, acting the upstream of ATG proteins. mTORC1 and mTORC2, 2 functionally distinct multiprotein complexes, are the main subunit of mTOR. However, only the rapamycin-sensitive mTORC1 directly controls cellular homeostasis via the inhibition of autophagy (Hosokawa et al., 2009) (Fig. 2). When growth factors like insulin-like growth factor bind to insulin-like growth factor receptors (IGF1R), class I PT3K pathway which catalyzes the conversion of phosphatidylinositol4,5-bisphosphate to phosphatidylinositol-3,4,5-trisphosphate, is activated. Phosphatidylinositol-3,4,5-trisphosphate recruits AKt and DDK1 to membrane, allowing the latter one to phosphorylate AKt (Meng et al., 2013; Petiot et al., 2000). The active AKt inhibits the activity of the TSC1 and/or TSC2 complex, whose activity can lead to an overall inhibition of mTORC1 signaling (Yoshida et al.,
Table 1 Major proteins involved in autophagy Mammalian autophagic proteins
Yeast homolog
Functions
Reference
Autophagosome initiation ULK1 Atg13 FIP200 Beclin 1
Atg1 Atg13 Atg17 Atg6
Kinase in ULK-Atg13-FIP200 complex, interfaces with mTORC1 Form Atg1 complex, mediates ULK1/2-FIP200 interactions Form Atg1 complex, mediates ULK1/2-FIP200 interactions PI3 K-III complex, Bcl-2ebinding protein; involved in autophagosome formation Form PI3 K-III complex
(Ravikumar et al., 2010) (Ravikumar et al., 2010) (Ravikumar et al., 2010) (Kang et al., 2011)
E2-like enzyme for pro-LC3 (Atg8) Cysteine protease cleaves Atg8 C-terminus, converts pro-LC3 (Atg8) to LC3-I, delipidates autophagosomal LC3-II Form Atg5-Atg12-Atg16 complex E1 ubiquitin conjugaseelike enzyme in both LC3 and Atg12-Atg5-Atg16 pathways Ubiquitin-like modifier conjugated to phosphatidyletanolamine Form Atg2-Atg9 complex, assists in autophagosomal assembly E2 ubiquitin ligase-like enzyme in Atg12-Atg5-Atg16 pathway Form Atg5-Atg12-Atg16 complex Form Atg5-Atg12-Atg16 complex
(Yang and Klionsky, 2010) (Yang and Klionsky, 2010)
(Yang and Klionsky, 2010) (Ravikumar et al., 2010) (Sarkar, 2013) (Sarkar, 2013) (Sarkar, 2013)
Involved in microtubular transport Regulate the fusion process Direct the trafficking of cargoes along microtubules
(B Mestre and I Colombo, 2013) (B Mestre and I Colombo, 2013) (Hyttinen et al., 2013)
Vps34 Autophagosome elongation Atg3 Atg4 Atg5 Atg7
Vps34 Atg3 Atg4 Atg5 Atg7
MAP1LC3A/B Atg8 Atg9A/B Atg9 Atg10 Atg10 Atg12 Atg12 Atg16 L1/L2 Atg16 Autophagosome maturation and fusion Dynein SNARE Rab proteins -
(Kang et al., 2011)
(Sarkar, 2013) (Sarkar, 2013)
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Fig. 1. The main steps in the autophagic process. The autophagy process involves the following steps: induction, initiation, elongation and completion, maturation and fusion, and degradation.
2011). By contrast, intracellular signals including nutrient starvation, low energy levels (increase of the AMP and/or ATP ratio), hypoxia, and DNA damage inhibit mTOR activity. P53, which is commonly mutated in human cancers, also regulates mTOR activity. Under conditions of oncogenic or genotoxic stress, nuclear P53 promotes translation of autophagy-including genes, sestrin 2 and dram, both of which stimulate autophagy by activating adenosine monophosphate-activated protein kinase (AMPK) (Tasdemir et al., 2008a). Conversely, cytosolic P53 inhibits autophagy through phosphorylation of AMPK, which weakens its kinase activity (Tasdemir et al., 2008b). Moreover, the mutation of the p53 ortholog CEP-1 exhibited a prolonged life span of Caenorhabditis elegans through an increase in baseline autophagy (Tavernarakis et al., 2008). In addition, p53 regulates expression of damageregulated autophagy modulator 1, which in turn induces autophagy. Recent study suggests that damage-regulated autophagy modulator 1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes, and clearance of autophagosomes (Zhang et al., 2013). Another recent finding reveals a new mechanism of regulation of mTORC1 signaling and autophagy, that is, quality control of proton-coupled amino-acid transporter 4 by Rab12. The knockdown of Rab12 can result in increased activity of mTORC1. The small GTPase Rab12 promotes constitutive degradation of proton-coupled amino-acid
transporter 4, whose accumulation in Rab12-knockdown cells modulates mTORC1 activity and autophagy (Matsui and Fukuda, 2013). In addition, cystatin C can also induce a fully functional autophagy via the mTOR inhibition in cells under basal conditions, and enhance the autophagic activation in cells exposed to nutritional deprivation and oxidative stress (Tizon et al., 2010). Several groups have correlated mTOR with autophagy regulation by finding that the ULK1eAtg13eFIP200 complex is a downstream target of mTOR. Active mTORC1 phosphorylates Atg13, whose inhibitory phosphorylation restrains autophagy by blocking the autophagosome formation. Once cellular energy is depleted, starvation results in rapid dephosphorylation of Atg13, assembly of the complex, and induction of autophagy (Kamada et al., 2010). Moreover, recent study demonstrated that TFEB, a master regulator of lysosomal biogenesis, is regulated by the lysosome via the mTORC1 pathway. TFEB localization is directly regulated by the mTORC1 signaling pathway via the activation state of amino acideRag GTPase (Settembre et al., 2012). 3.2. mTOR-independent signaling pathways Beside the canonical mTOR-dependent signaling pathway, pathways that act independently of mTOR also play an important role in regulating autophagy. Autophagy can be directly initiated by
Fig. 2. The autophagic pathway and potential drug targets. Two signaling pathways involve in the regulation of autophagy: mammalian target of rapamycin (mTOR)-dependent signaling pathways and mTOR-independent signaling pathways. Abbreviation: mTOR, mammalian target of rapamycin.
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AMPK, leading to direct phosphorylation of ULK1 and beclin-1 (Egan et al., 2011; Kim et al., 2011). Although activation of ULK1 by energy depletion occurs through a mechanism distinct to that mediated by amino acid starvation, the coordinated phosphorylation of ULK1 by mTORC1 and AMPK possibly control autophagic flux in response to metabolic requirements. In addition, the LKB1/ AMPK-dependent phosphorylation of p27 stabilizes this cyclindependent kinase inhibitor that allows cells to survive nutritional deprivation and oxidative stress by activating autophagy (Liang et al., 2007). Beclin-1, another relevant point worth mentioning, is bound to the anti-apoptotic protein Bcl-2 in nutrient-rich condition. However, when nutrients are scarce, Bcl-2 is phosphorylated by enzyme Jun N-terminal kinase 1 (JNK1), thereby achieving the dissociation of Bcl-2 from beclin-1 (Wei et al., 2008). Then these events promote the formation of the autophagy-stimulatory beclin-1ehVps34 complex. Although starvation also inhibits mTORC1, expression of constitutively active JNK1, however, does not interfere with mTORC1 activity. Moreover, rapamycin has no effect on JNK1 and Bcl-2 phosphorylation, an effect consistent with no alterations of these phospho-proteins in TSC2-deficient cells where mTORC1 is active (Sarkar et al., 2011). This evidence suggests that autophagy regulated by the JNK1/beclin-1/PI3KC3 and mTORC1 pathways are possibly independent of each other. Irrefutable evidence proves a cyclical pathway involving inositol and myoinositiol-1, 4,5-triphosphate (IP3), both of which inhibit autophagy (Sarkar et al., 2005). Intracellular cyclic adenosine monophosphate (cAMP) acts to increase IP3 production. IP3, in turn, binds to receptors on the ER membrane causing a release in Ca2þ from ER stores, which activates calpains that block autophagy. Sirtuin 1 (SIRT1), a phylogenetically conserved NADþ-dependent deacetylase, is another important autophagy regulator that is involved in aging and age-related diseases. Activation of the SIRT1 by 3 different approaches (overexpression, pharmacological activation with resveratrol, and depletion of its negative regulator nicotinamide) can prolong life span through the stimulation of autophagy basal rates in yeast, C. elegans, and flies (Morselli et al., 2010a). Evidence has emerged that sirtuin1 can form a molecular complex with several essential autophagic modulators, including autophagy related proteins (such as, Atg5, Atg7, and Atg8). It also can directly deacetylate these components (Morselli et al., 2010a). Thus, sirtuin-1 induces autophagy, an effect presumably mediated by the deacetylation of these essential autophagy proteins (Morselli et al., 2010b). 4. Autophagy in maintaining neuronal homeostasis The role of autophagy in maintaining neuronal homeostasis is particularly vital for postmitotic neurons, where the level of altered proteins and damaged organelles cannot be diluted by means of cell division. Moreover, the neurons possess the specialized cellular structures for intercellular communication. Thus, postmitotic neurons with specialized cellular structures have distinct mechanism of autophagic regulation compared with that of nonneuronal cells. In cultured primary neurons, blockage of autophagosome clearance by inhibiting cathepsins causes obviously rapid AVs accumulation without hindrance of autophagy induction (Boland et al., 2008). Thus, we can draw a conclusion that autophagy keeps constitutively active in neurons and that powerful clearance of autophagosome rather than the low level of autophagosome biosynthesis keeps their numbers low (Nixon and Yang, 2011). Neuronal highly specialized structures represent regions of high energy demand and protein turnover as a quality control for cell survival. Imbalance between the amount of altered protein and the capability of the quality control system contribute to the
accumulation of abnormal proteins and organelles, which ultimately end up with neuron degeneration and death. We can recognize the importance of basal autophagy in the maintenance of neuronal homeostasis through the classic experiment using mice deficient in the gene Atg5 or Atg7, which displayed neurobehavioral deficiencies (Zhang et al., 2009). Furthermore, inclusion bodies composed by polyubiquitinated proteins augment in both size and number with aging in these autophagy-lacking neurons. All these results demonstrate that constituent clearance of unpopular proteins through autophagy has a remarkable influence in the neuronal homeostasis. 5. A common series of events in neurodegeneration Neurodegenerative disorders display the dynamic variety in the diverse stages failures, regional susceptibility, and pathologic mechanisms. Nevertheless, almost all neurodegenerative diseases are age-dependent hereditary or sporadic disorders that are manifested by progressive loss of neural function. One common feature in their pathogenesis is the way in which neurons dispose the presence of pathogenic proteins. Many pathogenic proteins can be degraded through either the ubiquitin-proteasome or CMA only when they are in their soluble forms (Rothenberg et al., 2010). CMA, a type of selective autophagy, was different from other types of autophagy because of the unique way in which its substrate proteins are targeted one-by-one to the surface of the lysosomes, and then enter the digestive organelles (Cuervo et al., 2004). However, once organized into irreversible oligomers or multimeric complexes unable to undergo complete unfolding, proteins can only be eliminated via another feasible optiondmacroautophagy (Koga and Cuervo, 2011). It is too intricate to explain the exhaustive mechanisms that initiate the macroautophagy under these conditions clearly. This unsolved problem probably may be related to failure of UPS or CMA systems to degrade the aggregated proteins. Furthermore, this hypothesis is confirmed by the evidence that experimental blockage of proteolytic activity of these 2 systems has expressed up-regulation of macroautophagy (Bennett et al., 2005). Of course, macroautophagy does more than just work for the compensatory stage. It can be a major force in the clearance of pathogenic proteins. Nevertheless, macroautophagy is not that indestructible matrix upon which the altered proteins are eliminated in the natural course of neurodegenerative diseases. Consequently, once macroautophagy started to fail coupled with the further decreased activity of the UPS and of the CMA, it fails to complete the quality control task. As a result, the toxicity because of the presence of aberrant AVs often leads to neuronal dysfunction and ultimate neuronal death. 6. Autophagy in aging Almost all aging organisms share a gradual decrease in the activity of both the ubiquitinproteasome system and autophagy, which is believed to constitute an aging process. Numerous evidences now indicate that autophagy declines with age and this progressive reduction might have a causative role in the functional deterioration of biological systems during aging. A recent commendable mouse model, senescence accelerated mouse prone 8 (SAMP8), which is a non-genetically modified stain of mice with accelerated aging process share similar characteristics with aged humans and AD patients (Ma et al., 2011). In the aging process of SAMP8 mice, some remarkable autophagy-related changes with age including accumulated ubiquitinpositive proteins and decreased autophagy activity, exist in their brains (Ma et al., 2011). These results suggest that impaired autophagy may contribute to premature aging. We can draw the same conclusions from the studies in
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Fig. 3. Different types of autophagy coexisting in mammals and disease causing proteins affecting various types of autophagy in common neurodegenerative diseases. (A) Macroautophagy: a limiting membrane, termed a phagophore, forms engulfing cytosolic components and seals to form an autophagosome. Then this double membrane vesicle is delivered to lysosomes acquires hydrolytic enzymes by fusing with it for degradation of cargos. (B) Chaperone-mediated autophagy (CMA): unfolded substrate proteins are recruited by a cytosolic chaperone (hsc70) and its cochaperones to the surface of lysosomes. Upon binding to the receptor protein LAMP-2A, substrates cross the membrane assisted by a luminal chaperone (Lys-hsc70) and are then rapidly degraded in the lumen. (C) Microautophagy: through stimuli that remain poorly identified, invaginations at the lysosomal membrane trap cytosolic cargos that are internalized after the vesicles pinch off into the lysosomal lumen. Abbreviation: CMA, chaperone-mediated autophagy.
humans. The studies detected down-regulation of autophagy genes (ATG5, ATG7, and BECN1) in the brain of old persons as compared with young ones (Lipinski et al., 2010). Increasing findings indicate that conserved signaling pathways, such as the insulin/IGF-1 signaling pathway, mTOR signaling pathway, and AMP-activated protein kinase pathway converge on autophagy to extend lifespan. Although accumulating studies have established a tight connection between autophagy and aging, a one-way cause-and-effect relationship still remains obscure. Further studies should focus on the complete repertoire of interactions between autophagy and aging. 7. Autophagy in neurodegenerative diseases Autophagic dysfunctions are referred as a secondary pathologic mechanism for various neurodegenerative diseases, such as AD, PD, HD, and ALS (Nixon, 2006). Moreover, each neurodegenerative disease is related to defects in different steps of the autophagic pathway and in different types of autophagy (Fig. 3). 7.1. Autophagy in AD AD, the most common form of progressive dementia, is characterized by the presence of extracellular senile plagues and
intra-neuronal fibrillary tangles. The amyloid plagues are mainly composed of b-amyloid (Ab) peptides, a proteolytic product of a transmembrane protein-APP, whose sequential cleavage by b-secretase and g-secretase generates the senile Ab fragment (Jiang et al., 2013). The aggregated hyperphosphorylated tau, a microtubule-associated protein, is the main force contributing to the intracellular accumulation of neurofibrillary tangles (de Calignon et al., 2012). In the AD brain, dystrophic and degenerating neurites have an excess of autophagosomes and other types of AVs, which become a major intracellular reservoir of the toxic peptide (Nixon et al., 2005). Combined factors give rise to this massive accumulation of immature AVs within senile neurons, including elevated initiation of autophagy, retrograde transport of AVs and their deficits in the maturation to lysosomes (Boland et al., 2008; Li et al., 2010; Nixon and Yang, 2011) (Fig. 4A). It is the local accumulation of autophagosomes in dystrophic neurons that can contribute to Ab generation, which is achieved by both increased turnover of APP and enrichment of the g-secretase complex. Moreover, the study that used the APP/PS1 double transgenic Ab pathology-bearing model mouse demonstrates that the formation of AVs was mediated by activation of AMPK (Son et al., 2012). Autophagosome membranes are exactly rich in presenilin 1 whose mutations result in early-onset forms of autosomal
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Fig. 4. The impact of autophagy in neurodegeneration. (A) Autophagy in Alzheimer’s disease: combined factors give rise to massive accumulation of immature autophagic vacuoles (AVs), including elevated initiation of autophagy, retrograde transport of AVs, and their deficits in the maturation to lysosomes. (B) Autophagy in Parkinson’s disease: specific gene drawbacks result in autophagy failure, including dysfunction of chaperone-mediated autophagy (CMA), impaired mitophagy, and blockage of macroautophagy. (C) Autophagy in Huntington’s disease: the aberrant autophagosomal cargo recognition contributes to the accumulation of mutant huntingtin (Htt). Moreover, the dysfunction of macroautophagy may enhance the CMA activity, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to Huntington’s disease. (D) Autophagy in amyotrophic lateral sclerosis: during the autophagosome transport process, mutations in dynein or dynactin can cause defective autophagosome movement in the motor neurons, leading to the accumulation of mutant superoxide dismutase 1 (SOD1). Abbreviations: AVs, autophagic vacuoles; CMA, chaperone-mediated autophagy; Htt, huntingtin; SOD1, superoxide dismutase 1.
dominant AD. PS1 has diverse biological roles in cell adhesion, apoptosis, neurite outgrowth, calcium homeostasis, and synaptic plasticity. Although most of these roles involve PS1 as a component of the APP-cleaving g-secretase complex, PS1 also has g-secretaseindependent roles, which include a critical role in lysosome acidification that is essential for the activation of lysosomal proteases during autophagy. The decrease of PS1 function would lead to the accumulation of immature unglycosylated v-ATPase which is needed in the acidification of autolysosomes and/or lysosomes, the abnormal accumulation of late-stage autophagosomes with undigested contents can be discovered in PS1-null cells, just like the ultrastructures present in AD neurons (Zhu et al., 2013). Moreover, it was recently demonstrated that fibroblasts from presenilin 1 mutant AD patients express loss of macroautophagy, resulting in increased Ab accumulation caused by the impaired maturation of the V0a1 subunit of the bimodular v-type Hþ-ATPase proton pump that achieves autolysosomal acidification (Lee et al., 2010). Moreover, mounting studies revealed that the expression of beclin-1 is reduced in early AD (Lucin et al., 2013). The reduced beclin-1 protein levels following caspase-cleavage in midfrontal cortex gray matter from moderate to severe AD cases demonstrate that the autophagy-inducing protein beclin-1 is involved in AD (Pickford et al., 2008; Rohn et al., 2011). In APP transgenic mouse models of AD, beclin-1 reduced neuronal autophagy, disrupted lysosomes, promoted intracellular and extracellular Ab accumulation, and resulted in neurodegeneration. (Pickford et al., 2008; Sepe et al., 2014). By contrast, the exactly opposite pattern is observed in mice with increased expression of it (Jaeger et al., 2010; Wang et al., 2012). Moreover, recent studies position beclin-1 as a link between autophagy, retromer trafficking, and receptor-mediated phagocytosis (Lucin et al., 2013). In addition, the amelioration of lysosomal function in TgCRND8 markedly decreased extracellular Ab levels, and prevented the development of deficits of learning
and memory, which supports the involvement of autophagy clearance in the regulation of Ab metabolism (Yang et al., 2011). Autophagy is also partially involved in the regulation of the intracellular clearance of soluble tau and aggregated neurofibrillary tangles (NFTs). Inhibition of autophagy by 3-methylamphetamine (3-MA) and chloroquine delays tau clearance and result in tau aggregation (Hamano et al., 2008). Conversely, tau-overexpressing treated with rapamycin exhibits reduced aggregation of tau and tau-induced neurotoxicity in the 14-months-old PK//TauVLW mice (Rodriguez-Navarro et al., 2010). Moreover, blockage of phospholipase D1, acting as downstream of Vps34 to regulate autophagosome maturation, leads to higher levels of tau and p62 aggregates in the brain (Dall’Armi et al., 2010). Furthermore, autophagy was reported to take part in the degradation of a caspase cleaved form of tau (Metcalfe et al., 2012). In turn, the formation of tau oligomers and insoluble aggregates is also attributed to the failure of autophagylysosome system. The elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles (Rodriguez-Martin et al., 2013). In general, all these results highlight the pivotal role of autophagy in AD pathogenesis. 7.2. Autophagy in PD PD is the most common neurodegenerative movement disorder characterized by static tremor, bradykinesia, muscle rigidity, and postural instability. The heredofamilial forms of PD originate from mutations of at least 6 genes: a-synuclein (SNCA or PARK1), Parkin (PARK2), ubiquitin carboxy terminal hydrolase L1 (UCH-L1 or PARK5), PTEN-induced putative kinase 1 (PINK-1 or PARK6), DJ-1 (PARK7), and leucine-rich repeat kinase 2 (LRRK2 or PRAK8). Vast studies indicated that the proteins encoded by these specific genes have been described to regulate and/or interfere with lysosomal
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and/or autophagic pathways (Fig. 4B). For instance, a-synuclein can be degraded by different pathways including the ubiqutin proteosome pathway, CMA, and macroautophagy pathway (Luk et al., 2012). In turn, overexpression of wild type a-synuclein can interfere with different types of autophagy in mammalian cells, and mutant forms of a-synuclein A30P and A53T have been shown to inhibit CMA via a higher binding to the lysosomal-associated membrane protein 2A (LAMP-2A) than wild type a-synuclein (Malkus and Ischiropoulos, 2012). Moreover, these normal expressions of both mutant and wild type a-synuclein can induce macroautophagy for compensation (Shen et al., 2013). Once the compensation failed, a-synuclein aggregates in cells when present in protofibrils and/or oligomers, fibrils and large aggregates forms. Recent studies have determined the presence of oligomeric Syn, Syn-immunopositive aggregates, localized Syn, and alterations in membrane conductance which imply that leak channels may result in subsequent cytotoxicity in a dopaminergic-like cell line (Feng et al., 2010). Furthermore, it can impair the viability of adjacent cells and ultimately trigger neuronal death. This dynamic relationship is an important component of PD pathogenesis. Autophagy also participates in the turnover of mitochondria, whose dysfunction represents an important pathogenic mechanism of cell death in PD. The products of Parkin and PINK1 genes, the serine-threonine kinase PINK-1 and Parkin, are recruited to damaged mitochondria to provide convenience for mitophagy. In this way, Parkin and PINK-1 achieve the regulation of the elimination of the mitochondria by mitophagy (Narendra et al., 2008; Tanaka et al., 2010; Wang et al., 2011b). Hence, mutated forms of Parkin and PINK-1 impair ubiquitination of mitofusins and fail to mediate this degradation process, resulting in mitochondrial accumulation and excessive reactive oxygen species (ROS) production that damage neurons (Rakovic et al., 2011). In addition, the PINK-1 can interact with beclin-1 and significantly enhances basal and starvation-induced autophagy (Michiorri et al., 2010). Recent study reported that upon mitochondrial depolarization, PINK-1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to beclin1. PINK1-Bcl-xL interaction protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL, suggesting a novel mechanism through which PINK1 regulates cell survival (Arena et al., 2013). Functional deficiency of DJ-1, a ubiquitous redox-responsive protein with multiple functions, also contributes to altered autophagy in mouse and human cells, which might be associated to an increased autophagic flux (McCoy and Cookson, 2011). For the UCHL1, several studies have suggested that it might regulate CMA via the combination with LAMP-2A to mediate the translocation of ubiquitinated cargo inside lysosome (Cartier et al., 2012). UCH-L1 shows aberrant, increased binding to LAMP-2A, which inhibits asynuclein CMA and leads to its accumulation. Another study reported that lysosomal binding of several pathogenic mutant LRRK2 proteins interfered with the organization of the CMA translocation complex, resulting in defective CMA (Orenstein et al., 2013). Together, all these defects in both autophagy activation and lysosomal clearance may contribute to the pathogenesis in PD models. 7.3. Autophagy in HD HD is an autosomal dominant neurodegenerative disorder caused by the morbigenous huntingtin (Htt) proteins that are encoded by genes containing a repetitive DNA sequence (above 37 repeats in HD) consisting of the trinucleotide CAG in the Htt gene (Tsoi et al., 2012). Mutant huntingtin (mHtt), which forms perinuclear cytoplasmic aggregates and intranuclear inclusions in the neurons of patients with HD, can be degraded by the ubiquitin proteosome system or by
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autophagy pathway (Jeong et al., 2009). Several studies revealed that induction of autophagy leads to a decrease in both aggregated and soluble monomeric Htt species, and alleviates their toxicity in various models of HD (Ravikumar et al., 2004). Powerful supports for the involvement of autophagy in HD is provided by sequestration of mTOR in mHtt aggregates in cell models, transgenic mice, and human brains, which results in a decrease in mTOR activity and then induction of autophagy. However, these beneficial effects of rapamycin are autophagy dependent. Furthermore, it has no effects on autophagy and Htt accumulation where there is only inhibition of mTORC1. Under the combined inhibition of mTORC1 and mTORC2, autophagy can be initiated and Htt can be degraded (Roscic et al., 2011). This special phenomenon demonstrates that various molecules of the mTOR pathway may participate in the pathology of HD. In addition, the dysfunction of macroautophagy may enhance the CMA activity in the early stages of HD, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to cellular failure and the onset of pathologic manifestations (Fig. 4C). Despite the evident neuroprotective effect of autophagy in HD models, the precise mechanism that underlies autophagic dysfunction remains largely enigmatic. The aberrant autophagosomal cargo recognition has been considered to be responsible for impaired autophagy in HD (Martinez-Vicente et al., 2010). In these HD models and cells from HD patients, despite AVs formed at normal rates and were promptly removed by lysosomes, a primary defect in the ability of AVs to recognize cargo eventually resulted in Htt accumulation (Martinez-Vicente et al., 2010). In addition, another mechanism for impaired autophagy in HD is the sequestration of essential autophagy proteins, such as beclin-1, in mHtt aggregates (Wu et al., 2012). As the beclin-1 gene is insufficient in regulating autophagosome function, it has been suggested that an age-dependent reduction in beclin-1 expression might result in a decrease in autophagic activity, and then the accumulation of mutant Htt. 7.4. Autophagy in ALS ALS is a rapidly progressive neurodegenerative disorder that selectively affects motor neurons with uncertain pathogenesis. Studies of animal models and human postmortem tissues demonstrate that motor neuron degenerative processes consist of multiple pathologic events, such as oxidative stress, glutamate excitotoxicity, neuroinflammation, mitochondrial degeneration, cytoskeletal alterations, abnormalities in growth factors, and aggregation of proteins. Among these pathogenic mechanisms, plentiful aberrant protein accumulations in affected neuron that are associated with disturbances of protein homeostasis are outstanding, which insinuate that an impaired function of autophagy may be involved in the pathogenesis of ALS (Guo et al., 2010). Growing evidences certify this hypothesis. Indeed, the accumulation of autophagosomes was observed in the spinal cord of sporadic ALS patients (Morimoto et al., 2007), suggesting autophagic dysfunction in ALS. Moreover, previous study has found that autophagy is also significantly induced in the spinal cords of SOD1G93A mice (Li et al., 2008). In addition, recent studies demonstrated that the autophagic impairment in the ALS mice stem forms at the early stage of the disease, and becomes profound at the terminal stage (Zhang et al., 2011). For instance, increased autophagy can be detected during the presymptomatic stage (
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Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Review
Autophagy in aging and neurodegenerative diseases: implications for pathogenesis and therapy Chen-Chen Tan a, Jin-Tai Yu a, b, c, **, Meng-Shan Tan b, Teng Jiang c, Xi-Chen Zhu c, Lan Tan a, b, c, * a b c
Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, Qingdao, China Department of Neurology, Qingdao Municipal Hospital, College of Medicine and Pharmaceutics, Ocean University of China, Qingdao, China Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Qingdao, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 July 2013 Received in revised form 17 November 2013 Accepted 19 November 2013 Available online 28 November 2013
Neurodegenerative diseases, such as Alzheimer’s disease Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis, share a common cellular and molecular pathogenetic mechanism involving aberrant misfolded protein or peptide aggregation and deposition. Autophagy represents a major route for degradation of aggregated cellular proteins and dysfunctional organelles. Emerging studies have demonstrated that up-regulation of autophagy can lead to decreased levels of these toxic aggregateprone proteins, and is beneficial in the context of aging and various models of neurodegenerative diseases. Understanding the signaling pathways involved in the regulation of autophagy is crucial to the development of strategies for therapy. This review will discuss the cellular and molecular mechanisms of autophagy and its important role in the pathogenesis of aging and neurodegenerative diseases, and the ongoing drug discovery strategies for therapeutic modulation. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Autophagy Aging Alzheimer’s disease Parkinson’s disease Huntington’s disease Amyotrophic lateral sclerosis Lysosomal storage disorders Therapy
1. Introduction Autophagy (which means “self-eating”) is a lysosome degradation pathway by which cells capture intracellular proteins, lipids and organelles, and deliver them to the lysosomal compartment where they are degraded (Levine and Klionsky, 2004; Levine and Kroemer, 2008). It plays an important homeostatic role in cells and keeps the metabolic balance between synthesis, degradation, and subsequent turnover of cytoplasmic materials in stressful environment. Autophagy is classified into 3 types: macroautophagy, chaperone-mediated autophagy (CMA), and macroautophagy (Cuervo, 2004). Increasing studies have observed that autophagic vacuoles (AVs), the general term for autophagy-related vesicular structures, are abundant in neurons in an increasing * Corresponding author at: Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No. 5 Donghai Middle Road, Qingdao, Shandong Province 266071, China. Tel.: þ86 532 8890 5659; fax: þ86 532 8890 5659. ** Alternate corresponding author at: Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No. 5 Donghai Middle Road, Qingdao, Shandong Province 266071, China. Tel.: þ86 532 8890 5659; fax: þ86 532 8890 5659. E-mail addresses: [email protected] (J.-T. Yu), [email protected] (L. Tan). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.019
number of neurodegenerative disorders that are manifested by the presence of pathogenic proteins. Moreover, in the context of neurodegenerative disorders, the view that induction of autophagy is a neuroprotective response and that aberrant autophagy promotes neuronal cell death in most of these disorders has been widely accepted. Given these observations, autophagy as a potential therapeutic target in aging and neurodegenerative diseases is currently receiving more and more attention. In this review, we will focus on aging and neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and lysosomal storage disorders (LSDs), after briefly reviewing the mechanisms and function of autophagy. Furthermore, we also discuss how current knowledge about autophagy could be harnessed to open exciting new therapeutic perspectives for aging and neurodegenerative disorders. 2. The machinery of autophagy The proteins that are encoded by autophagy-related-genes (ATGs), ATG1-ATG35, organize into functional complexes that mediate the following steps in the autophagic processes: initiation,
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elongation, maturation and fusion, and degradation (Table 1) (Fig. 1). The process begins with the formation of a preautophagosomal structure or phagophore, which is probably derived from the lipid bilayer of the plasma membrane, including endoplasmic reticulum, golgi apparatus, plasma membrane, or mitochondria (Axe et al., 2008; Hailey et al., 2010; Ravikumar et al., 2010). Furthermore, recent studies suggest that the endoplasmic reticulum (ER)-mitochondria contact sites are involved in phagophore assembly (Hamasaki et al., 2013). This phagophore expands to engulf a region of cytoplasm or selected organelles, thereby sequestering the cargo in a double-membrane autophagosome. Several hetero-oligomeric protein complexes play an important role in the initiation stages. Nutrient depletion inhibits mammalian target of rapamycin complex 1 (mTORC1), which leads to dephosphorylation and activation of the ULK1-ATG13-FIP200 complexda crucial initiating step in autophagy (Ganley et al., 2009). Following closely, activation of this complex activates the beclin-1 interacting complex, which consists of beclin-1, BCL-2 family proteins, class III phosphatidylinositol 3-kinase (VPS34), and ATG14L (Li et al., 2012). Vps34 has a PI3K activity and produces phosphatidylinositol3phosphate, which promotes autophagosomal membrane nucleation (Juhasz et al., 2008). In addition, there are 2 specialized ubiquitin-like conjugation systems that control the elongation stages, the ATG5eATG12 conjugation system and the microtubuleassociated protein light chain 3 (LC3eATG8)- conjugation system. The first conjugation is facilitated by ATG7, ATG10, and ATG12 while the second one is facilitated by ATG3 and ATG4 (Hanada et al., 2007). LC3 (that originates from microtubularassociated protein-light chain) is cleaved at its C terminus by ATG4 to form LC3I, which then combined with lipid phosphatidyletanolamine to form Atg8-phosphatidyletanolamine or lapidated LC3-II, which localizes to the autophagosome membrane (Nakatogawa et al., 2007). As the autophagosome formation occurs at random sites in the cytoplasm, the autophagosome are transported along the microtubule toward the microtubule organizing-center, where lysosomes are plentiful to improve the opportunity for lysosomal fusion (Marambio et al., 2010). The protein, such as dynein and LC3-II, has the significant effect on the microtubular transport. During the transport, the autophagosome may fuse with a late endosome to generate an amphisome, which finally fuses with lysosome to
digest its cargo, or it may fuse immediately with lysosome to generate an autolysosome (Nixon and Yang, 2011). Then autophagosomal contents are degraded by lysosomal acid proteases and the amino acids and other by-products of degradation are released for metabolic recycling and for building of macromolecules. One of the most substantial molecules in the maturation of autophagosomes and/or endosomes is Rab7. It designates the maturation of autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes (Jager et al., 2004). In addition, recent data shows that Syntaxin17 is also recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster (Takats et al., 2013). During the proteolytic clearance via lysosomes, transcription factor EB (TFEB) modulates lysosomal biogenesis and autophagy by controlling the expression of lysosomal enzymes. Moreover, it has been shown that overexpression of TFEB leads to the generation of new lysosomes and increased numbers of autophagosomes in a variety of cell types (Sardiello et al., 2009; Settembre and Ballabio, 2011; Settembre et al., 2011).
3. The autophagic pathway 3.1. mTOR-dependent signaling pathways The mTOR is a primordial inhibitory signal, which participates in the initial process of signal transduction, acting the upstream of ATG proteins. mTORC1 and mTORC2, 2 functionally distinct multiprotein complexes, are the main subunit of mTOR. However, only the rapamycin-sensitive mTORC1 directly controls cellular homeostasis via the inhibition of autophagy (Hosokawa et al., 2009) (Fig. 2). When growth factors like insulin-like growth factor bind to insulin-like growth factor receptors (IGF1R), class I PT3K pathway which catalyzes the conversion of phosphatidylinositol4,5-bisphosphate to phosphatidylinositol-3,4,5-trisphosphate, is activated. Phosphatidylinositol-3,4,5-trisphosphate recruits AKt and DDK1 to membrane, allowing the latter one to phosphorylate AKt (Meng et al., 2013; Petiot et al., 2000). The active AKt inhibits the activity of the TSC1 and/or TSC2 complex, whose activity can lead to an overall inhibition of mTORC1 signaling (Yoshida et al.,
Table 1 Major proteins involved in autophagy Mammalian autophagic proteins
Yeast homolog
Functions
Reference
Autophagosome initiation ULK1 Atg13 FIP200 Beclin 1
Atg1 Atg13 Atg17 Atg6
Kinase in ULK-Atg13-FIP200 complex, interfaces with mTORC1 Form Atg1 complex, mediates ULK1/2-FIP200 interactions Form Atg1 complex, mediates ULK1/2-FIP200 interactions PI3 K-III complex, Bcl-2ebinding protein; involved in autophagosome formation Form PI3 K-III complex
(Ravikumar et al., 2010) (Ravikumar et al., 2010) (Ravikumar et al., 2010) (Kang et al., 2011)
E2-like enzyme for pro-LC3 (Atg8) Cysteine protease cleaves Atg8 C-terminus, converts pro-LC3 (Atg8) to LC3-I, delipidates autophagosomal LC3-II Form Atg5-Atg12-Atg16 complex E1 ubiquitin conjugaseelike enzyme in both LC3 and Atg12-Atg5-Atg16 pathways Ubiquitin-like modifier conjugated to phosphatidyletanolamine Form Atg2-Atg9 complex, assists in autophagosomal assembly E2 ubiquitin ligase-like enzyme in Atg12-Atg5-Atg16 pathway Form Atg5-Atg12-Atg16 complex Form Atg5-Atg12-Atg16 complex
(Yang and Klionsky, 2010) (Yang and Klionsky, 2010)
(Yang and Klionsky, 2010) (Ravikumar et al., 2010) (Sarkar, 2013) (Sarkar, 2013) (Sarkar, 2013)
Involved in microtubular transport Regulate the fusion process Direct the trafficking of cargoes along microtubules
(B Mestre and I Colombo, 2013) (B Mestre and I Colombo, 2013) (Hyttinen et al., 2013)
Vps34 Autophagosome elongation Atg3 Atg4 Atg5 Atg7
Vps34 Atg3 Atg4 Atg5 Atg7
MAP1LC3A/B Atg8 Atg9A/B Atg9 Atg10 Atg10 Atg12 Atg12 Atg16 L1/L2 Atg16 Autophagosome maturation and fusion Dynein SNARE Rab proteins -
(Kang et al., 2011)
(Sarkar, 2013) (Sarkar, 2013)
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Fig. 1. The main steps in the autophagic process. The autophagy process involves the following steps: induction, initiation, elongation and completion, maturation and fusion, and degradation.
2011). By contrast, intracellular signals including nutrient starvation, low energy levels (increase of the AMP and/or ATP ratio), hypoxia, and DNA damage inhibit mTOR activity. P53, which is commonly mutated in human cancers, also regulates mTOR activity. Under conditions of oncogenic or genotoxic stress, nuclear P53 promotes translation of autophagy-including genes, sestrin 2 and dram, both of which stimulate autophagy by activating adenosine monophosphate-activated protein kinase (AMPK) (Tasdemir et al., 2008a). Conversely, cytosolic P53 inhibits autophagy through phosphorylation of AMPK, which weakens its kinase activity (Tasdemir et al., 2008b). Moreover, the mutation of the p53 ortholog CEP-1 exhibited a prolonged life span of Caenorhabditis elegans through an increase in baseline autophagy (Tavernarakis et al., 2008). In addition, p53 regulates expression of damageregulated autophagy modulator 1, which in turn induces autophagy. Recent study suggests that damage-regulated autophagy modulator 1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes, and clearance of autophagosomes (Zhang et al., 2013). Another recent finding reveals a new mechanism of regulation of mTORC1 signaling and autophagy, that is, quality control of proton-coupled amino-acid transporter 4 by Rab12. The knockdown of Rab12 can result in increased activity of mTORC1. The small GTPase Rab12 promotes constitutive degradation of proton-coupled amino-acid
transporter 4, whose accumulation in Rab12-knockdown cells modulates mTORC1 activity and autophagy (Matsui and Fukuda, 2013). In addition, cystatin C can also induce a fully functional autophagy via the mTOR inhibition in cells under basal conditions, and enhance the autophagic activation in cells exposed to nutritional deprivation and oxidative stress (Tizon et al., 2010). Several groups have correlated mTOR with autophagy regulation by finding that the ULK1eAtg13eFIP200 complex is a downstream target of mTOR. Active mTORC1 phosphorylates Atg13, whose inhibitory phosphorylation restrains autophagy by blocking the autophagosome formation. Once cellular energy is depleted, starvation results in rapid dephosphorylation of Atg13, assembly of the complex, and induction of autophagy (Kamada et al., 2010). Moreover, recent study demonstrated that TFEB, a master regulator of lysosomal biogenesis, is regulated by the lysosome via the mTORC1 pathway. TFEB localization is directly regulated by the mTORC1 signaling pathway via the activation state of amino acideRag GTPase (Settembre et al., 2012). 3.2. mTOR-independent signaling pathways Beside the canonical mTOR-dependent signaling pathway, pathways that act independently of mTOR also play an important role in regulating autophagy. Autophagy can be directly initiated by
Fig. 2. The autophagic pathway and potential drug targets. Two signaling pathways involve in the regulation of autophagy: mammalian target of rapamycin (mTOR)-dependent signaling pathways and mTOR-independent signaling pathways. Abbreviation: mTOR, mammalian target of rapamycin.
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AMPK, leading to direct phosphorylation of ULK1 and beclin-1 (Egan et al., 2011; Kim et al., 2011). Although activation of ULK1 by energy depletion occurs through a mechanism distinct to that mediated by amino acid starvation, the coordinated phosphorylation of ULK1 by mTORC1 and AMPK possibly control autophagic flux in response to metabolic requirements. In addition, the LKB1/ AMPK-dependent phosphorylation of p27 stabilizes this cyclindependent kinase inhibitor that allows cells to survive nutritional deprivation and oxidative stress by activating autophagy (Liang et al., 2007). Beclin-1, another relevant point worth mentioning, is bound to the anti-apoptotic protein Bcl-2 in nutrient-rich condition. However, when nutrients are scarce, Bcl-2 is phosphorylated by enzyme Jun N-terminal kinase 1 (JNK1), thereby achieving the dissociation of Bcl-2 from beclin-1 (Wei et al., 2008). Then these events promote the formation of the autophagy-stimulatory beclin-1ehVps34 complex. Although starvation also inhibits mTORC1, expression of constitutively active JNK1, however, does not interfere with mTORC1 activity. Moreover, rapamycin has no effect on JNK1 and Bcl-2 phosphorylation, an effect consistent with no alterations of these phospho-proteins in TSC2-deficient cells where mTORC1 is active (Sarkar et al., 2011). This evidence suggests that autophagy regulated by the JNK1/beclin-1/PI3KC3 and mTORC1 pathways are possibly independent of each other. Irrefutable evidence proves a cyclical pathway involving inositol and myoinositiol-1, 4,5-triphosphate (IP3), both of which inhibit autophagy (Sarkar et al., 2005). Intracellular cyclic adenosine monophosphate (cAMP) acts to increase IP3 production. IP3, in turn, binds to receptors on the ER membrane causing a release in Ca2þ from ER stores, which activates calpains that block autophagy. Sirtuin 1 (SIRT1), a phylogenetically conserved NADþ-dependent deacetylase, is another important autophagy regulator that is involved in aging and age-related diseases. Activation of the SIRT1 by 3 different approaches (overexpression, pharmacological activation with resveratrol, and depletion of its negative regulator nicotinamide) can prolong life span through the stimulation of autophagy basal rates in yeast, C. elegans, and flies (Morselli et al., 2010a). Evidence has emerged that sirtuin1 can form a molecular complex with several essential autophagic modulators, including autophagy related proteins (such as, Atg5, Atg7, and Atg8). It also can directly deacetylate these components (Morselli et al., 2010a). Thus, sirtuin-1 induces autophagy, an effect presumably mediated by the deacetylation of these essential autophagy proteins (Morselli et al., 2010b). 4. Autophagy in maintaining neuronal homeostasis The role of autophagy in maintaining neuronal homeostasis is particularly vital for postmitotic neurons, where the level of altered proteins and damaged organelles cannot be diluted by means of cell division. Moreover, the neurons possess the specialized cellular structures for intercellular communication. Thus, postmitotic neurons with specialized cellular structures have distinct mechanism of autophagic regulation compared with that of nonneuronal cells. In cultured primary neurons, blockage of autophagosome clearance by inhibiting cathepsins causes obviously rapid AVs accumulation without hindrance of autophagy induction (Boland et al., 2008). Thus, we can draw a conclusion that autophagy keeps constitutively active in neurons and that powerful clearance of autophagosome rather than the low level of autophagosome biosynthesis keeps their numbers low (Nixon and Yang, 2011). Neuronal highly specialized structures represent regions of high energy demand and protein turnover as a quality control for cell survival. Imbalance between the amount of altered protein and the capability of the quality control system contribute to the
accumulation of abnormal proteins and organelles, which ultimately end up with neuron degeneration and death. We can recognize the importance of basal autophagy in the maintenance of neuronal homeostasis through the classic experiment using mice deficient in the gene Atg5 or Atg7, which displayed neurobehavioral deficiencies (Zhang et al., 2009). Furthermore, inclusion bodies composed by polyubiquitinated proteins augment in both size and number with aging in these autophagy-lacking neurons. All these results demonstrate that constituent clearance of unpopular proteins through autophagy has a remarkable influence in the neuronal homeostasis. 5. A common series of events in neurodegeneration Neurodegenerative disorders display the dynamic variety in the diverse stages failures, regional susceptibility, and pathologic mechanisms. Nevertheless, almost all neurodegenerative diseases are age-dependent hereditary or sporadic disorders that are manifested by progressive loss of neural function. One common feature in their pathogenesis is the way in which neurons dispose the presence of pathogenic proteins. Many pathogenic proteins can be degraded through either the ubiquitin-proteasome or CMA only when they are in their soluble forms (Rothenberg et al., 2010). CMA, a type of selective autophagy, was different from other types of autophagy because of the unique way in which its substrate proteins are targeted one-by-one to the surface of the lysosomes, and then enter the digestive organelles (Cuervo et al., 2004). However, once organized into irreversible oligomers or multimeric complexes unable to undergo complete unfolding, proteins can only be eliminated via another feasible optiondmacroautophagy (Koga and Cuervo, 2011). It is too intricate to explain the exhaustive mechanisms that initiate the macroautophagy under these conditions clearly. This unsolved problem probably may be related to failure of UPS or CMA systems to degrade the aggregated proteins. Furthermore, this hypothesis is confirmed by the evidence that experimental blockage of proteolytic activity of these 2 systems has expressed up-regulation of macroautophagy (Bennett et al., 2005). Of course, macroautophagy does more than just work for the compensatory stage. It can be a major force in the clearance of pathogenic proteins. Nevertheless, macroautophagy is not that indestructible matrix upon which the altered proteins are eliminated in the natural course of neurodegenerative diseases. Consequently, once macroautophagy started to fail coupled with the further decreased activity of the UPS and of the CMA, it fails to complete the quality control task. As a result, the toxicity because of the presence of aberrant AVs often leads to neuronal dysfunction and ultimate neuronal death. 6. Autophagy in aging Almost all aging organisms share a gradual decrease in the activity of both the ubiquitinproteasome system and autophagy, which is believed to constitute an aging process. Numerous evidences now indicate that autophagy declines with age and this progressive reduction might have a causative role in the functional deterioration of biological systems during aging. A recent commendable mouse model, senescence accelerated mouse prone 8 (SAMP8), which is a non-genetically modified stain of mice with accelerated aging process share similar characteristics with aged humans and AD patients (Ma et al., 2011). In the aging process of SAMP8 mice, some remarkable autophagy-related changes with age including accumulated ubiquitinpositive proteins and decreased autophagy activity, exist in their brains (Ma et al., 2011). These results suggest that impaired autophagy may contribute to premature aging. We can draw the same conclusions from the studies in
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Fig. 3. Different types of autophagy coexisting in mammals and disease causing proteins affecting various types of autophagy in common neurodegenerative diseases. (A) Macroautophagy: a limiting membrane, termed a phagophore, forms engulfing cytosolic components and seals to form an autophagosome. Then this double membrane vesicle is delivered to lysosomes acquires hydrolytic enzymes by fusing with it for degradation of cargos. (B) Chaperone-mediated autophagy (CMA): unfolded substrate proteins are recruited by a cytosolic chaperone (hsc70) and its cochaperones to the surface of lysosomes. Upon binding to the receptor protein LAMP-2A, substrates cross the membrane assisted by a luminal chaperone (Lys-hsc70) and are then rapidly degraded in the lumen. (C) Microautophagy: through stimuli that remain poorly identified, invaginations at the lysosomal membrane trap cytosolic cargos that are internalized after the vesicles pinch off into the lysosomal lumen. Abbreviation: CMA, chaperone-mediated autophagy.
humans. The studies detected down-regulation of autophagy genes (ATG5, ATG7, and BECN1) in the brain of old persons as compared with young ones (Lipinski et al., 2010). Increasing findings indicate that conserved signaling pathways, such as the insulin/IGF-1 signaling pathway, mTOR signaling pathway, and AMP-activated protein kinase pathway converge on autophagy to extend lifespan. Although accumulating studies have established a tight connection between autophagy and aging, a one-way cause-and-effect relationship still remains obscure. Further studies should focus on the complete repertoire of interactions between autophagy and aging. 7. Autophagy in neurodegenerative diseases Autophagic dysfunctions are referred as a secondary pathologic mechanism for various neurodegenerative diseases, such as AD, PD, HD, and ALS (Nixon, 2006). Moreover, each neurodegenerative disease is related to defects in different steps of the autophagic pathway and in different types of autophagy (Fig. 3). 7.1. Autophagy in AD AD, the most common form of progressive dementia, is characterized by the presence of extracellular senile plagues and
intra-neuronal fibrillary tangles. The amyloid plagues are mainly composed of b-amyloid (Ab) peptides, a proteolytic product of a transmembrane protein-APP, whose sequential cleavage by b-secretase and g-secretase generates the senile Ab fragment (Jiang et al., 2013). The aggregated hyperphosphorylated tau, a microtubule-associated protein, is the main force contributing to the intracellular accumulation of neurofibrillary tangles (de Calignon et al., 2012). In the AD brain, dystrophic and degenerating neurites have an excess of autophagosomes and other types of AVs, which become a major intracellular reservoir of the toxic peptide (Nixon et al., 2005). Combined factors give rise to this massive accumulation of immature AVs within senile neurons, including elevated initiation of autophagy, retrograde transport of AVs and their deficits in the maturation to lysosomes (Boland et al., 2008; Li et al., 2010; Nixon and Yang, 2011) (Fig. 4A). It is the local accumulation of autophagosomes in dystrophic neurons that can contribute to Ab generation, which is achieved by both increased turnover of APP and enrichment of the g-secretase complex. Moreover, the study that used the APP/PS1 double transgenic Ab pathology-bearing model mouse demonstrates that the formation of AVs was mediated by activation of AMPK (Son et al., 2012). Autophagosome membranes are exactly rich in presenilin 1 whose mutations result in early-onset forms of autosomal
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Fig. 4. The impact of autophagy in neurodegeneration. (A) Autophagy in Alzheimer’s disease: combined factors give rise to massive accumulation of immature autophagic vacuoles (AVs), including elevated initiation of autophagy, retrograde transport of AVs, and their deficits in the maturation to lysosomes. (B) Autophagy in Parkinson’s disease: specific gene drawbacks result in autophagy failure, including dysfunction of chaperone-mediated autophagy (CMA), impaired mitophagy, and blockage of macroautophagy. (C) Autophagy in Huntington’s disease: the aberrant autophagosomal cargo recognition contributes to the accumulation of mutant huntingtin (Htt). Moreover, the dysfunction of macroautophagy may enhance the CMA activity, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to Huntington’s disease. (D) Autophagy in amyotrophic lateral sclerosis: during the autophagosome transport process, mutations in dynein or dynactin can cause defective autophagosome movement in the motor neurons, leading to the accumulation of mutant superoxide dismutase 1 (SOD1). Abbreviations: AVs, autophagic vacuoles; CMA, chaperone-mediated autophagy; Htt, huntingtin; SOD1, superoxide dismutase 1.
dominant AD. PS1 has diverse biological roles in cell adhesion, apoptosis, neurite outgrowth, calcium homeostasis, and synaptic plasticity. Although most of these roles involve PS1 as a component of the APP-cleaving g-secretase complex, PS1 also has g-secretaseindependent roles, which include a critical role in lysosome acidification that is essential for the activation of lysosomal proteases during autophagy. The decrease of PS1 function would lead to the accumulation of immature unglycosylated v-ATPase which is needed in the acidification of autolysosomes and/or lysosomes, the abnormal accumulation of late-stage autophagosomes with undigested contents can be discovered in PS1-null cells, just like the ultrastructures present in AD neurons (Zhu et al., 2013). Moreover, it was recently demonstrated that fibroblasts from presenilin 1 mutant AD patients express loss of macroautophagy, resulting in increased Ab accumulation caused by the impaired maturation of the V0a1 subunit of the bimodular v-type Hþ-ATPase proton pump that achieves autolysosomal acidification (Lee et al., 2010). Moreover, mounting studies revealed that the expression of beclin-1 is reduced in early AD (Lucin et al., 2013). The reduced beclin-1 protein levels following caspase-cleavage in midfrontal cortex gray matter from moderate to severe AD cases demonstrate that the autophagy-inducing protein beclin-1 is involved in AD (Pickford et al., 2008; Rohn et al., 2011). In APP transgenic mouse models of AD, beclin-1 reduced neuronal autophagy, disrupted lysosomes, promoted intracellular and extracellular Ab accumulation, and resulted in neurodegeneration. (Pickford et al., 2008; Sepe et al., 2014). By contrast, the exactly opposite pattern is observed in mice with increased expression of it (Jaeger et al., 2010; Wang et al., 2012). Moreover, recent studies position beclin-1 as a link between autophagy, retromer trafficking, and receptor-mediated phagocytosis (Lucin et al., 2013). In addition, the amelioration of lysosomal function in TgCRND8 markedly decreased extracellular Ab levels, and prevented the development of deficits of learning
and memory, which supports the involvement of autophagy clearance in the regulation of Ab metabolism (Yang et al., 2011). Autophagy is also partially involved in the regulation of the intracellular clearance of soluble tau and aggregated neurofibrillary tangles (NFTs). Inhibition of autophagy by 3-methylamphetamine (3-MA) and chloroquine delays tau clearance and result in tau aggregation (Hamano et al., 2008). Conversely, tau-overexpressing treated with rapamycin exhibits reduced aggregation of tau and tau-induced neurotoxicity in the 14-months-old PK//TauVLW mice (Rodriguez-Navarro et al., 2010). Moreover, blockage of phospholipase D1, acting as downstream of Vps34 to regulate autophagosome maturation, leads to higher levels of tau and p62 aggregates in the brain (Dall’Armi et al., 2010). Furthermore, autophagy was reported to take part in the degradation of a caspase cleaved form of tau (Metcalfe et al., 2012). In turn, the formation of tau oligomers and insoluble aggregates is also attributed to the failure of autophagylysosome system. The elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles (Rodriguez-Martin et al., 2013). In general, all these results highlight the pivotal role of autophagy in AD pathogenesis. 7.2. Autophagy in PD PD is the most common neurodegenerative movement disorder characterized by static tremor, bradykinesia, muscle rigidity, and postural instability. The heredofamilial forms of PD originate from mutations of at least 6 genes: a-synuclein (SNCA or PARK1), Parkin (PARK2), ubiquitin carboxy terminal hydrolase L1 (UCH-L1 or PARK5), PTEN-induced putative kinase 1 (PINK-1 or PARK6), DJ-1 (PARK7), and leucine-rich repeat kinase 2 (LRRK2 or PRAK8). Vast studies indicated that the proteins encoded by these specific genes have been described to regulate and/or interfere with lysosomal
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and/or autophagic pathways (Fig. 4B). For instance, a-synuclein can be degraded by different pathways including the ubiqutin proteosome pathway, CMA, and macroautophagy pathway (Luk et al., 2012). In turn, overexpression of wild type a-synuclein can interfere with different types of autophagy in mammalian cells, and mutant forms of a-synuclein A30P and A53T have been shown to inhibit CMA via a higher binding to the lysosomal-associated membrane protein 2A (LAMP-2A) than wild type a-synuclein (Malkus and Ischiropoulos, 2012). Moreover, these normal expressions of both mutant and wild type a-synuclein can induce macroautophagy for compensation (Shen et al., 2013). Once the compensation failed, a-synuclein aggregates in cells when present in protofibrils and/or oligomers, fibrils and large aggregates forms. Recent studies have determined the presence of oligomeric Syn, Syn-immunopositive aggregates, localized Syn, and alterations in membrane conductance which imply that leak channels may result in subsequent cytotoxicity in a dopaminergic-like cell line (Feng et al., 2010). Furthermore, it can impair the viability of adjacent cells and ultimately trigger neuronal death. This dynamic relationship is an important component of PD pathogenesis. Autophagy also participates in the turnover of mitochondria, whose dysfunction represents an important pathogenic mechanism of cell death in PD. The products of Parkin and PINK1 genes, the serine-threonine kinase PINK-1 and Parkin, are recruited to damaged mitochondria to provide convenience for mitophagy. In this way, Parkin and PINK-1 achieve the regulation of the elimination of the mitochondria by mitophagy (Narendra et al., 2008; Tanaka et al., 2010; Wang et al., 2011b). Hence, mutated forms of Parkin and PINK-1 impair ubiquitination of mitofusins and fail to mediate this degradation process, resulting in mitochondrial accumulation and excessive reactive oxygen species (ROS) production that damage neurons (Rakovic et al., 2011). In addition, the PINK-1 can interact with beclin-1 and significantly enhances basal and starvation-induced autophagy (Michiorri et al., 2010). Recent study reported that upon mitochondrial depolarization, PINK-1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to beclin1. PINK1-Bcl-xL interaction protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL, suggesting a novel mechanism through which PINK1 regulates cell survival (Arena et al., 2013). Functional deficiency of DJ-1, a ubiquitous redox-responsive protein with multiple functions, also contributes to altered autophagy in mouse and human cells, which might be associated to an increased autophagic flux (McCoy and Cookson, 2011). For the UCHL1, several studies have suggested that it might regulate CMA via the combination with LAMP-2A to mediate the translocation of ubiquitinated cargo inside lysosome (Cartier et al., 2012). UCH-L1 shows aberrant, increased binding to LAMP-2A, which inhibits asynuclein CMA and leads to its accumulation. Another study reported that lysosomal binding of several pathogenic mutant LRRK2 proteins interfered with the organization of the CMA translocation complex, resulting in defective CMA (Orenstein et al., 2013). Together, all these defects in both autophagy activation and lysosomal clearance may contribute to the pathogenesis in PD models. 7.3. Autophagy in HD HD is an autosomal dominant neurodegenerative disorder caused by the morbigenous huntingtin (Htt) proteins that are encoded by genes containing a repetitive DNA sequence (above 37 repeats in HD) consisting of the trinucleotide CAG in the Htt gene (Tsoi et al., 2012). Mutant huntingtin (mHtt), which forms perinuclear cytoplasmic aggregates and intranuclear inclusions in the neurons of patients with HD, can be degraded by the ubiquitin proteosome system or by
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autophagy pathway (Jeong et al., 2009). Several studies revealed that induction of autophagy leads to a decrease in both aggregated and soluble monomeric Htt species, and alleviates their toxicity in various models of HD (Ravikumar et al., 2004). Powerful supports for the involvement of autophagy in HD is provided by sequestration of mTOR in mHtt aggregates in cell models, transgenic mice, and human brains, which results in a decrease in mTOR activity and then induction of autophagy. However, these beneficial effects of rapamycin are autophagy dependent. Furthermore, it has no effects on autophagy and Htt accumulation where there is only inhibition of mTORC1. Under the combined inhibition of mTORC1 and mTORC2, autophagy can be initiated and Htt can be degraded (Roscic et al., 2011). This special phenomenon demonstrates that various molecules of the mTOR pathway may participate in the pathology of HD. In addition, the dysfunction of macroautophagy may enhance the CMA activity in the early stages of HD, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to cellular failure and the onset of pathologic manifestations (Fig. 4C). Despite the evident neuroprotective effect of autophagy in HD models, the precise mechanism that underlies autophagic dysfunction remains largely enigmatic. The aberrant autophagosomal cargo recognition has been considered to be responsible for impaired autophagy in HD (Martinez-Vicente et al., 2010). In these HD models and cells from HD patients, despite AVs formed at normal rates and were promptly removed by lysosomes, a primary defect in the ability of AVs to recognize cargo eventually resulted in Htt accumulation (Martinez-Vicente et al., 2010). In addition, another mechanism for impaired autophagy in HD is the sequestration of essential autophagy proteins, such as beclin-1, in mHtt aggregates (Wu et al., 2012). As the beclin-1 gene is insufficient in regulating autophagosome function, it has been suggested that an age-dependent reduction in beclin-1 expression might result in a decrease in autophagic activity, and then the accumulation of mutant Htt. 7.4. Autophagy in ALS ALS is a rapidly progressive neurodegenerative disorder that selectively affects motor neurons with uncertain pathogenesis. Studies of animal models and human postmortem tissues demonstrate that motor neuron degenerative processes consist of multiple pathologic events, such as oxidative stress, glutamate excitotoxicity, neuroinflammation, mitochondrial degeneration, cytoskeletal alterations, abnormalities in growth factors, and aggregation of proteins. Among these pathogenic mechanisms, plentiful aberrant protein accumulations in affected neuron that are associated with disturbances of protein homeostasis are outstanding, which insinuate that an impaired function of autophagy may be involved in the pathogenesis of ALS (Guo et al., 2010). Growing evidences certify this hypothesis. Indeed, the accumulation of autophagosomes was observed in the spinal cord of sporadic ALS patients (Morimoto et al., 2007), suggesting autophagic dysfunction in ALS. Moreover, previous study has found that autophagy is also significantly induced in the spinal cords of SOD1G93A mice (Li et al., 2008). In addition, recent studies demonstrated that the autophagic impairment in the ALS mice stem forms at the early stage of the disease, and becomes profound at the terminal stage (Zhang et al., 2011). For instance, increased autophagy can be detected during the presymptomatic stage (
