Vesicular degradation pathways in neurons: at the crossroads of autophagy and endo-lysosomal degradation

Abstract

Autophagy and endo-lysosomal degradation are two parallel degradation pathways essential for maintaining neuronal health and function. Autophagosomes and endosomes sequester cellular cargo through different mechanisms, but these pathways converge upon fusion with lysosomes. Both pathways are spatially regulated, with distinct features evident in the soma, axons and dendrites, possibly as an adaptation to the unique morphology of neurons and the specific demands of each compartment. Relatively little is known about how autophagy and endo-lysosomal degradation interact and how their activities may be coordinated. We review our current understanding of autophagy and endo-lysosomal degradation in neurons, highlighting common features and differences as well as the intersection of these two essential cellular pathways.

Introduction

Neurons are highly dependent on intracellular degradation pathways to prevent the accumulation of dysfunctional organelles and proteins. Lysosomal degradation in neurons is mediated by two major pathways: macroautophagy (hereafter autophagy) and endo-lysosomal degradation. Both pathways are essential for neuronal health. Genetic disruption of either pathway results in protein aggregation and neurodegeneration [1–4]. While autophagy and endo-lysosomal degradation both lead to the degradation of cargo via fusion with lysosomes, they differ in mechanisms of cargo uptake. Autophagy begins with the engulfment of cytoplasmic cargo into a double-membrane vesicle called an autophagosome. Endo-lysosomal degradation initiates during endosome maturation, when membrane proteins are sorted into vesicles that bud from the membrane into the lumen of the endosome. Endosomes can also take up cytosolic cargo into intralumenal vesicles through a process called endosomal microautophagy [5].

Recent reviews have addressed either autophagy or endo-lysosomal degradation [6–9]. Here, we focus on the intersection and interplay between these pathways, highlighting both overlapping and unique functions.

Mechanisms of cargo uptake into autophagosomes and late endosomes

Autophagosomes engulf cargo in bulk or in a selective manner. Autophagosome formation is orchestrated by a conserved pathway of autophagy-related (ATG) proteins. Knockout of key ATG proteins, such as ATG5 or ATG7, are sufficient to induce neurodegeneration in a cell-autonomous manner [3,4], indicating the importance of this pathway for neuronal health.

Bulk autophagy (Figure 1A) initiates at ER subdomains that are enriched for phosphatidylinositol 3-phosphate (PI(3)P). Two key protein complexes interact in the initiation of autophagosome formation: the ULK complex and the phosphatidylinositol 3-kinase complex. The ULK complex activates the phosphatidylinositol 3-kinase complex by phosphorylation. The activated PI(3)-kinase complex then generates localized PI(3)P on the ER membrane, resulting in a PI(3)P-enriched subdomain called the omegasome. The local enrichment of PI(3)P recruits the effector protein WIPI2B, which in turn recruits the ATG12–5-16L E3-like complex that mediates elongation of the autophagosomal membrane to a cup-shaped structure called the phagophore. Following elongation, the phagophore membrane seals to form a vesicle that completely sequesters the engulfed material. LC3, a canonical marker for autophagosomes, is lipidated and incorporated into the growing autophagosome membrane [6]. There are multiple related proteins in the LC3/GABARAP family that are expressed in vertebrate neurons, including LC3A, LC3B, LC3C (LC3C is expressed in humans but not mice), Gamma-aminobutyric acid receptor-associated protein (GABARAP), GABARAP-like 1 (GABARAPL1/GEC1) and GABARAP-like 2 (GABARAPL2/GATE16) [10], although it remains unclear whether these proteins have overlapping or distinct functions in neuronal autophagy.

Figure 1: Mechanisms of cargo uptake into autophagosomes and endosomes.

(A) Bulk Autophagy. Autophagosome formation starts in ER subdomains enriched for PI(3)P (Initiation). The PI(3)P effector protein WIPI2B recruits downstream effector proteins such as the ATG12–5-16L complex, and thereby mediates elongation of the autophagosomal membrane (Elongation). Autophagosomal cargo is delivered to lysosomes for degradation (Fusion). (B) Selective Autophagy. Selective autophagy of mitochondria (mitophagy) is initiated via the PINK1/Parkin pathway. Parkin mediates ubiquitination of mitochondrial outer membrane proteins. Autophagy adaptors recognize the polyubiquitin chains and link the damaged mitochondrion to LC3 in the growing autophagosomal membrane. (C) Uptake of membrane-associated cargo in the endo-lysosomal pathway. During maturation of RAB5 positive early endosomes, the ESCRT complex mediates inward-budding of ubiquitinated parts of the endosomal membrane. The resulting late endosome fuses with lysosomes for degradation of the intralumenal vesicles. (D) Endosomal Microautophagy. The ESCRT mediates invagination of the endosomal membrane for uptake of cytosolic cargo. Endosomal microautophagy selectively sequesters cytosolic proteins with a KFERQ-like motif that are bound to Hsc70. Damaged mitochondria can be invaginated into endosomes via a Parkin- and ESCRT-dependent mechanism.

Bulk autophagy occurs constitutively in neurons throughout development, both in vitro [11,12] and in vivo [13,14], in a spatially-localized manner in the growth cone and at synaptic sites [14,15]. Engulfed cargos include mitochondrial fragments and aggregated proteins [12,16], but there is no evidence for selective uptake or the involvement of specific receptors.

Selective autophagy (Figure 1B) in neurons occurs via a pathway that is distinct both molecularly and spatially from the bulk uptake characteristic of axonal autophagy [17]. The selective removal of protein aggregates starts with their polyubiquitination. Autophagy receptors such as p62 and optineurin (OPTN) recognize polyubiquitin chains and drive autophagosome formation around the aggregate by linking polyubiquitinated substrates to LC3/GABARAPs in the growing autophagosomal membrane [18].

The selective degradation of dysfunctional mitochondria is known as mitophagy. PINK1 is a kinase that is usually rapidly degraded but accumulates more stably on the outer membrane of damaged mitochondria. The PINK1-dependent phosphorylation of ubiquitin recruits the E3-ubiquitin ligase Parkin, which mediates the polyubiquitination of mitochondrial outer membrane proteins. Autophagy receptors including OPTN, NPD52, and TAX1BP1 recognize the resulting polyubiquitin chains [19–21] and induce the formation of an autophagosome around a damaged mitochondrion [21,22]. In neurons, PINK1- and Parkin-dependent mitophagy has been observed in the axon [23], but is more commonly observed in the soma [11,17].

Endo-lysosomal degradation (Figure 1C) starts with RAB5-positive early endosomes. Endosomal membrane proteins are targeted for degradation by ubiquitination. During maturation of early endosomes to RAB7-positive late endosomes, the Endosomal Sorting Complex Required for Transport (ESCRT) machinery segregates ubiquitinated cargo into domains of the endosomal membrane and generates inward-budding vesicles [24].

In addition, cytosolic cargo can be internalized into the late endosome by endosomal microautophagy (Figure 1D). The ESCRT machinery mediates invagination of the endosomal membrane into intralumenal vesicles that contain trapped cytosolic cargo. Endosomal microautophagy of cytosolic proteins is mediated by binding of heat shock cognate 70 kDa protein (Hsc70) to a KFERQ-like pentapeptide motif in the substrate protein [5,25]. Neuronal endosomal microautophagy of synaptic proteins has been described in Drosophila [26]. Interestingly, a study in mouse embryonic fibroblasts reported that RAB5-positive endosomes can also sequester damaged mitochondria via the ESCRT machinery and deliver them for lysosomal degradation [27]. It is currently not known whether endosome-mediated degradation of mitochondria also occurs in neurons.

Degradative pathways in the developing axon

Autophagosomal and endo-lysosomal degradation occur in parallel in developing axons [28]. Live-imaging studies showed continuous autophagosome formation in the growth cone of DRG neurons, likely driving the constitutive bulk removal of cytosolic proteins and organelles from this high-turnover compartment [12,15,29]. Distal formation of axonal autophagosomes has been confirmed in vivo in Drosophila and C. elegans, where bulk autophagy is required for the assembly of pre-synaptic compartments [13,14]. Interestingly, autophagy negatively affects the length of the axon during neurodevelopment. Loss-of-function mutations in several ATG genes increased outgrowth of the nociceptive sensory neuron PVD in C. elegans [13]. Similarly, inhibition of autophagy by ATG7 knockdown caused elongation of axons in isolated rat cortical neurons, while activation of autophagy by rapamycin suppressed axon growth [30]. A possible explanation is that knockdown of autophagy decreases the bulk removal of cytoplasmic components via autophagosomes and thereby increases the amount of available cellular material for axon outgrowth at the distal tip. It is unclear whether inhibition of autophagy in outgrowing axons has any detrimental effects, such as accumulation of defective organelles or proteins.

Selective macroautophagy also plays a vital role in developing axons. The autophagy-linked FYVE protein (Alfy) is a scaffold protein that selectively recruits the autophagic machinery to ubiquitinated proteins [31]. Loss of Alfy results in axon guidance defects and perinatal lethality in mice [31]. This suggests that loss of selective protein degradation via autophagy cannot easily be compensated through the endo-lysosomal pathway.

Recent observations have shed new light on the spatially-specific regulation of endo-lysosomal degradation in developing neurons. Live-imaging of the developing visual system of the Drosophila brain demonstrated the sorting of synaptic vesicle (SV) proteins and plasma membrane proteins into two distinct content-specific ‘hub’ compartments [28]. Both hubs were located at axon terminals and underwent continuous budding of smaller vesicles that were subsequently transported retrogradely towards the cell soma. The low pH and the presence of the Cathepsin-L-like protease CP1 suggest that these hub compartments may have degradative properties [28,32]. Remarkably, autophagosomes formed in parallel in the distal axon but did not show any interaction with the endosomal hubs [28]. These findings suggest that a subset of endosomal cargo is degraded locally in the distal axon, in contrast to autophagic cargos which have been shown to be transported back to the cell soma, maturing en route to autophagolyosomes competent to degrade cargos [12,16].

Local degradation of membrane proteins could help recycling membrane compartments at the distal tip and compensate for the high need of lipids to support membrane extension during axonal outgrowth. It is unclear whether the observed hub compartments are specific for developing axons or if they can also be detected in synaptically connected neurons. It is also unknown if there is a sorting or signaling mechanism that determines whether proteins are degraded in hubs, engulfed by autophagosomes or sequestered into late endosomes and transported towards the cell body. Further work is needed to clarify the specific role of each degradation pathway during neuronal development.

Degradation in the presynaptic compartment

Mitochondria are critical sources of energy in the presynaptic compartment. Defective mitochondria are removed via selective mitophagy through the PINK1/Parkin pathway [17]. In neurons, the primary site for mitophagy remains controversial. Ashrafi et al. observed PINK1/Parkin dependent translocation of the autophagy machinery to damaged mitochondria in the axon [23]. Thus, one would expect accumulation of mitochondria in the axonal compartment upon PINK1/Parkin knockout. However, mitochondria in Parkin-deficient flies do not accumulate in the axon but in the cell body [33]. Findings from Lin et al. [34] are in line with mitophagy primarily occurring in the soma. Lin et al. observed that stressed mitochondria release the anchoring protein syntaphilin and are subsequently transported to the cell body [34].

Recent work in mouse embryonic fibroblasts suggests that RAB5-positive endosomes can take up defective mitochondria and deliver them for lysosomal degradation. Upon Parkin-dependent ubiquitination, mitochondria are engulfed through ESCRT-mediated invagination of the endosomal membrane [27]. Interestingly, activation of endosome-mediated clearance of mitochondria preceded activation of mitophagy. Inhibition of the endosomal pathway led to increased autophagic activity. This suggests that autophagy may compensate for impairment of endosomal mitochondria degradation [27]. However, it is yet unclear whether degradation of mitochondria via endosomes also takes place in neurons.

Both autophagy and endo-lysosomal degradation are involved in the degradation of SV proteins (Figure 2) [35]. Using Botulinum Neurotoxin as a marker for SVs, Wang et al. showed that synaptic activity increased autophagosome formation at the synapse and stimulated SV retrograde transport to the cell soma [36]. Several synaptic proteins are involved in autophagosome formation. Synaptojanin and Endophilin A are mostly known for their role in endocytosis and SV recycling. Studies revealed an additional function of both proteins in autophagosome formation. Upon phosphorylation, Endophilin A mediates the formation of highly curved membranes that function as docking sites for core proteins of autophagosome biogenesis [37,38]. Synaptojanin has been shown to remove the PI(3)P effector WIPI2B from growing autophagosomes. Normally, WIPI2B is first recruited to the growing autophagosome but needs to be released back into the cytosol at later stages of autophagosome formation. Synaptojanin-deficient neurons accumulate WIPI2B-positive nascent autophagosomes that cannot mature into functional autophagosomes [39]. Together, these studies indicate that a specialized molecular machinery controls autophagosome formation at the presynapse and adjusts autophagosomal degradation to the changing local demands.

Figure 2: Autophagy and endo-lysosmal degradation in the presynapse.

(A) Schematic representation of autophagy in the presynaptic compartment. Bassoon regulates the level of presynaptic activity by sequestering ATG5 from autophagosome formation. Endophilin A (EndoA) mediates the formation of highly curved membranes that serve as docking sites for core proteins of autophagosome biogenesis. Synaptojanin removes WIPI2B from nascent autophagosomes. This step is necessary for autophagosome maturation. (B) Endo-lysosomal degradation in the presynaptic compartment. RAB35 recruits the ESCRT machinery and mediates inward-budding of ubiquitinated parts of the endosomal membrane as intralumenal vesicles. The active zone proteins Bassoon and Piccolo control the ubiquitination of SV proteins by inhibiting the E3 ubiquitin ligase Siah1. Cytosolic proteins at the synapse with a KFERQ-like motif can be degraded via endosomal microautophagy.

There is compelling evidence that in addition to presynaptic autophagy, the endo-lysosomal pathway also plays an important role in mediating basal and activity-dependent turnover of SV proteins (Figure 2B). RAB35 and its GTPase activating protein Skywalker are key players in sorting SV proteins for endo-lysosomal degradation [40]. Synaptic stimulation activates RAB35, which recruits the ESCRT machinery and mediates the formation of intraluminal vesicles [41]. Notably, neuronal activity only increased degradation of a subset of SV proteins. It augmented degradation of SV2 and VAMP2 whereas degradation of Synaptotagmin1 and SNAP-25 remained unchanged [41]. This suggests that SV proteins are not randomly selected for endo-lysosomal degradation. It is interesting to speculate that SV2 and VAMP2 may become particularly vulnerable to structural damage through frequent use and are therefore preferentially ubiquitinated. It is unclear whether RAB35 and Skywalker are only involved in targeting SV proteins for endo-lysosomal degradation or if they also participate in the sorting of other plasma membrane proteins [42].

The presynaptic active zone proteins Bassoon and Piccolo control the ubiquitination and endo-lysosomal degradation of SV proteins. Bassoon and Piccolo inhibit the E3 ubiquitin ligase Siah1 via their zinc finger domains [43]. Double knockdown of Bassoon and Piccolo causes aberrant degradation of multiple SV proteins and synapse degeneration [43]. Interestingly, Bassoon also controls autophagy at the presynaptic site. Binding of the Bassoon CC2 domain to ATG5 inhibits the induction of autophagy. Loss of Bassoon causes overactivation of presynaptic autophagy [44]. These studies suggest that Bassoon negatively regulates both autophagy and endo-lysosomal degradation at the presynaptic site.

While it is evident that both autophagy and endo-lysosomal degradation participate in the removal of SV proteins, it is unclear how their activities are coordinated. Are certain SV proteins selectively removed by the autophagosomal or by the endo-lysosomal pathway? If so, can one pathway compensate for the loss of the other? Given that inhibition of either pathway results in neurodegeneration, complete compensation is unlikely, but partial compensation may be possible.

Turnover in dendritic and postsynaptic compartments

Yap et al. investigated the spatial organization of the endo-lysosomal system in dendrites and soma [45]. Degradative Cathepsin B/D positive lysosomes were found to be concentrated in the soma and proximal dendritic segment (< 25 μm from the soma boundary) while early endosomes and late endosomes were present throughout the dendrite [45]. Accumulation of degradative lysosomes in the neuronal soma was also observed by other groups [46–48]. Yap et al. proposed a model in which RAB7-positive late endosomes transport cargo retrogradely to proximal dendrite and soma, where degradation occurs upon fusion with Cathepsin B/D-positive lysosomes [45]. In contrast, two other studies described degradative lysosomes also in distal parts of the dendrite [49,50]. Both studies observed Lyso-Tracker positive organelles throughout the dendrite and confirmed their identity as degradative lysosomes using GPN, a substrate for Cathepsin C that induces osmotic lysis of lysosomes upon cleavage [49,50]. The different markers used (Cathepsin B/D staining vs. Lyso-Tracker and GPN) may explain the seemingly contradictory findings. Degradative lysosomes in the distal dendrite could represent a specialized population that serves specific functions in synaptically connected dendrites [49].

There are remarkable differences between dendritic and axonal autophagy. While autophagosomes form constitutively in the distal axon and are processively transported in the retrograde direction, autophagosomes form infrequently in dendrites and are mostly stationary or move bidirectionally [15,29]. The distinct behavior of autophagosomes in dendrites and the axon could be related to different functions in each compartment. A specific function of dendritic autophagy is to regulate the morphology and density of dendritic spines. Tang et al. showed that autophagy is essential for postnatal pruning of dendritic spines [51]. Inhibition of autophagy causes an increased spine density, resembling the synaptic pathology of autism spectrum disorders [51]. Similarly, Schäffner et al. found that in adult-born hippocampal neurons, impairment of autophagosomal flux led to altered dendritic morphology, increased spine density and aberrant spine positioning [52]. Nikoletopoulou et al. identified three scaffold proteins of dendritic spines (PSD-95, SHANK3 and PICK1) as autophagosomal cargo in the mature brain [53]. Aberrant activation of autophagy caused defects in long-term potentiation, indicating a role for dendritic autophagy in the process of learning and memory formation. Notably, the endo-lysosomal system has also been shown to be involved in regulating dendritic spine plasticity. Back-propagating dendritic action potentials caused calcium release from lysosomes, leading to exocytosis of lysosomal Cathepsin B and activation of extracellular matrix metalloproteinase 9 (MMP9). MMP9-mediated remodeling of the extracellular matrix allowed enlargement of dendritic spine heads for long-lasting structural plasticity [50].

Open questions

Both autophagy and the endo-lysosomal pathway converge at the point of cargo delivery to lysosomes. While our knowledge about each pathway is increasing, relatively little is known about how autophagy and endo-lysosomal degradation are related to each other. How big is the overlap in cargo content? Does each pathway have a specific function, or can autophagy and endo-lysosomal degradation compensate for each other? Further work is necessary to study the coordination of autophagy and endo-lysosomal degradation and elucidate their distinct roles in the different neural compartments. This will improve our understanding of how their dysfunction contributes to the pathogenesis of neurodegenerative diseases.

Highlights.

• Autophagy and endo-lysosomal degradation are essential for neuronal health.

• Autophagosomes and endosomes both deliver cargo for lysosomal degradation.

• Both pathways show distinct features in the neuronal soma, dendrites and axon.

• The two pathways have overlapping and unique functions in neurons.