Autophagosome dynamics in neurodegeneration at a glance

ABSTRACT

Autophagy is an essential homeostatic process for degrading cellular cargo. Aging organelles and protein aggregates are degraded by the autophagosome-lysosome pathway, which is particularly crucial in neurons. There is increasing evidence implicating defective autophagy in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease and Huntington's disease. Recent work using live-cell imaging has identified autophagy as a predominantly polarized process in neuronal axons; autophagosomes preferentially form at the axon tip and undergo retrograde transport back towards the cell body. Autophagosomes engulf cargo including damaged mitochondria (mitophagy) and protein aggregates, and subsequently fuse with lysosomes during axonal transport to effectively degrade their internalized cargo. In this Cell Science at a Glance article and the accompanying poster, we review recent progress on the dynamics of the autophagy pathway in neurons and highlight the defects observed at each step of this pathway during neurodegeneration.

Introduction

Autophagy is a cellular degradation process in which cytosolic cargos are degraded by lysosomes, and it can be divided into three subtypes – microautophagy, chaperone-mediated autophagy and macroautophagy. In microautophagy, cytosolic cargo is directly engulfed by the lysosome through invagination of the lysosomal membrane (Li et al., 2012), whereas in chaperone-mediated autophagy, cargo is selectively recognized by cytosolic chaperones that deliver the cargo to a translocation complex on the lysosomal membrane (Cuervo and Wong, 2014).

In contrast, macroautophagy (hereafter referred to as autophagy) involves the formation of the autophagosome, a double-membraned organelle that forms around cytosolic cargo and subsequently degrades the cargo by lysosomal fusion (Yang and Klionsky, 2010). In addition to non-selective cargo degradation during cellular stress, autophagosomes can selectively degrade protein aggregates, mitochondria (mitophagy), endoplasmic reticulum (ER-phagy) and peroxisomes (pexophagy) (Rogov et al., 2014). Selective autophagy is mediated by autophagy receptors that preferentially bind ubiquitylated organelles or other cargos, and subsequently recruit the autophagosome protein light chain 3 (LC3; encoded by MAP1LC3A–C) through their LC3-interaction region (LIR) motif (Stolz et al., 2014; Wild et al., 2014).

There is increasing evidence implicating defective autophagy in neurodegeneration (Harris and Rubinsztein, 2012; Nixon, 2013; Wong and Cuervo, 2010). However, the unique dynamics of autophagosomes in neurons have only recently been studied using live-cell imaging, revealing the preferential formation of autophagosomes at the axon tip and their subsequent retrograde axonal transport towards the cell body (Lee et al., 2011; Maday et al., 2012). These studies provide a new model for studying neuronal autophagy and its potential defects during neurodegeneration.

This Cell Science at a Glance article will summarize recent work on the autophagy pathway in neurons and the defects observed at each step of the process in ALS and Alzheimer's, Parkinson's and Huntington's diseases. These steps include autophagosome formation at the axon tip, cargo engulfment of protein aggregates and damaged mitochondria (mitophagy), axonal transport of autophagosomes, lysosomal fusion and cargo degradation.

Autophagy is an essential homeostatic pathway in neurons

Like other cell types, neurons accumulate protein aggregates and damaged organelles such as mitochondria that must be degraded by autophagy to maintain cellular homeostasis. However, neurons are post-mitotic cells that are highly polarized with both dendritic and axonal compartments, which can extend over distances many times greater than their cell soma. Consequently, neurons require motor proteins to actively supply proteins and organelles to these distal processes, and to drive the transport of signaling molecules and degradative compartments back to the cell body. Neurons also have high energetic demands due to their elaborate morphologies and their need to maintain active electrochemical signaling. They require efficient recycling of proteins and organelles at the synapse, as well as throughout the axon, dendrites and cell soma, making autophagic degradation particularly crucial in neurons. Autophagy is also required for neuronal development and the maintenance of axonal homeostasis (Fimia et al., 2007; Komatsu et al., 2007; Wang et al., 2006).

Not surprisingly, defective autophagy induces protein aggregation and neurodegeneration (Hara et al., 2006; Komatsu et al., 2006). Although autophagy is efficient in younger neurons (Boland et al., 2008), autophagy proteins such as beclin-1, Atg5 and Atg7 decline with age (Lipinski et al., 2010; Shibata et al., 2006), potentially contributing to the late onset of many neurodegenerative diseases (Rubinsztein et al., 2011).

Autophagosome biogenesis in neurons

Neurons exhibit robust constitutive autophagy, with autophagosomes forming preferentially at the axon tip (Maday et al., 2012). This observation suggests that autophagy is required for protein and organelle turnover distally, perhaps to balance the net flux of proteins and organelles arriving by anterograde transport (Maday et al., 2014). Distal biogenesis might also be coupled to synaptic function. There is also evidence that autophagosome formation can be induced along the axon in order to clear damaged mitochondria (Ashrafi et al., 2014).

Autophagosome biogenesis at the axon tip begins with the dynamic recruitment of Atg13 and Atg5 to double-FYVE containing protein 1 (DFCP-1, also known as ZYVE1) omegasomes, a phosphatidylinositol 3-phosphate (PI3P)-enriched omega-shaped ER structure that serves as a platform for autophagosome biogenesis (Maday and Holzbaur, 2014). This recruitment is followed by the incorporation of lipidated LC3 into the developing autophagosome over a period of 4–6 minutes (Maday et al., 2012) (see poster).

Autophagosome formation does not appear to be impaired in the majority of neurodegenerative diseases, and might even be upregulated. Autophagy is transcriptionally upregulated in Alzheimer's disease patient brains, potentially by an amyloid-β-mediated increase in reactive oxygen species (ROS) production and phosphoinositide 3-kinase (PI3K) activity (Lipinski et al., 2010). ALS patients also exhibit increased levels of the autophagy initiation proteins beclin-1 and the Atg5–Atg12 complex (Hetz et al., 2009) and an overall increase in autophagosomes (Sasaki, 2011). In Huntington's disease models, autophagosome formation at the axon tip and density along the axon are not altered (Baldo et al., 2013; Martinez-Vicente et al., 2010; Wong and Holzbaur, 2014a).

By contrast, in Parkinson's disease, α-synuclein overexpression inhibits autophagy by inhibition of Rab1a, leading to Atg9 mislocalization and defective omegasome and autophagosome formation (Winslow et al., 2010). A Parkinson's-disease-associated mutation in VPS35, a member of the retromer complex, also disrupts autophagosome formation owing to abnormal Atg9 trafficking (Zavodszky et al., 2014). Another Parkinson's-disease-associated protein, leucine-rich repeat kinase 2 (LRRK2), also localizes to autophagosome membranes, and the loss of LRRK2 disrupts autophagic induction (Schapansky et al., 2014).

Certain disease-associated proteins directly regulate autophagic formation. Myristoylation of a caspase-3-cleaved fragment of huntingtin promotes membrane curvature during autophagosome formation (Martin et al., 2014), while ubiquilin 2, an ALS-associated protein that binds to LC3, promotes autophagosome formation (Rothenberg et al., 2010). With recent advances in our understanding of neuronal autophagy, it will be crucial to examine the role of localized autophagosome formation in neurodegeneration.

Cargo loading of disease-associated proteins

Autophagy is the predominant degradation pathway for protein aggregates, including mutant α-synuclein (Webb et al., 2003), mutant superoxide dismutase 1 (SOD1) (Kabuta et al., 2006) and polyglutamine expansions in huntingtin (polyQ-htt) (Ravikumar et al., 2002; Ravikumar et al., 2004). Autophagy can also regulate the turnover of other disease-associated proteins, including amyloid-β (Parr et al., 2012), wild-type α-synuclein (Webb et al., 2003) and transactive response DNA binding protein 43 kDa (TDP-43) (Scotter et al., 2014).

Protein aggregates can be degraded by selective autophagy, which involves the recruitment of autophagy receptor proteins, including SQSTM1 (also known as p62), neighbor of BRCA1 gene 1 (NBR1), optineurin, nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2), and TAX1BP1 (also known as TRAF6-binding protein, T6BP), to ubiquitylated proteins (Birgisdottir et al., 2013; Rogov et al., 2014; Stolz et al., 2014) (see poster). Interestingly, mutations in both p62 and optineurin are causative for rare forms of familial ALS (Fecto et al., 2011; Maruyama et al., 2010). The activities of these receptors are regulated by different kinases. Phosphorylation of p62 by casein-kinase 2 (CK2) at S403 increases its affinity for polyubiquitin chains (Matsumoto et al., 2011), whereas phosphorylation of optineurin at S177 by TANK-binding kinase 1 (TBK1) increases its affinity for LC3 (Wild et al., 2011). Although recent work has begun to identify the autophagy receptors responsible for selective autophagy of various disease-associated proteins, the dynamics of their recruitment and whether different receptors might have redundant roles remain unclear.

Alzheimer's disease pathology is characterized by the formation of hyperphosphorylated tau tangles and amyloid-β plaques. Autophagic degradation of tau is regulated by nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated induction of the autophagy receptor NDP52 (Jo et al., 2014) and the Alzheimer's-disease-associated locus phosphatidylinositol binding clathrin assembly protein (PICALM, also known as CALM), which controls the endocytosis of soluble NSF attachment protein receptors (SNAREs) that are involved in the autophagy pathway (Moreau et al., 2014). Autophagy also regulates amyloid-β production (Yu et al., 2005), and defective autophagy leads to the accumulation of intraneuronal amyloid precursor protein (APP) (Nilsson et al., 2013). In microglia, amyloid-β is degraded by autophagy through the autophagy receptor optineurin (Cho et al., 2014) (see poster).

In Parkinson's disease, α-synuclein accumulates and forms Lewy body inclusions. Mutant forms of α-synuclein (A53T and A30P) are degraded predominantly by autophagy, rather than by chaperone-mediated autophagy (Cuervo et al., 2004; Webb et al., 2003). By contrast, chaperone-mediated autophagy degrades both wild-type α-synuclein and LRRK2, but becomes impaired by Parkinson's-disease-associated mutations in either protein (Cuervo et al., 2004; Orenstein et al., 2013).

In ALS, autophagy induction enhances TDP-43 turnover and increases survival of ALS neuronal models (Barmada et al., 2014; Caccamo et al., 2009). Cytoplasmic TDP-43 and fused in sarcoma (FUS) localize to stress granules, which are degraded by selective autophagy in a process termed granulophagy (Buchan et al., 2013; Ryu et al., 2014). Although soluble TDP-43 is primarily degraded by the proteasome, aggregated TDP-43 is cleared by autophagy (Scotter et al., 2014). ALS-associated mutant SOD1 is predominantly cleared by autophagy (Kabuta et al., 2006) that involves the autophagy receptor optineurin (Korac et al., 2013).

Huntington's disease is caused by polyQ-htt. Autophagy is crucial for degrading polyQ-htt (Qin et al., 2003; Ravikumar et al., 2002) and, accordingly, upregulation of autophagy increases survival in Huntington's disease models (Ravikumar et al., 2004). The autophagy receptor p62 is recruited to the vicinity of aggregated polyQ-htt, and reduced p62 levels lead to increased polyQ-htt-mediated cell death (Bjørkøy et al., 2005). Other autophagy receptors for polyQ-htt include optineurin (Korac et al., 2013) and Tollip, a member of CUET ubiquitin-binding adaptor proteins (Lu et al., 2014b). PolyQ-htt could be targeted to autophagosomes by several mechanisms, including its acetylation at K444 by CREB-binding protein (CBP) (Jeong et al., 2009) and activation of the insulin receptor substrate-2 (Yamamoto et al., 2006).

Using live-cell imaging in cultured neurons, recent studies have found that autophagosomes that undergo axonal transport engulf both mutant SOD1 (Maday et al., 2012) and polyQ-htt (Wong and Holzbaur, 2014a), further demonstrating that autophagy contributes to protein turnover in the axon. Thus, autophagy might play a crucial role in regulating aggregate formation both at synapses and along the axon.

Dynamics of mitochondrial degradation in neurodegenerative disease

Mitochondrial degradation through parkin-mediated mitophagy has drawn much attention, with many key discoveries made within the past year (Box 1). Parkin-mediated mitophagy has been primarily implicated in Parkinson's disease, as this pathway involves PTEN-induced putative kinase 1 (PINK1; also known as PARK6) and parkin (PARK2), two genes linked to familial Parkinson's disease (Kitada et al., 1998; Valente et al., 2004).

Box 1. Recent insights into the PINK1–parkin mitophagy pathway.

Recent work on mitophagy has filled important gaps in our understanding of the PINK1–parkin pathway. PINK1 binds to phosphoglycerate mutase family member 5 (PGAM5) in the inner mitochondrial membrane (Lu et al., 2014a) but, upon cleavage, translocates to the cytosol for proteasomal degradation (Fedorowicz et al., 2014; Yamano and Youle, 2013). Upon mitochondrial damage, parkin is recruited to damaged mitochondria downstream of PINK1, which phosphorylates Ser65 on the ubiquitin-like (UBL) domain of parkin (Kondapalli et al., 2012; Shiba-Fukushima et al., 2012) and Ser65 on ubiquitin, leading to parkin activation (Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Ordureau et al., 2014; Zhang et al., 2014).

Several E2 ubiquitin-conjugating enzymes (UBE2) were recently identified for parkin, including UBE2D2, UBE2D3 and UBE2L3, which charge parkin with ubiquitin, and UBE2N, which regulates mitochondrial clustering (Fiesel et al., 2014; Geisler et al., 2014). Three different deubiquitylating enzymes (DUBs) were also recently found to regulate mitophagy, with USP30 and USP15 opposing mitophagy by deubiquitylating parkin substrates (Bingol et al., 2014; Cornelissen et al., 2014), and USP8 promoting mitophagy by deubiquitylating parkin itself (Durcan et al., 2014). Another E3 ubiquitin ligase, Gp78 (also known as AMFR), also ubiquitylates mitofusin1 and mitofusin2 to induce mitophagy (Fu et al., 2013).

Downstream of mitochondrial ubiquitylation, our recent work identifies optineurin as an autophagy receptor, which recruits autophagosomes to ubiquitylated mitochondria, leading to mitochondrial degradation (Wong and Holzbaur, 2014b). Cardiolipin, an inner mitochondrial membrane phospholipid, also binds the autophagosome protein LC3 (Chu et al., 2013), suggesting that there might be alternative mechanisms of autophagosome recruitment. As many of these studies were performed in non-neuronal cells, it will be important to establish which of these steps also occur in neurons and might be defective during neurodegeneration. Robust basal mitophagy occurs in neurons (Maday et al., 2012), but it is unclear whether this constitutive process occurs through PINK1- and parkin-dependent or independent pathways (Allen et al., 2013; Bingol et al., 2014; Grenier et al., 2013; Strappazzon et al., 2014; Webster et al., 2013). By contrast, locally induced mitophagy in the axon has been shown to be dependent on both PINK1 and parkin (Ashrafi et al., 2014).

PINK1 is normally cleaved by the protease presenilin-associated rhomboid-like protein (PARL) on the inner mitochondrial membrane, leading to its degradation (Deas et al., 2011; Greene et al., 2012; Jin et al., 2010; Meissner et al., 2011; Whitworth et al., 2008). Upon mitochondrial damage, such as depolarization (Narendra et al., 2008), increased ROS production (Yang and Yang, 2013), activation of the mitochondrial unfolded protein response (mtUPR) (Jin and Youle, 2013) or expression of the short mitochondrial isoform of ARF (smARF, encoded by CDKN2A) (Grenier et al., 2014), PINK1 accumulates on the outer mitochondrial membrane and subsequently recruits the E3 ubiquitin ligase parkin to ubiquitylate outer mitochondrial membrane proteins (Matsuda et al., 2010; Narendra et al., 2010b; Sarraf et al., 2013; Vives-Bauza et al., 2010) (see poster). Parkinson's-disease-associated mutations in PINK1 and parkin disrupt their respective kinase and ubiquitylating activities (Lee et al., 2010a; Song et al., 2013; Sriram et al., 2005), leading to defective mitochondrial degradation. Depletion of DJ-1, another Parkinson's-disease-associated gene, disrupts both mitochondrial dynamics and morphology (Hao et al., 2010; Irrcher et al., 2010; Thomas et al., 2011). In addition, two other Parkinson's-disease-associated proteins, F-box domain-containing protein (Fbxo7; also known as PARK15) and sterol regulatory element binding transcription factor (SREBF1) are also implicated in mitophagy (Burchell et al., 2013; Ivatt et al., 2014).

Recent progress now implicates parkin-mediated mitochondrial degradation in ALS. We recently found that the ALS-associated protein optineurin is a novel autophagy receptor for damaged mitochondria that undergo parkin-mediated mitophagy (Wong and Holzbaur, 2014b). Optineurin is dynamically recruited to ubiquitylated mitochondria through its ubiquitin-binding domain (UBAN) downstream of parkin recruitment. Recruitment of optineurin leads to autophagosome formation, mediated by the interaction between its LIR motif (Wild et al., 2011) and LC3, resulting in the engulfment and degradation of damaged mitochondria. An ALS-associated E478G mutation in the UBAN of optineurin (Maruyama et al., 2010) inhibits optineurin recruitment to damaged mitochondria, resulting in inefficient mitochondrial degradation (Wong and Holzbaur, 2014b).

Another ALS-associated protein, valosin containing protein (VCP, also known as p97) (Johnson et al., 2010), also translocates to damaged mitochondria downstream of parkin, and it aids in the proteasomal degradation of ubiquitylated mitofusins Mfn1 and Mfn2 (Kim et al., 2013; Tanaka et al., 2010). Disease-associated VCP mutants are inefficiently recruited to mitochondria and disrupt mitochondrial clearance (Kim et al., 2013; Kimura et al., 2013). In addition, p62 is linked to ALS (Fecto et al., 2011) and regulates mitochondrial clustering through its Phox and Bem1p (PB1) oligomerization domain (Narendra et al., 2010a; Okatsu et al., 2010). Taken together, these studies demonstrate a role for the ALS-associated proteins optineurin, VCP and p62 in mitophagy, and further implicate mitochondrial damage in ALS pathogenesis. Of note, loss of either TDP-43 or FUS, two RNA-binding proteins involved in ALS, leads to decreased levels of parkin, which might also disrupt mitophagy initiation (Lagier-Tourenne et al., 2012; Polymenidou et al., 2011).

Defective mitochondrial dynamics have also been implicated in Huntington's disease (Costa and Scorrano, 2012). These defects might be exacerbated by inefficient mitophagy due to defective autophagic recognition of mitochondrial cargo (Martinez-Vicente et al., 2010), or disrupted autophagosome transport along the axon leading to inhibition of cargo degradation (Wong and Holzbaur, 2014a). Thus, defective mitophagy, once thought to be a hallmark of familial Parkinson's disease, might be a common characteristic of multiple neurodegenerative diseases and, in fact, might represent a point of vulnerability in the neuron.

Live-cell imaging studies of mitophagy in neurons initially found that, upon mitochondrial depolarization of the whole cell, autophagosome engulfment preferentially occurred around mitochondria in the cell soma (Cai et al., 2012). However, localized damage to mitochondria in neuronal axons was also found to induce autophagosome formation locally (Ashrafi et al., 2014). As healthy mitochondria normally undergo bidirectional transport in axons, damaged mitochondria might be stalled in axons owing to release of the Milton/kinesin motor protein complex from mitochondria by PINK1 and parkin-dependent degradation of the adaptor Miro (Wang et al., 2011). In addition, neurons demonstrate a robust pathway of constitutive basal mitophagy at the axon tip (Maday et al., 2012); this pathway is impaired by expression of polyQ-htt (Wong and Holzbaur, 2014a).

Regulation of autophagosome axonal transport in disease

Live-cell imaging studies in cultured neurons reveal robust retrograde transport of autophagosomes from the axon tip towards the cell body (Lee et al., 2011; Maday and Holzbaur, 2014; Maday et al., 2012). This transport is driven by the retrograde motor dynein (Katsumata et al., 2010; Lee et al., 2011; Maday et al., 2012) and its activator dynactin (Ikenaka et al., 2013) (see poster). Not surprisingly, dynein mutations in both fly and mouse models lead to impaired autophagic clearance of polyQ-htt (Ravikumar et al., 2005). The axonal transport of autophagosomes is further regulated by the motor adaptors huntingtin (Wong and Holzbaur, 2014a) and JNK-interacting protein 1 (JIP1) (Fu et al., 2014), which interact with motor proteins on autophagosomes to regulate their activity (Box 2).

Box 2. Regulation of the axonal transport of autophagosomes.

Vesicles and organelles undergo transport along the axon on microtubule tracks, moving in either the retrograde (towards the soma) or anterograde (away from the soma) direction. Retrograde movement is driven by the motor dynein and its adaptor dynactin, whereas anterograde transport is driven by kinesins. Scaffold proteins, such as JIP1, JIP3, huntingtin, Milton/TRAKs and Hook-1, bind motor proteins and regulate their activity, and thus are crucial for regulating organelle transport (Fu and Holzbaur, 2014). Cargos move differentially along the axon; whereas mitochondria and lysosomes move bidirectionally, APP and dense core vesicles move predominantly in the anterograde direction, and signaling endosome transport is primarily retrograde. Efficient axonal transport is crucial to supply the distal synapse with newly synthesized material, and to clear damaged proteins and organelles (Maday et al., 2014; Perlson et al., 2010). Not surprisingly, mutations in motor proteins lead to neurodegeneration (Farrer et al., 2009; Puls et al., 2003; Zhao et al., 2001).

Autophagosome transport along the axon is primarily retrograde and driven by dynein–dynactin (Ikenaka et al., 2013; Katsumata et al., 2010; Lee et al., 2011; Maday et al., 2012). Autophagosome transport is regulated by huntingtin (Wong and Holzbaur, 2014a), a scaffolding protein that binds dynein directly (Caviston et al., 2007) and also interacts with dynactin (p150^Glued subunit) and kinesin through Huntingtin-associated protein 1 (HAP-1) (Engelender et al., 1997; Li et al., 1995; Li et al., 1998; McGuire et al., 2006; Twelvetrees et al., 2010). Huntingtin localizes to the outer membrane of autophagosomes (Atwal et al., 2007; Martinez-Vicente et al., 2010) and regulates autophagosome transport through its interactions with dynein and HAP-1 (Wong and Holzbaur, 2014a). Autophagosome axonal transport is also regulated by JIP1, which binds dynactin and kinesin-1 in a phosphorylation-dependent manner (Fu and Holzbaur, 2013), and also binds LC3 through its LIR domain. JIP1 binding to LC3 competitively disrupts JIP1-mediated activation of kinesin, inhibiting anterograde motility and favoring retrograde transport of autophagosomes (Fu et al., 2014). Thus, similar to other organelles, autophagosome axonal transport is tightly regulated by scaffolding proteins to sustain efficient transport, which is crucial for maintaining neuronal homeostasis.

Defective lysosomal and autolysosomal transport have been observed upon inhibition of lysosomal proteolysis, which leads to dystrophic swellings that are immunopositive for APP and characteristic of Alzheimer's disease (Lee et al., 2011). In neurons harboring α-synuclein aggregates induced by the addition of preformed α-synuclein fibrils, autophagosomes demonstrate decreased motility (Volpicelli-Daley et al., 2014). In Huntington's disease models, polyQ-htt disrupts autophagosome axonal transport, resulting in defects in compartment acidification and defective degradation of engulfed mitochondrial fragments (Wong and Holzbaur, 2014a).

By contrast, autophagosome transport was not disrupted in sensory neurons from early- or late-stage ALS model SOD1^G93A mice, despite the formation of SOD1^G93A aggregates along the axon (Maday et al., 2012). Thus, defects in autophagosome transport might be specific to certain neurodegenerative diseases. Alternatively, autophagosome transport might be disrupted in disease-specific neuronal populations (e.g. motor neurons in ALS). As defective transport leads to disrupted autophagosome maturation (Fu et al., 2014; Wong and Holzbaur, 2014a), transport defects might contribute to neuronal accumulation of autophagic cargo, such as damaged mitochondria or protein aggregates.

Autophagosome maturation and lysosomal fusion in disease

Autophagosome maturation involves fusion of the autophagosome with LAMP1-positive lysosomes that contain cathepsin proteases, leading to the formation of autolysosomes (Lee et al., 2011; Maday et al., 2012). Autophagosomes also gradually acidify as they undergo axonal transport, likely owing to lysosomal fusion and acquisition of the proton pump v-ATPase; the resulting acidification is necessary for cathepsin activation (Lee et al., 2011).

In brains of Alzheimer's disease patients, immature autophagosomes accumulate in dystrophic neurites (Nixon et al., 2005), potentially caused by defective cathepsin-mediated proteolysis or impaired autophagosome transport (Boland et al., 2008; Lee et al., 2011). Presenilin 1 mutations, the most common cause of familial Alzheimer's disease (Sherrington et al., 1995), disrupt the targeting of the v-ATPase V0a1 subunit from the ER to lysosomes (Lee et al., 2010b). As the v-ATPase is necessary for efficient acidification of both lysosomes and autophagosomes, this further implicates defects in the cellular degradation machinery in the pathogenesis of familial Alzheimer's disease.

In Parkinson's disease, wild-type α-synuclein aggregates impair autophagy by delaying autophagosome maturation (Tanik et al., 2013) and mutant α-synuclein A53T causes accumulation of autophagic-vesicular structures and impaired lysosomal hydrolysis (Stefanis et al., 2001). Parkinson's-disease-linked mutations in another gene, ATP13A2 (PARK9) that encode a lysosomal P-type ATPase, also lead to defects in lysosomal acidification, the processing of lysosomal enzymes and the clearance of autophagic substrates (Dehay et al., 2012; Usenovic et al., 2012). Finally, the Parkinson's-disease-associated protein LRRK2, which has been implicated in endosomal-lysosomal trafficking, localizes to autophagosomes and activates a Ca²⁺-dependent pathway that leads to increased autophagosome formation and a decreased number of acidic lysosomes (Alegre-Abarrategui et al., 2009; Gómez-Suaga et al., 2012; MacLeod et al., 2013).

By contrast, the ALS-associated protein ALS2 (also known as alsin) regulates endolysosomal trafficking, potentially by mediating the fusion between endosomes and autophagosomes (Hadano et al., 2010), and another ALS-associated protein, VCP, also regulates autophagosome maturation (Ju et al., 2009; Tresse et al., 2010). Furthermore, ALS-associated mutations in the protein CHMP2B disrupt autophagosome maturation by inhibiting the fusion of autophagosomes with multivesicular bodies (Cox et al., 2010; Filimonenko et al., 2007; Lee et al., 2007). More recently, the ALS and frontotemporal-dementia-associated protein C9ORF72 (DeJesus-Hernandez et al., 2011; Renton et al., 2011) has been implicated in endosomal trafficking (Farg et al., 2014).

As efficient lysosomal fusion and protease activity are crucial for autophagy, defects in this final step of autophagy can disrupt cargo degradation, even if previous steps in the autophagy pathway are functional. Thus, it will be important to further study the dynamics of autophagosome maturation and lysosomal fusion in neurons and whether these dynamics are impaired during neurodegeneration.

Conclusion

Recent studies demonstrate that constitutive autophagosome formation in neurons follows an ordered and spatially regulated pathway, with preferential formation at the axon tip. As cellular architectures differ among neuronal populations, the spatially restricted formation of autophagosomes might contribute to selective neuronal vulnerability during neurodegeneration. Autophagosomes subsequently mature by fusion with axonal lysosomes as they undergo retrograde axonal transport towards the cell body. This new model of neuronal autophagy provides a novel framework for understanding how defects in this pathway might contribute to neurodegeneration. As there is evidence for localized mitophagy along the axon and in the soma, it will also be interesting to further examine the regulation of locally induced autophagosome biogenesis in neurons.

Several key questions in the field of neuronal autophagy and degeneration still remain unresolved. Does the autophagosome initiation pathway that has been established in non-neuronal cells follow the same pathway in neurons? Does selective autophagy of lipids or ER occur in neurons? How might synaptic activity regulate the location and rate of autophagosome formation? Is autophagy differentially regulated in axons and dendrites? How effectively can autophagy be upregulated in response to protein aggregation or mitochondrial dysfunction? How is mitophagy regulated in neurons? What factors mediate autophagosome-lysosome fusion along the axon, and are these steps disrupted in disease? It will also be important to further study neuronal autophagy in vivo to better understand whether misregulated autophagy is upstream or downstream of the initial neurodegenerative insult and, ultimately, to determine whether modulation of autophagy is a viable therapeutic target for the treatment of neurodegenerative diseases.