In 2010, Ralph Nixon’s lab published a beautiful study demonstrating the involvement of presenilin-1 (PS1) in autophagy function and lysosome acidification (Lee et al., 2010). They were able to show that certain PS1 mutations, found in familial Alzheimer's disease (AD) cases, lead to the mistargeting of the v-ATPase V0a1 subunit, and thus cause diminished lysosomal protein degradation (see ARF related news story). This current study is a very exciting extension of this work, demonstrating that both PS1 and PS2 are required for the correct functioning of autophagosomal-lysosomal protein degradation and that this PS involvement appears to reach well beyond the inhibition of lysosomal acidification.

Neely and colleagues use PS1, PS2, and PS1 and 2 knockout cells and PS siRNAs to probe the effects of reduced PS levels on autophagy. They find increased levels of LC3-II, a common marker for mature autophagosomes and decreased phospho-mTOR, normally a key inhibitor of autophagy activation, and conclude that autophagy...  Read more

In 2010, Ralph Nixon’s lab published a beautiful study demonstrating the involvement of presenilin-1 (PS1) in autophagy function and lysosome acidification (Lee et al., 2010). They were able to show that certain PS1 mutations, found in familial Alzheimer's disease (AD) cases, lead to the mistargeting of the v-ATPase V0a1 subunit, and thus cause diminished lysosomal protein degradation (see ARF related news story). This current study is a very exciting extension of this work, demonstrating that both PS1 and PS2 are required for the correct functioning of autophagosomal-lysosomal protein degradation and that this PS involvement appears to reach well beyond the inhibition of lysosomal acidification.

Neely and colleagues use PS1, PS2, and PS1 and 2 knockout cells and PS siRNAs to probe the effects of reduced PS levels on autophagy. They find increased levels of LC3-II, a common marker for mature autophagosomes and decreased phospho-mTOR, normally a key inhibitor of autophagy activation, and conclude that autophagy activity appears elevated. The authors then demonstrate accumulation of EGFP-LC3-positive vacuoles in PS-knockout cells and interpret that as a buildup of autophagosomes. Additionally, they show that this effect is independent of γ-secretase activity of the presenilins. To test if this activated autophagy is, in fact, able to properly degrade long-lived proteins, Neely and colleagues perform a pulse-chase protein degradation assay. Interestingly, they discover that protein degradation is impaired in PS-knockout cells, despite the abundant presence of autophagosomes and apparent markers of autophagy activation. Further experiments indicate that this degradative impairment in PS-knockout cells is neither due to inhibition of the ubiquitin-proteasome system, nor could it be rescued by stimulating autophagy even further, using pharmacological compounds. The authors conclude that presenilins play an important role in regulating autophagosomal-lysosomal protein degradation in a γ-secretase independent manner.

This study is an exciting step forward in our understanding of the complex machinery that regulates autophagy initiation, elongation, and autophagosomal-lysosomal fusion. It demonstrates that the large protein factories that orchestrate intracellular protein and vesicle sorting may have an important role in the development of neurodegenerative diseases, especially in proteinopathies. Presenilins, previously mostly known for their involvement in amyloid-β pathology of AD, now appear onstage again in a γ-secretase-independent pathway. We think the reduction of beclin-1 in the PS double-knockout cells is particularly interesting: Beclin-1 is a key autophagy-regulating protein with a known involvement in AD (Pickford et al., 2008). Here, Neely and colleagues do not see this reduction in the short-term siRNA experiments, and they propose that in long-term disease stages and stably transfected cell lines, loss of PS function could cause the accumulation of undegradable autophagosomes and reduced beclin-1 levels. This is in good agreement with data from our laboratory, where the inhibition of autophagosomal-lysosomal fusion led to decreased levels of beclin-1, and beclin-1 was indeed reduced in brains of sporadic AD patients (Jaeger et al., 2010). There, we showed in vitro and in vivo that beclin-1 can be reduced at the same time while LC3-II accumulates, similar to the findings of Neely and coauthors after PS-knockout.

A complex protein network appears to exist that regulates autophagy initiation and autophagosomal-lysosomal fusion, and this complex might involve both, presenilins and beclin-1. Recent publications have successfully mapped interaction partners for PS1 (Wakabayashi et al., 2009) and, while not directly detecting beclin-1, found many proteins involved in membrane trafficking. One particularly interesting protein is VCP/p97, a protein implicated in autophagy regulation, protein degradation, and mutations that are associated with neurodegenerative diseases (Ju et al., 2009; Hirabayashi et al., 2001). It will be very exciting to further investigate the protein machinery that controls the assembly of intracellular degradation and trafficking vacuoles. The identification of novel proteins and protein interactions, such as in this study, will allow us to enhance our understanding of neurological disorders that appear to suffer from two seemingly exclusive features: too much autophagy activation and too little protein turnover.

References:
Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, Wolfe DM, Martinez-Vicente M, Massey AC, Sovak G, Uchiyama Y, Westaway D, Cuervo AM, Nixon RA. Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010 Jun 25;141(7):1146-58. Epub 2010 Jun 10. Abstract

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. Abstract

Jaeger PA, Pickford F, Sun CH, Lucin KM, Masliah E, Wyss-Coray T. Regulation of amyloid precursor protein processing by the Beclin 1 complex. PLoS One. 2010 Jun 15;5(6):e11102. Abstract

Wakabayashi T, Craessaerts K, Bammens L, Bentahir M, Borgions F, Herdewijn P, Staes A, Timmerman E, Vandekerckhove J, Rubinstein E, Boucheix C, Gevaert K, De Strooper B. Analysis of the γ-secretase interactome and validation of its association with tetraspanin-enriched microdomains. Nat Cell Biol. 2009 Nov;11(11):1340-6. Epub 2009 Oct 18. Abstract

Ju JS, Fuentealba RA, Miller SE, Jackson E, Piwnica-Worms D, Baloh RH, Weihl CC. Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease. J Cell Biol. 2009 Dec 14;187(6):875-88. Abstract

Hirabayashi M, Inoue K, Tanaka K, Nakadate K, Ohsawa Y, Kamei Y, Popiel AH, Sinohara A, Iwamatsu A, Kimura Y, Uchiyama Y, Hori S, Kakizuka A. VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration. Cell Death Differ. 2001 Oct;8(10):977-84. Abstract

View all comments by Philipp Jaeger

In this study, Lee and colleagues describe a novel function of presenilin-1 (PS1), a protein previously found and best characterized as being involved in γ-secretase cleavage of amyloid precursor protein (APP) and Notch. The authors report that PS1 knockout cells exhibit a marked reduction in autolysosomal protein degradation in response to autophagy activation induced by serum starvation. On the subcellular level, these PS1 knockout cells present a phenotype that resembles histopathological changes in Alzheimer disease brains: the accumulation of numerous membrane-bound vesicles of the autolysosomal pathway (autophagosomes, early and late autolysosomes) that are filled with amorphous, undigested, electron-dense material.

Furthermore, the authors provide evidence for impaired maturation of cathepsin D, an important lysosomal protease, in the PS1 knockout cells. They show that this deficiency is due to reduced acidification of the lysosomal lumen. In a comprehensive attempt to identify the underlying mechanistic defects, Lee and colleagues discovered the involvement of PS1 in...  Read more

In this study, Lee and colleagues describe a novel function of presenilin-1 (PS1), a protein previously found and best characterized as being involved in γ-secretase cleavage of amyloid precursor protein (APP) and Notch. The authors report that PS1 knockout cells exhibit a marked reduction in autolysosomal protein degradation in response to autophagy activation induced by serum starvation. On the subcellular level, these PS1 knockout cells present a phenotype that resembles histopathological changes in Alzheimer disease brains: the accumulation of numerous membrane-bound vesicles of the autolysosomal pathway (autophagosomes, early and late autolysosomes) that are filled with amorphous, undigested, electron-dense material.

Furthermore, the authors provide evidence for impaired maturation of cathepsin D, an important lysosomal protease, in the PS1 knockout cells. They show that this deficiency is due to reduced acidification of the lysosomal lumen. In a comprehensive attempt to identify the underlying mechanistic defects, Lee and colleagues discovered the involvement of PS1 in the maturation and localization of v-ATPase V0a1. This proton pump is important to establish a low intra-lysosomal pH. PS1 directly binds to the ATPase, modulates its glycosylation state, and in this way modifies the ATPase's maturation, degradation, and subcellular localization. The authors conclude that PS1 knockout causes decreased levels of mature v-ATPase V0a1, which in turn leads to impaired lysosomal acidification and decreased lysosomal proteolysis. They continue to support this hypothesis with data from PS1 hypomorphic mice and from AD patients' fibroblasts. In the hypomorphic mice PS1 levels are high enough to sustain Notch cleavage and prevent developmental defects, but appear insufficient to maintain normal lysosomal protein turnover. In the AD fibroblasts, certain PS1 mutations seem to strongly inhibit proteolysis, indicating that these mutations likely play a role in PS1-v-ATPase V0a1 interactions. Lee and colleagues thus propose a novel function of PS1 in lysosomal acidification, based on v-ATPase V0a1 maturation, which could be contributing to the observed accumulation of aberrant autophagosomes and lysosomes in AD patients’ brain tissue.

View all comments by Philipp Jaeger

The recent report from Randy Nixon and colleagues is an interesting development in the story of familial Alzheimer disease (FAD) and its molecular roots. It is established that autophagy is deficient in the neurons of Alzheimer disease patients and that increased or induced autophagy can reverse these deficits. However, until now, the underlying mechanism of the deficient autophagy has not been clear. Nixon and colleagues have identified a defect in the acidification of the lysosome organelle specifically associated with mutations in PS1 found in FAD. While PS1 mutations have long been associated with increases in Aβ, this paper identifies a function for the holoprotein as a chaperone in the ER. Furthermore, the researchers were able to identify the ATPase complex that is dissociated in PS1 mutants. These important findings could lead to new avenues of therapies that target the ATPase complex by targeting the chaperone function of PS1.

View all comments by Eliezer Masliah

Nixon and colleagues show the involvement of PS1 in autophagy/lysosomal function, and indicate that PS1 mutations in familial AD cause its impairment. We believe that the autophagic/lysosomal pathway is a key therapeutic target, and it is important to investigate further if improving its function would be beneficial for decreasing amyloid-β production in vivo.

View all comments by Toshiyuki Nakagawa

The study by Nixon and colleagues is an absolutely gorgeous paper. It is cell biology at its best. It is interesting that our studies manipulating beclin-1 arrive at a very similar pathology as the lack of presenilin, both in vivo and in cell culture, causing an abnormal accumulation of lysosomes and autolysosomes (see also Pickford et al., 2008). Indeed, beclin-1 may have a role not only in the initiation of autophagy, but as an increasing number of studies suggest, in vesicle trafficking as well. Whether beclin-1 and presenilins interact at some level will be interesting to explore in the future.

However, we could have a friendly debate about whether autophagy really requires presenilin as stated in the title, since the study does not actually manipulate the autophagy process. Rather, it interferes with the final degradative step and shows very nicely that presenilins are necessary for lysosomal degradation.

If autophagy is the process of manufacturing garbage bags, filling them with trash, and hauling them to the dump,...  Read more

The study by Nixon and colleagues is an absolutely gorgeous paper. It is cell biology at its best. It is interesting that our studies manipulating beclin-1 arrive at a very similar pathology as the lack of presenilin, both in vivo and in cell culture, causing an abnormal accumulation of lysosomes and autolysosomes (see also Pickford et al., 2008). Indeed, beclin-1 may have a role not only in the initiation of autophagy, but as an increasing number of studies suggest, in vesicle trafficking as well. Whether beclin-1 and presenilins interact at some level will be interesting to explore in the future.

However, we could have a friendly debate about whether autophagy really requires presenilin as stated in the title, since the study does not actually manipulate the autophagy process. Rather, it interferes with the final degradative step and shows very nicely that presenilins are necessary for lysosomal degradation.

If autophagy is the process of manufacturing garbage bags, filling them with trash, and hauling them to the dump, lysosomal degradation would be the incineration of the trash-filled bags. For the incinerator to run, you need fuel and pipes that bring the fuel into the incinerator (the ATPase). If the incinerator does not run, you will naturally accumulate the bags filled with trash, but it would not follow that garbage bag production is impaired.

Furthermore, it is likely that endosomal trafficking and multivesicular bodies are equally affected by lysosomal degradation, but the paper does not discuss these possibilities.

View all comments by Tony Wyss-Coray

Reply to comment by Tony Wyss-Coray
Since its very early descriptions, autophagy has been defined as the lysosomal digestion of a cell’s own cytoplasmic material and not simply the sequestration of these components. Implied by this definition, and generally accepted in the autophagy field, is the central concept that lysosomal proteolysis is required to complete autophagy. This is a critical point, especially because autophagy failure in disease states, as measured by the diminished turnover of specific autophagy substrates, can result from failure of substrate sequestration, autophagosome formation, fusion of autophagosomes with a lysosome, or digestion of the substrate. Distinguishing which step in autophagy is defective in different neurodegenerative diseases has become important and usually involves evaluating not only autophagosome formation, but also autophagic flux, which reflects the balance between substrate sequestration and proteolytic clearance (1). Narrowly defining autophagy as only the sequestration step during autophagy, as proposed by Tony...  Read more

Reply to comment by Tony Wyss-Coray
Since its very early descriptions, autophagy has been defined as the lysosomal digestion of a cell’s own cytoplasmic material and not simply the sequestration of these components. Implied by this definition, and generally accepted in the autophagy field, is the central concept that lysosomal proteolysis is required to complete autophagy. This is a critical point, especially because autophagy failure in disease states, as measured by the diminished turnover of specific autophagy substrates, can result from failure of substrate sequestration, autophagosome formation, fusion of autophagosomes with a lysosome, or digestion of the substrate. Distinguishing which step in autophagy is defective in different neurodegenerative diseases has become important and usually involves evaluating not only autophagosome formation, but also autophagic flux, which reflects the balance between substrate sequestration and proteolytic clearance (1). Narrowly defining autophagy as only the sequestration step during autophagy, as proposed by Tony Wyss-Coray, does not recognize the well-accepted key role of the lysosome in this self-digestion process.

Tony points out that our results imply, but do not show, that cargoes delivered to lysosomes by endocytosis should also be affected by lysosomal failure. Indeed, we present data in our paper demonstrating that lysosomal turnover of endosomal substrates is decreased, as one might predict. Thus, in addition to being required for autophagy, presenilin-1 is required for the turnover of endosomal substrates.

References:
1. Esther Wong & Ana Maria Cuervo. Autophagy gone awry in neurodegenerative diseases. Nature Neuroscience. 2010 July; 13(7).

View all comments by Ana Maria Cuervo
View all comments by Ralph Nixon