Axonal autophagosomes use the ride-on service for retrograde transport toward the soma

Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process in cells. Neurons are highly polarized cells with long axons, thus facing special challenges to transport AVs generated at distal processes toward the soma where mature acidic lysosomes are relatively enriched. We recently revealed a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. Late endosome (LE)-loaded dynein-SNAPIN motor-adaptor complexes mediate the retrograde transport of autophagosomes upon their fusion with LEs in distal axons. This motor-adaptor sharing mechanism enables neurons to maintain effective autophagic clearance in the soma, thus reducing autophagic stress in axons. Therefore, our study reveals a new cellular mechanism underlying the removal of distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases.     

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.     

Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp^0/0) hippocampal neuronal cells in comparison to cellular prion protein (PrP^C)-expressing (Prnp^+/+) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrP^C is associated with autophagy using Prnp^0/0 hippocampal neuronal cells under hydrogen peroxide (H₂O₂)-induced oxidative stress. We found that Prnp^0/0 cells were more susceptible to oxidative stress than Prnp^+/+ cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H₂O₂-treated Prnp^0/0 cells compared with H₂O₂-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H₂O₂-treated Prnp^0/0 cells, while the H₂O₂-treated Prnp^+/+ cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H₂O₂. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp^+/+ cells contributes to the prosurvival effect of autophagy against H₂O₂ cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrP^C may impair autophagic flux via H₂O₂-induced oxidative stress.     

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP⁺RFP⁺ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.     

Sortilin,a novel APOE receptor implicated in Alzheimer disease

In the brain, apolipoprotein E (APOE) delivers cholesterol-rich lipoproteins to neurons to support synaptogenesis and maintenance of synaptic connections. Three APOE alleles exist in the human population with ε4 being an Alzheimer disease (AD) risk gene and ε2 being protective relative to the common ε3 variant. Many hypotheses have been advanced concerning allele-specific effects of APOE on neurodegeneration including effects on Aβ clearance, synaptic transmission, or neurotoxicity. Central to most proposed APOE functions is its interaction with receptors that mediate cellular uptake of this ligand. Several members of the LDL receptor gene family have been implicated as APOE receptors in the (patho)physiology of APOE in the brain, yet their specific modes of action in AD remain controversial. Recently, the pro-neurotrophin receptor sortilin has been identified as a novel APOE receptor in neurons. Ablation of sortilin expression in mice results in accumulation of APOE and Aβ in the brain. Moreover, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/Aβ complexes. Despite increased brain APOE levels, sortilin-deficient animals recapitulate anomalies in brain lipid homeostasis seen in APOE null mice, indicating functional deficiency in APOE uptake pathways. Taken together, these findings suggest a link between Aβ catabolism and pro-neurotrophin signaling converging on this receptor pathway.     

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

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.     

Exploring the multifaceted roles of heat shock protein B8 (HSPB8) in diseases

HSPB8 is a member of ubiquitous small heat shock protein (sHSP) family, whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. Investigation of HSPB8 structure indicated that HSPB8 belongs to the group of so-called intrinsically disordered proteins and possesses a highly flexible structure. Unlike most other sHSPs, HSPB8 tends to form small-molecular-mass oligomers and exhibits substrate-dependent chaperone activity. In cooperation with BAG3, the chaperone activity of HSPB8 was reported to be involved in the delivery of misfolded proteins to the autophagy machinery. Through this way, HSPB8 interferes with pathological processes leading to neurodegenerative diseases. Accordingly, published studies have identified genetic links between mutations of HSPB8 and some kind of neuromuscular diseases, further supporting its important role in neurodegenerative disorders. In addition to their anti-aggregation properties, HSPB8 is indicated to interact with a wide range of client proteins, modulating their maturations and activities, and therefore, regulates a large repertoire of cellular functions, including apoptosis, proliferation, inflammation and etc. As a result, HSPB8 has key roles in cancer biology, autoimmune diseases, cardiac diseases and cerebral vascular diseases.