Autophagy, the process by which cells dispose unwanted proteins, is essential for neuronal survival, and faulty autophagy is linked to Alzheimer’s and other age-related diseases. In the February 23 Journal of Neuroscience, Frank LaFerla and colleagues at the University of California, Irvine, report data that further implicate presenilins in this cellular housekeeping system. The research broadens the field’s understanding of presenilins as multifunctional proteins that do far more than slice amyloid-β out of its precursor.

Long interested in the role of presenilins (PS) in calcium signaling, co-corresponding author Kim Green discovered that these membrane proteins activate the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump (ARF related news story on Green et al., 2008). Shortly thereafter, Green heard a talk by Ralph Nixon, Nathan Kline Institute for Psychiatric Research, New York, on how PS mutations disrupt lysosomal function in AD patients. That got Green’s mind off calcium long enough to run a few initial experiments hinting at whether wild-type presenilins might also play a role in regulating protein degradation pathways. He and graduate student Kara Neely looked at expression of autophagy-related proteins in embryonic fibroblasts from PS1-deficient, PS2-deficient, and PS double-knockout mice. Comparing them to wild-type mouse cells, “we were shocked to see such huge differences in autophagic markers, particularly LC3-II (a marker for autophagosomes),” Green said. “Protein degradation is so important for aging, and for all these different diseases, that it was something we felt we needed to understand—how presenilins played a role in this.”

That realization launched first author Neely on a series of additional experiments to address the role of PS in autophagy. She looked at autophagy markers in PS-deficient fibroblasts and in human neuroblastoma cells treated with small-interfering RNAs to knock down PS1 or PS2. Her Western blots showed changes, relative to wild-type cells, in several autophagy-related proteins—upregulated LC3-II, reduced levels of phosphorylated mTOR (an autophagy inhibitor) and faster-migrating LAMP2a (a lysosomal protein). Backing the biochemical data, PS double-knockout fibroblasts had more autophagosomes than wild-type cells. Interestingly, the changes seemed independent of PS’s catalytic activity, as treatment with two different γ-secretase inhibitors did not affect levels of key autophagic markers.

Philipp Jaeger, who works with Tony Wyss-Coray at Stanford University in Palo Alto, California, called the study “an exciting step forward”—in part because “presenilins, mainly known for their involvement in amyloid-β pathology of AD now appear onstage again in a γ-secretase-independent pathway.” (See full comment below).

As fate would have it, the current paper was submitted for publication just days before Nixon’s group reported similar findings in Cell (ARF related news story on Lee et al., 2010). Whereas the Irvine researchers studied both presenilins, Nixon and colleagues focused on PS1, identifying its role in glycosylation of a proton pump required for lysosome acidification. For the present paper, reviewers asked Green and colleagues to also examine lysosome acidity in their PS-deficient cells. The scientists stained double-knockout fibroblasts with red LysoTracker dye that enters acidic compartments and fluoresces more in lower pH. The authors found more red-staining puncta per cell in the PS-null cells compared with controls, and took that to indicate “no deficits in lysosomal acidification, despite robust impairments in autophagic degradation,” they wrote.

These findings contrast with Nixon’s study, which found less LysoTracker fluorescence in PS1-null cells, relative to wild-type. (The New York scientists also confirmed the acidification defect with several other methods, including direct pH measurement using a radiometric lysosensor.) The discrepancy between the two studies might stem from the different cell types used (i.e., PS double-knockouts by the Irvine group, PS1-deficient cells by the New York researchers). Nixon’s lab has unpublished data suggesting that PS2 may have “a completely different impact on cells than PS1,” and that the two proteins could compensate for each other in some regard, complicating the final readout, Nixon told ARF. “When you combine the deletions of both in the same cells, you end up with a hybrid where certain aspects of the phenotype are rescued by the PS2 deletion, and others are retained. Both deletions aren’t doing the same thing. Trying to sort out the roles of PS1 or PS2 alone in the double-knockout is just not possible, in our opinion, without confirming what you’re showing in the single-knockout,” Nixon added.

From an overall standpoint, however, the Cell paper and current study “agree almost completely about the basic idea that PS has a fundamental role in autophagy,” Nixon said. “The question then becomes whether there are purely deficits in clearance and lysosomal proteolysis, or whether there are also alterations at the front end of the pathway, i.e., induction,” he said (see Nixon and Yang, 2011, review).

This is where recent work by Wyss-Coray’s lab may come into play. The Stanford researchers found that reduction of the early autophagy protein beclin-1 worsened Aβ deposition and synaptic defects in AD transgenic mice. They also found reduced beclin-1 levels in postmortem brain tissue from AD patients (ARF related news story on Pickford et al., 2008). More recently, Jaeger and colleagues fleshed out this link with cell biology experiments showing that beclin-1 regulates turnover of amyloid precursor protein (ARF related news story on Jaeger et al., 2010).

Neely and colleagues’ current study found lower beclin-1 levels in PS-null cells. On the whole, though, its findings do not tie in with APP processing or tau pathology. Rather, they reveal “the physiological role of PS in an important protein degradation pathway,” Green noted. “Whether this has any implications for sporadic AD we don’t know.”

Besides trying to understand exactly how PS figures in autophagy, the Irvine team is now examining cells with PS mutations associated with the age-related disease frontotemporal dementia to see if autophagy in those cells differs from that of cells with PS mutations that cause familial AD.—Esther Landhuis.

Reference:
Neely KM, Green KN, LaFerla FM. Presenilin is necessary for efficient proteolysis through the autophagy-lysosome system in a γ-secretase-independent manner. J. Neurosci. 23 Feb 2011;31(8):2781-2791. Abstract

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  1. 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:

    . Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010 Jun 25;141(7):1146-58. PubMed.

    . 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. PubMed.

    . Regulation of amyloid precursor protein processing by the Beclin 1 complex. PLoS One. 2010;5(6):e11102. PubMed.

    . Analysis of the gamma-secretase interactome and validation of its association with tetraspanin-enriched microdomains. Nat Cell Biol. 2009 Nov;11(11):1340-6. PubMed.

    . Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease. J Cell Biol. 2009 Dec 14;187(6):875-88. PubMed.

    . VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration. Cell Death Differ. 2001 Oct;8(10):977-84. PubMed.

References

News Citations

  1. Pump It Up—Presenilins Linked to ER SERCA Activity
  2. Death of the Neatnik: Neurons Perish When Trash Clutters Their Space?
  3. Autophagy Regulator Helps Neurons Stomach Excess Aβ, Resist AD

Paper Citations

  1. . SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production. J Cell Biol. 2008 Jun 30;181(7):1107-16. PubMed.
  2. . Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010 Jun 25;141(7):1146-58. PubMed.
  3. . Autophagy failure in Alzheimer's disease-locating the primary defect. Neurobiol Dis. 2011 Jul;43(1):38-45. PubMed.
  4. . 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. PubMed.
  5. . Regulation of amyloid precursor protein processing by the Beclin 1 complex. PLoS One. 2010;5(6):e11102. PubMed.
  6. . Presenilin is necessary for efficient proteolysis through the autophagy-lysosome system in a γ-secretase-independent manner. J Neurosci. 2011 Feb 23;31(8) PubMed.

Further Reading

Papers

  1. . Presenilin is necessary for efficient proteolysis through the autophagy-lysosome system in a γ-secretase-independent manner. J Neurosci. 2011 Feb 23;31(8) PubMed.
  2. . Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010 Jun 25;141(7):1146-58. PubMed.
  3. . 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. PubMed.
  4. . Regulation of amyloid precursor protein processing by the Beclin 1 complex. PLoS One. 2010;5(6):e11102. PubMed.
  5. . Autophagy failure in Alzheimer's disease-locating the primary defect. Neurobiol Dis. 2011 Jul;43(1):38-45. PubMed.

Primary Papers

  1. . Presenilin is necessary for efficient proteolysis through the autophagy-lysosome system in a γ-secretase-independent manner. J Neurosci. 2011 Feb 23;31(8) PubMed.