Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons

Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis.     

Snapin deficiency is associated with developmental defects of the central nervous system

The autophagy-lysosomal pathway is an intracellular degradation process essential for maintaining neuronal homoeostasis. Defects in this pathway have been directly linked to a growing number of neurodegenerative disorders. We recently revealed that Snapin plays a critical role in co-ordinating dynein-driven retrograde transport and late endosomal-lysosomal trafficking, thus maintaining efficient autophagy-lysosomal function. Deleting snapin in neurons impairs lysosomal proteolysis and reduces the clearance of autolysosomes. The role of the autophagy-lysosomal system in neuronal development is, however, largely uncharacterized. Here, we report that snapin deficiency leads to developmental defects in the central nervous system. Embryonic snapin-/- mouse brain showed reduced cortical plates and intermediate zone cell density, increased apoptotic death in the cortex and third ventricle, enhanced membrane-bound LC3-II staining associated with autophagic vacuoles and an accumulation of polyubiquitinated proteins in the cortex and hippocampus. Thus our results provide in vivo evidence for the essential role of late endocytic transport and autophagy-lysosomal function in maintaining neuronal survival and development of the mammalian central nervous system. In addition, our study supports the existence of a functional interplay between the autophagy-lysosome and ubiquitin-proteasome systems in the protein quality-control process.     

Nudel Promotes Axonal Lysosome Clearance and Endo-lysosome Formation via Dynein-Mediated Transport

Axonal transport is critical for neuronal function and survival. Cytoplasmic dynein and its accessory complex dynactin form a microtubule minus end-directed motor in charge of retrograde transport. In this study, we show that Nudel, a dynein regulator, was highly expressed in dorsal root ganglion (DRG) neurons. Microinjection of anti-Nudel antibody into cultured DRG neurons abolished retrograde transport of membranous organelles in the axon and led to dispersions of Golgi cisternae in the soma. As a result, lysosomes, which are normally enriched in the soma, moved persistently into and thus accumulated in axons. Endo-lysosome formation was also markedly delayed. As anterograde motility of mitochondria was not inhibited, the antibody apparently did not abolish retrograde transport by destructing axonal microtubule tracks. Similar results were obtained by microinjecting N-terminal Nudel, anti-dynein antibody or a p150^Glued mutant capable of abrogating the dynein–dynactin association. These results indicate a critical role of Nudel in dynein-mediated axonal transport. Moreover, the effects of dynein on endolysosome formation and regional sequestration of lysosomes may contribute to defects in the endocytic pathway seen in neurons of patients or animals with malfunction of dynein.     

Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons

Mitochondria are essential for neuronal survival and function. Proper degradation of aged and damaged mitochondria through mitophagy is a key cellular pathway for mitochondrial quality control. Recent studies have indicated that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Translocation of Parkin to damaged mitochondria induces mitophagy in many nonneuronal cell types. However, evidence showing Parkin translocation in primary neurons is controversial, leaving unanswered questions as to how and where Parkin-mediated mitophagy occurs in neurons. Here, we report the unique process of dissipating mitochondrial Δψ(m)-induced and Parkin-mediated mitophagy in mature cortical neurons. Compared with nonneuronal cells, neuronal mitophagy is a much slower and compartmentally restricted process, coupled with reduced anterograde mitochondrial transport. Parkin-targeted mitochondria are accumulated in the somatodendritic regions where mature lysosomes are predominantly located. Time-lapse imaging shows dynamic formation and elimination of Parkin- and LC3-ring-like structures surrounding depolarized mitochondria through the autophagy-lysosomal pathway in the soma. Knocking down Parkin in neurons impairs the elimination of dysfunctional mitochondria. Thus, our study provides neuronal evidence for dynamic and spatial Parkin-mediated mitophagy, which will help us understand whether altered mitophagy contributes to pathogenesis of several major neurodegenerative diseases characterized by mitochondrial dysfunction and impaired transport.     

Axonal autophagosomes recruit dynein for retrograde transport through fusion with late endosomes

Efficient degradation of autophagic vacuoles (AVs) via lysosomes is an important cellular homeostatic process. This is particularly challenging for neurons because mature acidic lysosomes are relatively enriched in the soma. Although dynein-driven retrograde transport of AVs was suggested, a fundamental question remains how autophagosomes generated at distal axons acquire dynein motors for retrograde transport toward the soma. In this paper, we demonstrate that late endosome (LE)–loaded dynein–snapin complexes drive AV retrograde transport in axons upon fusion of autophagosomes with LEs into amphisomes. Blocking the fusion with syntaxin17 knockdown reduced recruitment of dynein motors to AVs, thus immobilizing them in axons. Deficiency in dynein–snapin coupling impaired AV transport, resulting in AV accumulation in neurites and synaptic terminals. Altogether, our study provides the first evidence that autophagosomes recruit dynein through fusion with LEs and reveals a new motor–adaptor sharing mechanism by which neurons may remove distal AVs engulfing aggregated proteins and dysfunctional organelles for efficient degradation in the soma.     

Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons

Neurotrophin signaling is crucial for neuron growth. While the "signaling endosomes" hypothesis is one of the accepted models, the molecular machinery that drives retrograde axonal transport of TrkB signaling endosomes is largely unknown. In particular, mechanisms recruiting dynein to TrkB signaling endosomes have not been elucidated. Here, using snapin deficient mice and gene rescue experiments combined with compartmentalized cultures of live cortical neurons, we reveal that Snapin, as a dynein adaptor, mediates retrograde axonal transport of TrkB signaling endosomes. Such a role is essential for dendritic growth of cortical neurons. Deleting snapin or disrupting Snapin-dynein interaction abolishes TrkB retrograde transport, impairs BDNF-induced retrograde signaling from axonal terminals to the nucleus, and decreases dendritic growth. Such defects were rescued by reintroducing the snapin gene. Our study indicates that Snapin-dynein coupling is one of the primary mechanisms driving BDNF-TrkB retrograde transport, thus providing mechanistic insights into the regulation of neuronal growth and survival.     

A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events

Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders—Niemann-Pick type C, mucolipidosis type IV, and Sandhoff’s disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.     

Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport

Cellular homeostasis in neurons requires that the synthesis and anterograde axonal transport of protein and membrane be balanced by their degradation and retrograde transport. To address the nature and regulation of retrograde transport in cultured sympathetic neurons, I analyzed the behavior, composition, and ultrastructure of a class of large, phase-dense organelles whose movement has been shown to be influenced by axonal growth (Hollenbeck, P. J., and D. Bray. 1987. J. Cell Biol. 105:2827-2835). In actively elongating axons these organelles underwent both anterograde and retrograde movements, giving rise to inefficient net retrograde transport. This could be shifted to more efficient, higher volume retrograde transport by halting axonal outgrowth, or conversely shifted to less efficient retrograde transport with a larger anterograde component by increasing the intracellular cyclic AMP concentration. When neurons were loaded with Texas red- dextran by trituration, autophagy cleared the label from an even distribution throughout the neuronal cytosol to a punctate, presumably lysosomal, distribution in the cell body within 72 h. During this process, 100% of the phase-dense organelles were fluorescent, showing that they contained material sequestered from the cytosol and indicating that they conveyed this material to the cell body. When 29 examples of this class of organelle were identified by light microscopy and then relocated using correlative electron microscopy, they had a relatively constant ultrastructure consisting of a bilamellar or multilamellar boundary membrane and cytoplasmic contents, characteristic of autophagic vacuoles. When neurons took up Lucifer yellow, FITC-dextran, or Texas red-ovalbumin from the medium via endocytosis at the growth cone, 100% of the phase-dense organelles became fluorescent, demonstrating that they also contain products of endocytosis. Furthermore, pulse-chase experiments with fluorescent endocytic tracers confirmed that these organelles are formed in the most distal region of the axon and undergo net retrograde transport. Quantitative ratiometric imaging with endocytosed 8-hydroxypyrene-1,3,6- trisulfonic acid showed that the mean pH of their lumena was 7.05. These results indicate that the endocytic and autophagic pathways merge in the distal axon, resulting in a class of predegradative organelles that undergo regulated transport back to the cell body.     

Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR.

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TeNT HC). Morphological and kinetic analysis of TeNT HC retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT HC carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor p75NTR. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.     

Lysosomal dysfunction in neurodegeneration

Neuronal homeostasis and survival critically depend on an efficient autophagy-lysosomal degradation pathway, especially since neurons cannot reduce the concentration of misfolded proteins and damaged organelles by cell division. While increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative disorders, the molecular underpinnings of the role of lysosomes in neurodegeneration remain largely unknown. To this end, studies of neurodegenerative disorders caused by mutations in lysosomal proteins offer an opportunity to elucidate such mechanisms and potentially identify specific therapeutic targets. One of these disorders is Kufor-Rakeb syndrome, caused by mutations in the lysosomal protein ATP13A2/PARK9 and characterized by early-onset Parkinsonism, pyramidal degeneration and dementia. We found that loss of ATP13A2 function results in impaired lysosomal function and, consequently, accumulation of SNCA/α-synuclein and neurotoxicity. Our results suggest that targeting of ATP13A2 to lysosomes to enhance lysosomal function may result in neuroprotection in Kufor-Rakeb syndrome. From a broader perspective, these findings, together with other recent studies of lysosomal dysfunction in neurodegeneration, suggest that strategies to upregulate lysosomal function in neurons represent a promising therapeutic approach for neurodegenerative disorders.     

Lysosomal dysfunction in neurodegeneration: The role of ATP13A2/PARK9

Neuronal homeostasis and survival critically depend on an efficient autophagy-lysosomal degradation pathway, especially since neurons cannot reduce the concentration of misfolded proteins and damaged organelles by cell division. While increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative disorders, the molecular underpinnings of the role of lysosomes in neurodegeneration remain largely unknown. To this end, studies of neurodegenerative disorders caused by mutations in lysosomal proteins offer an opportunity to elucidate such mechanisms and potentially identify specific therapeutic targets. One of these disorders is Kufor-Rakeb syndrome, caused by mutations in the lysosomal protein ATP13A2/PARK9 and characterized by early-onset Parkinsonism, pyramidal degeneration and dementia. We found that loss of ATP13A2 function results in impaired lysosomal function and, consequently, accumulation of SNCA/α-synuclein and neurotoxicity. Our results suggest that targeting of ATP13A2 to lysosomes to enhance lysosomal function may result in neuroprotection in Kufor-Rakeb syndrome. From a broader perspective, these findings, together with other recent studies of lysosomal dysfunction in neurodegeneration, suggest that strategies to upregulate lysosomal function in neurons represent a promising therapeutic approach for neurodegenerative disorders.     

Lysosomal vitamin E accumulation in Niemann-Pick type C disease

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.     

Lysosomal vitamin E accumulation in Niemann-Pick type C disease

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.     

Accumulation of sialic acid in endocytic compartments interferes with the formation of mature lysosomes. Impaired proteolytic processing of cathepsin B in fibroblasts of patients with lysosomal sialic acid storage disease.

The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.     

Palmitoyl protein thioesterase 1 (PPT1) deficiency causes endocytic defects connected to abnormal saposin processing

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of the childhood caused by mutations in the gene encoding palmitoyl protein thioesterase 1 (PPT1). PPT1 localizes to late endosomes/lysosomes of non-neuronal cells and in neurons also to presynaptic areas. PPT1-deficiency causes massive death of cortical neurons and most tissues show an accumulation of saposins A and D. We have here studied endocytic pathways, saposin localization and processing in PPT1-deficient fibroblasts to elucidate the cellular defects resulting in accumulation of specific saposins. We show that PPT1-deficiency causes a defect in fluid-phase and receptor-mediated endocytosis, whereas marker uptake and recycling endocytosis remain intact. Furthermore, we show that saposins A and D are more abundant and relocalized in PPT-deficient fibroblasts and mouse primary neurons. Metabolic labeling and immunoprecipitation analyses revealed hypersecretion and abnormal processing of prosaposin, implying that the accumulation of saposins may result from endocytic defects. We show for the first time a connection between saposin storage and a defect in the endocytic pathway of INCL cells. These data provide new insights into the metabolism of PPT1-deficient cells and offer a basis for further studies on cellular processes causing neuronal death in INCL and other neurodegenerative diseases.     

Retrograde axonal transport and motor neuron disease

Transport of material between extensive neuronal processes and the cell body is crucial for neuronal function and survival. Growing evidence shows that deficits in axonal transport contribute to the pathogenesis of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we review recent data indicating that defects in dynein-mediated retrograde axonal transport are involved in ALS etiology. We discuss how mutant copper-zinc superoxide dismutase (SOD1) and an aberrant interaction between mutant SOD1 and dynein could perturb retrograde transport of neurotrophic factors and mitochondria. A possible contribution of axonal transport to the aggregation and degradation processes of mutant SOD1 is also reviewed. We further consider how the interference with axonal transport and protein turnover by mutant SOD1 could influence the function and viability of motor neurons in ALS.     

Action in the axon: generation and transport of signaling endosomes

Neurons extend axonal processes over long distances, necessitating efficient transport mechanisms to convey target-derived neurotrophic survival signals from remote distal axons to cell bodies. Retrograde transport, powered by dynein motors, supplies cell bodies with survival signals in the form of 'signaling endosomes'. In this review, we will discuss new advances in our understanding of the motor proteins that bind to and move signaling components in a retrograde direction and discuss mechanisms that might specify distinct neuronal responses to spatially restricted neurotrophin signals. Disruption of retrograde transport leads to a variety of neurodegenerative diseases, highlighting the role of retrograde transport of signaling endosomes for axonal maintenance and the importance of efficient transport for neuronal survival and function.     

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.     

Syntaxin 7 mediates endocytic trafficking to late endosomes

The lysosome functions are ensured by accurate membrane trafficking in the cell. We found that mouse syntaxin 7 could complement yeast vam3 and pep12 mutants defective in docking/fusion to vacuolar and prevacuolar membranes, respectively. Immunohistochemical studies showed that syntaxin 7 is localized to late endosomes, but not to early endosomes. Induced expression of mutant syntaxin 7 blocked endocytic transport from early to late endosomes but did not block the transport of cathepsin D and lamp-2 from the trans-Golgi network to lysosomes. Thus, syntaxin 7 mediates the endocytic trafficking from early endosomes to late endosomes and lysosomes. These results also suggest that the biosynthetic pathway utilizes a different machinery from that of the endocytic pathway in the docking/fusion to late endosomes.