Nilsson P, Loganathan K, Sekiguchi M, Matsuba Y, Hui K, Tsubuki S, Tanaka M, Iwata N, Saito T, Saido TC.
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The intracellular world of endosomal-autophagic-lysosomal (EAL) proteolysis is a complex and dynamic system, where boundaries between homotypic and heterotypic organelle classification often become blurred. In the past month, three research studies have been published that suggest a direct connection between the process of macroautophagy and the degradative clearance of Aβ (Li et al., 2013; Nilsson et al., 2013; Tian et al., 2013). Although results presented in each of these studies stemmed from innovative experimental approaches, I have some reservation accepting their shared interpretation that autophagosomes (the macroautophagic branch of the EAL system) directly alter Aβ precursor protein (APP) or Aβ metabolism. Rather than comment on specific experiments from these papers, below I have outlined three inclusion criteria that I believe should be met before a direct relationship between macroautophagy and APP metabolism is inferred:
1) Endogenous APP must be metabolized by pure autophagy activation. Many studies that report changes in APP metabolism during starvation-induced autophagy do not consider the likelihood that they arise due to alterations in endocytosis. For almost 25 years, it has been known that a large proportion of APP and APP metabolites are catabolized within the endosomal-lysosomal system (Benowitz et al., 1989; Cole et al., 1989). However, studies proposing autophagosomal APP metabolism fail to demonstrate an exclusion of the endosomal-lysosomal component of APP metabolism. In our experience, we found that rapamycin-induced autophagy increased LC3-II-positive autophagosomes without altering endogenous APP metabolism in wild-type rat cortical neurons (Boland et al., 2010). Although some studies conducted in APP-overexpressing cells have reported changes in APP metabolism during rapamycin-induced autophagy, the full impact of APP overexpression on EAL proteolysis is currently unknown. Considering that APP overexpression places more catabolic stress on lysosomes, it is likely that lysosomes from these cells have reduced capacity for clearing APP and its metabolites.
2) APP or APP metabolites must be localized to autophagosomes. Many studies reporting a colocalization of APP, APP-C-terminal fragments (CTFs), or Aβ with LC3-II assume that amyloidogenic cargo is actively engulfed into autophagosomes. How a non-cytosolic protein such as APP becomes engulfed into an autophagosome is a key aspect of this debate that has yet to be resolved. While it may be possible that some endosome- or endoplasmic reticulum-derived membrane may enter autophagosomes, no studies have conclusively shown that APP makes its way into neuronal autophagosomes through these routes. Considering that lysosomes receive cargo from endocytic and autophagic routes, it is highly likely that LC3-II-positive organelles containing APP-derived material are merely autolysosomes that have received cargo from autophagic (LC3-II) and endocytic (APP) routes. This is particularly relevant to Aβ-engulfment studies, such as the recent report suggesting Aβ-induced impairment of lysosomal flux led to an accumulation of dysfunctional autolysosomes in NG2 cells (Li et al., 2013). In addition to immunocytochemical colocalization studies, a number of subcellular fractionation studies have shown APP and APP metabolites colocalized with LC3-II, although none have provided clear evidence of APP localization to homotypic autophagosomes devoid of lysosomal membrane proteins, e.g. LC3-II (+ve)/LAMP-1 (–ve). Therefore, more consideration needs to be given to the possibility that endosomal routes are chiefly responsible for the delivery of amyloidogenic material to lysosomes and/or autolysosomes.
3) Determine changes in lysosomal APP metabolism in cells devoid of macroautophagy. Studies claiming that macroautophagy directly regulates APP metabolism provide convincing evidence that APP and APP metabolites increase in the absence of macroautophagic processes caused by a loss of Beclin 1 (Pickford et al., 2008), ATG5 (Tian et al., 2013), or ATG 7 (Nilsson et al., 2013). What these studies have not considered is the cellular impact that the loss of macroautophagy has on lysosomal flux. We know that in the absence of macroautophagy, a large accumulation of p62-positive protein aggregates and other poly-ubiquitinated proteins occurs, however, little attention is given to the potentially dramatic impact this has on lysosomal flux. As shown recently by Nilsson et al., a knockdown of ATG 7 dramatically decreased extracellular amyloid, yet intracellular amyloid staining increased. Therefore, these findings not only indicate that amyloidogenesis can proceed in the absence of macroautophagy, they also suggest that impaired degradation and subsequent accumulation of Aβ within overburdened lysosomes may be more detrimental to neuron viability than amounts of extracellular amyloid plaques.
In summary: All of the studies cited above highlight the important neuroprotective role of efficient lysosomal flux, which may serve as a prophylactic against the development of AD. Therapies capable of specifically modulating endocytic and autophagic branches of the EAL pathway are potentially very important, but can only be developed once a deep understanding of each branch is complete. It is therefore recommended that a more rigorous delineation of these pathways be performed to determine their individual contribution within the overall context of lysosomal flux. Research in this area is now at a crossroads: Some researchers advocate the simulation of macroautophagy to remove intracellular protein aggregates common among neurodegenerative diseases, while others remain cautious about the possible downsides of heightened autophagy on aged neurons with an already-compromised lysosomal system.
Ultimately, most researchers in this field agree that the maintenance of efficient lysosomal flux is a key requirement of neuron longevity. Therefore, therapies that can sustain this function throughout old age are likely to provide wide-ranging health benefits, including protection against the onset and progression of AD.
Benowitz LI, Rodriguez W, Paskevich P, Mufson EJ, Schenk D, Neve RL.
The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects.
Exp Neurol. 1989 Dec;106(3):237-50.
Boland B, Smith DA, Mooney D, Jung SS, Walsh DM, Platt FM.
Macroautophagy is not directly involved in the metabolism of amyloid precursor protein.
J Biol Chem. 2010 Nov 26;285(48):37415-26.
Cole GM, Huynh TV, Saitoh T.
Evidence for lysosomal processing of amyloid beta-protein precursor in cultured cells.
Neurochem Res. 1989 Oct;14(10):933-9.
Li W, Tang Y, Fan Z, Meng Y, Yang G, Luo J, Ke ZJ.
Autophagy is involved in oligodendroglial precursor-mediated clearance of amyloid peptide.
Mol Neurodegener. 2013;8:27.
Pickford F, Masliah E, Britschgi M, Lucin K, Narasimhan R, Jaeger PA, Small S, Spencer B, Rockenstein E, Levine B, Wyss-Coray T.
The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice.
J Clin Invest. 2008 Jun;118(6):2190-9.
Tian Y, Chang JC, Fan EY, Flajolet M, Greengard P.
Adaptor complex AP2/PICALM, through interaction with LC3, targets Alzheimer's APP-CTF for terminal degradation via autophagy.
Proc Natl Acad Sci U S A. 2013 Oct 15;110(42):17071-6.