Defective retrograde transport impairs autophagic clearance in Alzheimer disease neurons

Macroautophagy/autophagy plays a key role in cellular quality control by eliminating protein aggregates and damaged organelles, which is essential for the maintenance of neuronal homeostasis. Defective autophagy has been implicated in the pathogenesis of Alzheimer disease (AD). In AD brains, autophagic vacuoles (AVs) accumulate massively within dystrophic neurites. This raises a fundamental question as to whether impaired autophagic clearance contributes to AD-associated autophagic stress. We recently revealed that AD neurons display defective retrograde transport and accumulation of amphisomes predominantly in axons and presynaptic terminals. Amyloid β (Aβ) oligomers are enriched in axons and interact with dynein motors. This interaction interferes with the coupling of the dynein motor with its adaptor SNAPIN. Such deficits disrupt dynein-driven retrograde transport of amphisomes, thus trapping them in distal axons and impairing their degradation in the soma. Therefore, our study provides new mechanistic insights into AD-linked autophagic pathology, and builds a foundation for developing potential AD therapeutic strategies by rescuing retrograde transport of amphisomes.     

A role of GCC88 in the retrograde transport of CI‐M6PR and the maintenance of lysosomal activity

GCC88 is a golgin coiled‐coil protein at the trans‐Golgi (TGN) that functions as a tethering factor for the endosome‐derived retrograde transport vesicles. Here, we demonstrate that GCC88 is required for the endosome‐to‐TGN retrograde transport of the cation‐independent mannose 6‐phosphate receptor (CI‐M6PR). The knockout of GCC88 perturbs the retrieval of CI‐M6PR and decreases its cellular level at the steady state, which causes the improper processing of newly synthesized cathepsin‐D, a lysosomal hydrolase dependent on CI‐M6PR for its delivery to lysosomes. At the whole cell level, the knockout of GCC88 reduces the lysosomal proteolytic capacity but does not impair of the efficiency of autophagy within these cells.     

Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/microm2, whereas isolated lysosomes had about 2000 particles/microm2). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/microm2), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearence of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi- or multilamellar autophagic vacuoles appeared to engage in such fusions.     

Progressive endolysosomal deficits impair autophagic clearance beginning at early asymptomatic stages in fALS mice

Autophagy is an important homeostatic process that functions by eliminating defective organelles and aggregated proteins over a neuron''s lifetime. One pathological hallmark in amyotrophic lateral sclerosis (ALS)-linked motor neurons (MNs) is axonal accumulation of autophagic vacuoles (AVs), thus raising a fundamental question as to whether reduced autophagic clearance due to an impaired lysosomal system contributes to autophagic stress and axonal degeneration. We recently revealed progressive lysosomal deficits in spinal MNs beginning at early asymptomatic stages in fALS-linked mice expressing the human (Hs) SOD1^G93A protein. Such deficits impair the degradation of AVs engulfing damaged mitochondria from distal axons. These early pathological changes are attributable to mutant HsSOD1, which interferes with dynein-driven endolysosomal trafficking. Elucidation of this pathological mechanism is broadly relevant, because autophagy-lysosomal deficits are associated with several major neurodegenerative diseases. Therefore, enhancing autophagic clearance by rescuing endolysosomal trafficking may be a potential therapeutic strategy for ALS and perhaps other neurodegenerative diseases.     

A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.     

The dynein inhibitor Ciliobrevin D inhibits the bidirectional transport of organelles along sensory axons and impairs NGF‐mediated regulation of growth cones and axon branches

The axonal transport of organelles is critical for the development, maintenance, and survival of neurons, and its dysfunction has been implicated in several neurodegenerative diseases. Retrograde axon transport is mediated by the motor protein dynein. In this study, using embryonic chicken dorsal root ganglion neurons, we investigate the effects of Ciliobrevin D, a pharmacological dynein inhibitor, on the transport of axonal organelles, axon extension, nerve growth factor (NGF)‐induced branching and growth cone expansion, and axon thinning in response to actin filament depolymerization. Live imaging of mitochondria, lysosomes, and Golgi‐derived vesicles in axons revealed that both the retrograde and anterograde transport of these organelles was inhibited by treatment with Ciliobrevin D. Treatment with Ciliobrevin D reversibly inhibits axon extension and transport, with effects detectable within the first 20 min of treatment. NGF induces growth cone expansion, axonal filopodia formation and branching. Ciliobrevin D prevented NGF‐induced formation of axonal filopodia and branching but not growth cone expansion. Finally, we report that the retrograde reorganization of the axonal cytoplasm which occurs on actin filament depolymerization is inhibited by treatment with Ciliobrevin D, indicating a role for microtubule based transport in this process, as well as Ciliobrevin D accelerating Wallerian degeneration. This study identifies Ciliobrevin D as an inhibitor of the bidirectional transport of multiple axonal organelles, indicating this drug may be a valuable tool for both the study of dynein function and a first pass analysis of the role of axonal transport. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 757–777, 2015