Retrograde Axonal Autophagy and Endocytic Pathways Are Parallel and Separate in Neurons.

Autophagy and endocytic trafficking are two key pathways that regulate the composition and integrity of the neuronal proteome. Alterations in these pathways are sufficient to cause neurodevelopmental and neurodegenerative disorders. Thus, defining how autophagy and endocytic pathways are organized in neurons remains a key area of investigation. These pathways share many features and converge on lysosomes for cargo degradation, but what remains unclear is the degree to which the identity of each pathway is preserved in each compartment of the neuron. Here, we elucidate the degree of intersection between autophagic and endocytic pathways in axons of primary mouse cortical neurons of both sexes. Using microfluidic chambers, we labeled newly-generated bulk endosomes and signaling endosomes in the distal axon, and systematically tracked their trajectories, molecular composition, and functional characteristics relative to autophagosomes. We find that newly-formed endosomes and autophagosomes both undergo retrograde transport in the axon, but as distinct organelle populations. Moreover, these pathways differ in their degree of acidification and association with molecular determinants of organelle maturation. These results suggest that the identity of autophagic and newly endocytosed organelles is preserved for the length of the axon. Lastly, we find that expression of a pathogenic form of α-synuclein, a protein enriched in presynaptic terminals, increases merging between autophagic and endocytic pathways. Thus, aberrant merging of these pathways may represent a mechanism contributing to neuronal dysfunction in Parkinson's disease (PD) and related α-synucleinopathies.SIGNIFICANCE STATEMENT Autophagy and endocytic trafficking are retrograde pathways in neuronal axons that fulfill critical degradative and signaling functions. These pathways share many features and converge on lysosomes for cargo degradation, but the extent to which the identity of each pathway is preserved in axons is unclear. We find that autophagosomes and endosomes formed in the distal axon undergo retrograde transport to the soma in parallel and separate pathways. These pathways also have distinct maturation profiles along the mid-axon, further highlighting differences in the potential fate of transported cargo. Strikingly, expression of a pathogenic variant of α-synuclein increases merging between autophagic and endocytic pathways, suggesting that mis-sorting of axonal cargo may contribute to neuronal dysfunction in Parkinson's disease (PD) and related α-synucleinopathies.

Brain-derived neurotrophic factor stimulates the retrograde pathway for axonal autophagy.

Autophagy is a lysosomal degradation pathway important for neuronal development, function, and survival. How autophagy in axons is regulated by neurotrophins to impact neuronal viability and function is poorly understood. Here, we use live-cell imaging in primary neurons to investigate the regulation of axonal autophagy by the neurotrophin brain-derived neurotrophic factor (BDNF) and elucidate whether autophagosomes carry BDNF-mediated signaling information. We find that BDNF induces autophagic flux in primary neurons by stimulating the retrograde pathway for autophagy in axons. We observed an increase in autophagosome density and retrograde flux in axons, and a corresponding increase in autophagosome density in the soma. However, we find little evidence of autophagosomes comigrating with BDNF. In contrast, BDNF effectively engages its cognate receptor TrkB to undergo retrograde transport in the axon. These compartments, however, are distinct from LC3-positive autophagic organelles in the axon. Together, we find that BDNF stimulates autophagy in the axon, but retrograde autophagosomes do not appear to carry BDNF cargo. Thus, autophagosomes likely do not play a major role in relaying neurotrophic signaling information across the axon in the form of active BDNF/TrkB complexes. Rather, BDNF likely stimulates autophagy as a consequence of BDNF-induced processes that require canonical roles for autophagy in degradation.

The Endolysosomal System and Proteostasis: From Development to Degeneration.

How do neurons adapt their endolysosomal system to address the particular challenge of membrane transport across their elaborate cellular landscape and to maintain proteostasis for the lifetime of the organism? Here we review recent findings that address this central question. We discuss the cellular and molecular mechanisms of endolysosomal trafficking and the autophagy pathway in neurons, as well as their role in neuronal development and degeneration. These studies highlight the importance of understanding the basic cell biology of endolysosomal trafficking and autophagy and their roles in the maintenance of proteostasis within the context of neurons, which will be critical for developing effective therapies for various neurodevelopmental and neurodegenerative disorders.

Compartment-Specific Regulation of Autophagy in Primary Neurons.

UNLABELLED: Autophagy is an essential degradative pathway that maintains neuronal homeostasis and prevents axon degeneration. Initial observations suggest that autophagy is spatially regulated in neurons, but how autophagy is regulated in distinct neuronal compartments is unclear. Using live-cell imaging in mouse hippocampal neurons, we establish the compartment-specific mechanisms of constitutive autophagy under basal conditions, as well as in response to stress induced by nutrient deprivation. We find that at steady state, the cell soma contains populations of autophagosomes derived from distinct neuronal compartments and defined by differences in maturation state and dynamics. Axonal autophagosomes enter the soma and remain confined within the somatodendritic domain. This compartmentalization likely facilitates cargo degradation by enabling fusion with proteolytically active lysosomes enriched in the soma. In contrast, autophagosomes generated within the soma are less mobile and tend to cluster. Surprisingly, starvation did not induce autophagy in either the axonal or somatodendritic compartment. While starvation robustly decreased mTORC1 signaling in neurons, this decrease was not sufficient to activate autophagy. Furthermore, pharmacological inhibition of mammalian target of rapamycin with Torin1 also was not sufficient to markedly upregulate neuronal autophagy. These observations suggest that the primary physiological function of autophagy in neurons may not be to mobilize amino acids and other biosynthetic building blocks in response to starvation, in contrast to findings in other cell types. Rather, constitutive autophagy in neurons may function to maintain cellular homeostasis by balancing synthesis and degradation, especially within distal axonal processes far removed from the soma. SIGNIFICANCE STATEMENT: Autophagy is an essential homeostatic process in neurons, but neuron-specific mechanisms are poorly understood. Here, we compare autophagosome dynamics within neuronal compartments. Axonal autophagy is a vectorial process that delivers cargo from the distal axon to the soma. The soma, however, contains autophagosomes at different maturation states, including input received from the axon combined with locally generated autophagosomes. Once in the soma, autophagosomes are confined to the somatodendritic domain, facilitating cargo degradation and recycling of biosynthetic building blocks to primary sites of protein synthesis. Neuronal autophagy is not robustly upregulated in response to starvation or mammalian target of rapamycin inhibition, suggesting that constitutive autophagy in neurons maintains homeostasis by playing an integral role in regulating the quality of the neuronal proteome.

Ordered recruitment of dynactin to the microtubule plus-end is required for efficient initiation of retrograde axonal transport.

Long-range retrograde axonal transport in neurons is driven exclusively by the microtubule motor cytoplasmic dynein. The efficient initiation of dynein-mediated transport from the distal axon is critical for normal neuronal function, and neurodegenerative disease-associated mutations have been shown to specifically disrupt this process. Here, we examine the role of dynamic microtubules and microtubule plus-end binding proteins (+TIPs) in the initiation of dynein-mediated retrograde axonal transport using live-cell imaging of cargo motility in primary mouse dorsal root ganglion neurons. We show that end-binding (EB)-positive dynamic microtubules are enriched in the distal axon. The +TIPs EB1, EB3, and cytoplasmic linker protein-170 (CLIP-170) interact with these dynamic microtubules, recruiting the dynein activator dynactin in an ordered pathway, leading to the initiation of retrograde transport by the motor dynein. Once transport has initiated, however, neither the EBs nor CLIP-170 are required to maintain transport flux along the mid-axon. In contrast, the +TIP Lis1 activates transport through a distinct mechanism and is required to maintain processive organelle transport along both the distal and mid-axon. Further, we show that the EB/CLIP-170/dynactin-dependent mechanism is required for the efficient initiation of transport from the distal axon for multiple distinct cargos, including mitochondria, Rab5-positive early endosomes, late endosomes/lysosomes, and TrkA-, TrkB-, and APP-positive organelles. Our observations indicate that there is an essential role for +TIPs in the regulation of retrograde transport initiation in the neuron.

Molecular cascade reveals sequential milestones underlying hippocampal neural stem cell development into an adult state.

Quiescent adult neural stem cells (NSCs) in the mammalian brain arise from proliferating NSCs during development. Beyond acquisition of quiescence, an adult NSC hallmark, little is known about the process, milestones, and mechanisms underlying the transition of developmental NSCs to an adult NSC state. Here, we performed targeted single-cell RNA-seq analysis to reveal the molecular cascade underlying NSC development in the early postnatal mouse dentate gyrus. We identified two sequential steps, first a transition to quiescence followed by further maturation, each of which involved distinct changes in metabolic gene expression. Direct metabolic analysis uncovered distinct milestones, including an autophagy burst before NSC quiescence acquisition and cellular reactive oxygen species level elevation along NSC maturation. Functionally, autophagy is important for the NSC transition to quiescence during early postnatal development. Together, our study reveals a multi-step process with defined milestones underlying establishment of the adult NSC pool in the mammalian brain.

Beyond housekeeping: autophagy regulates PKA signaling at synapses.

Autophagy modulates synaptic function and plasticity, but the molecular basis for this process is largely unknown. A recent tour de force study by Overhoff and colleagues identifies a novel role for autophagy in regulating PKA signaling at synapses to modulate the organization of the postsynaptic proteome and neuronal excitability.

Proteasomal inhibition preferentially stimulates lysosome activity relative to autophagic flux in primary astrocytes.

Neurons and astrocytes face unique demands on their proteome to enable proper function and survival of the nervous system. Consequently, both cell types are critically dependent on robust quality control pathways such as macroautophagy (hereafter referred to as autophagy) and the ubiquitin-proteasome system (UPS). We previously reported that autophagy is differentially regulated in astrocytes and neurons in the context of metabolic stress, but less is understood in the context of proteotoxic stress induced by inhibition of the UPS. Dysfunction of the proteasome or autophagy has been linked to the progression of various neurodegenerative diseases. Therefore, in this study, we explored the connection between autophagy and the proteasome in primary astrocytes and neurons. Prior studies largely in non-neural models report a compensatory relationship whereby inhibition of the UPS stimulates autophagy. To our surprise, inhibition of the proteasome did not robustly upregulate autophagy in astrocytes or neurons. In fact, the effects on autophagy are modest particularly in comparison to paradigms of metabolic stress. Rather, we find that UPS inhibition in astrocytes induces formation of Ub-positive aggregates that harbor the selective autophagy receptor, SQSTM1/p62, but these structures were not productive substrates for autophagy. By contrast, we observed a significant increase in lysosomal degradation in astrocytes in response to UPS inhibition, but this stimulation was not sufficient to reduce total SQSTM1 levels. Last, UPS inhibition was more toxic in neurons compared to astrocytes, suggesting a cell type-specific vulnerability to proteotoxic stress.Abbreviations: Baf A1: bafilomycin A1; CQ: chloroquine; Epox: epoxomicin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; p-ULK1: phospho-ULK1; SQSTM1/p62: sequestosome 1; Ub: ubiquitin; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system.

Autophagy: Identification of MTMR5 as a neuron-enriched suppressor.

A puzzle of autophagy in neurons is that, unlike in other cells, it is not robustly induced by inhibition of mammalian target of rapamycin (mTOR). A new study now solves this conundrum and establishes that myotubularin-related phosphatase 5 limits the induction of neuronal autophagy by mTOR inhibitors.

Organization of the autophagy pathway in neurons.

Macroautophagy (hereafter referred to as autophagy) is an essential quality-control pathway in neurons, which face unique functional and morphological challenges in maintaining the integrity of organelles and the proteome. To overcome these challenges, neurons have developed compartment-specific pathways for autophagy. In this review, we discuss the organization of the autophagy pathway, from autophagosome biogenesis, trafficking, to clearance, in the neuron. We dissect the compartment-specific mechanisms and functions of autophagy in axons, dendrites, and the soma. Furthermore, we highlight examples of how steps along the autophagy pathway are impaired in the context of aging and neurodegenerative disease, which underscore the critical importance of autophagy in maintaining neuronal function and survival.

Synaptic activity controls autophagic vacuole motility and function in dendrites.

Macroautophagy (hereafter "autophagy") is a lysosomal degradation pathway that is important for learning and memory, suggesting critical roles for autophagy at the neuronal synapse. Little is known, however, about the molecular details of how autophagy is regulated with synaptic activity. Here, we used live-cell confocal microscopy to define the autophagy pathway in primary hippocampal neurons under various paradigms of synaptic activity. We found that synaptic activity regulates the motility of autophagic vacuoles (AVs) in dendrites. Stimulation of synaptic activity dampens AV motility, whereas silencing synaptic activity induces AV motility. Activity-dependent effects on dendritic AV motility are local and reversible. Importantly, these effects are compartment specific, occurring in dendrites and not in axons. Most strikingly, synaptic activity increases the presence of degradative autolysosomes in dendrites and not in axons. On the basis of our findings, we propose a model whereby synaptic activity locally controls AV dynamics and function within dendrites that may regulate the synaptic proteome.

A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells.

The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1's arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.

Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways.

NgCAM is a cell adhesion molecule that is largely axonal in neurons and apical in epithelia. In Madin-Darby canine kidney cells, NgCAM is targeted to the apical surface by transcytosis, being first inserted into the basolateral domain from which it is internalized and transported to the apical domain. Initial basolateral transport is mediated by a sequence motif (Y(33)RSL) decoded by the AP-1B clathrin adaptor complex. This motif is a substrate in vitro for tyrosine phosphorylation by p60src, a modification that disrupts NgCAM's ability to interact with clathrin adaptors. Based on the behavior of various NgCAM mutants, it appears that after arrival at the basolateral surface, the AP-1B interaction site is silenced by phosphorylation of Tyr(33). This slows endocytosis and inhibits basolateral recycling from endosomes, resulting in NgCAM transcytosis due to a cryptic apical targeting signal in its extracellular domain. Thus, transcytosis of NgCAM and perhaps other membrane proteins may reflect the spatial regulation of recognition by adaptors such as AP-1B.

Differential regulation of autophagy during metabolic stress in astrocytes and neurons.

Macroautophagy/autophagy is a key homeostatic process that targets cytoplasmic components to the lysosome for breakdown and recycling. Autophagy plays critical roles in glia and neurons that affect development, functionality, and viability of the nervous system. The mechanisms that regulate autophagy in glia and neurons, however, are poorly understood. Here, we define the molecular underpinnings of autophagy in primary cortical astrocytes in response to metabolic stress, and perform a comparative study in primary hippocampal neurons. We find that inducing metabolic stress by nutrient deprivation or pharmacological inhibition of MTOR (mechanistic target of rapamycin kinase) robustly activates autophagy in astrocytes. While both paradigms of metabolic stress dampen MTOR signaling, they affect the autophagy pathway differently. Further, we find that starvation-induced autophagic flux is dependent on the buffering system of the starvation solution. Lastly, starvation conditions that strongly activate autophagy in astrocytes have less pronounced effects on autophagy in neurons. Combined, our study reveals the complexity of regulating autophagy in different paradigms of metabolic stress, as well as in different cell types of the brain. Our findings raise important implications for how neurons and glia may collaborate to maintain homeostasis in the brain. ABBREVIATIONS: ACSF: artificial cerebrospinal fluid; baf A1: bafilomycin A1; EBSS: earle's balanced salt solution; GFAP: glial fibrillary acidic protein; Glc: glucose; GM: glial media; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; p-RPS6: phospho-RPS6; p-ULK1: phospho-ULK1; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; ULK1: unc-51-like kinase 1.

The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains.

Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD mu1A or mu1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B-containing membranes that were both distinct from AP-1A-positive TGN elements and more closely apposed to transferrin receptor-positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.

Methods for Imaging Autophagosome Dynamics in Primary Neurons.

Autophagy is an essential degradative pathway that maintains neuronal homeostasis and prevents axon degeneration. However, the mechanisms of autophagy in neurons are only beginning to be understood. To address this fundamental gap in knowledge, we have established several key methodologies for live-cell imaging and quantitative analysis of autophagy in primary hippocampal neurons. Using these methods, we have defined compartment-specific dynamics of autophagy in real-time under basal versus stress conditions. For example, we have characterized autophagosome biogenesis in the distal axon and subsequent retrograde transport to the soma for degradation. Autophagosomes are also generated locally within the soma. In contrast to the axon, the majority of autophagosomes in dendrites are stationary, while some exhibit bidirectional movement. These studies establish an initial road map for autophagosome dynamics in each compartment of the neuron and set the stage for a more detailed understanding of neuronal autophagy in stress and disease.

Neuronal autophagy and intercellular regulation of homeostasis in the brain.

Neurons are particularly dependent on robust quality control pathways to maintain cellular homeostasis and functionality throughout their extended lifetime. Failure to regulate protein and organelle integrity is linked to devastating neurodegenerative diseases. Autophagy is a lysosomal degradation pathway that maintains homeostasis by recycling damaged or aged cellular components. Autophagy has important functions in development of the nervous system, as well as in neuronal function and survival. In fact, defects in autophagy underlie neurodegeneration in mice and humans. Here, we review the compartment-specific dynamics and functions for autophagy in neurons. Emerging evidence suggests novel pathways for the intercellular coordination of quality control pathways between neurons and glia to maintain homeostasis in the brain.

Neuronal endosomes to lysosomes: A journey to the soma.

How are lysosomal degradation pathways spatially organized in the complex landscape of a neuron? Cheng et al. (2018. J Cell Biol. https://doi.org/10.1083/jcb.201711083) and Yap et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201711039) characterize the distribution and function of endolysosomal organelles in neurons, providing insights into compartment-specific mechanisms regulating the neuronal proteome.

Compartment-specific dynamics and functions of autophagy in neurons.

Autophagy is a lysosomal degradation pathway that is critical to maintaining neuronal homeostasis and viability. Autophagy sequesters damaged and aged cellular components from the intracellular environment, and shuttles these diverse macromolecules to lysosomes for destruction. This active surveillance of the quality of the cytoplasm and organelles is essential in neurons to sustain their long-term functionality and viability. Indeed, defective autophagy is linked to neurodevelopmental abnormalities and neurodegeneration in mammals. Here, we review the mechanisms of autophagy in neurons and functional roles for autophagy in neuronal homeostasis. We focus on the compartment-specific dynamics of autophagy in neurons, and how autophagy might perform non-canonical functions critical for neurons. We suggest the existence of multiple populations of autophagosomes with compartment-specific functions important for neural activity and function. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 298-310, 2018.

Mechanisms of neuronal homeostasis: Autophagy in the axon.

Autophagy is an evolutionarily conserved lysosomal degradation pathway that removes damaged organelles and protein aggregates from the cytoplasm. Being post-mitotic, neurons are particularly vulnerable to the accumulation of proteotoxins and are thus heavily dependent on autophagy to maintain homeostasis. In fact, CNS-specific and neuron-specific loss of autophagy is sufficient to cause neurodegeneration in mice. Further, mutations in genes that encode PINK1 and Parkin, proteins that selectively remove damaged mitochondria, cause Parkinson's disease, linking defective autophagy with neurodegenerative disease in humans. This review provides an overview of the mechanisms of autophagy in the axon and the role of neuronal autophagy in axonal homeostasis and degeneration. The pathway for autophagosome biogenesis and maturation along the axon will be discussed as well as several key insights revealing the diverse functions of axonal autophagy. Evidence linking altered autophagy with axonal degeneration and neuronal death will be presented. Appropriate manipulation of autophagy may lead to promising therapeutics for neurodegenerative diseases. This article is part of a Special Issue entitled SI:Autophagy.