The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. - AlzForum Alzheimer Research Paper of the Week

The Pickford et al. study adds strong support to an emerging view that autophagic-lysosomal impairment in AD can contribute to Aβ pathology and also to neurodegeneration through additional Aβ-independent mechanisms, which might be shared by other neurological diseases across the lifespan (1). The deficiencies Pickford and colleagues identified in the initial “sequestration” stages of autophagy compound other defects. We previously reported the clearance of Aβ-generating autophagic vacuoles that lead to vacuole accumulation—even in the presence of possibly slowed autophagosome formation as implied by the current findings.

Protein/vesicular trafficking defects in AD tend to be viewed from the focused perspective of how APP metabolism is altered, but, as this and other recent studies imply, the trafficking/handling of many proteins is affected by alterations of endosomes, autophagic compartments, and lysosomes, which are increasingly being linked to AD-related genetic factors (e.g., presenilin, SorLA, APP duplication, etc.). These more global effects on neuronal function are... Read more

Another Herpes Virus-Alzheimer’s Disease Connection: Beclin Beckons
This study not only strengthens the link between AD and autophagy by relating it to a reduced beclin 1 activity in the diseased brain. It also strengthens, indirectly, another link which we proposed (1), namely, among herpes simplex virus type 1 (HSV1), autophagy, and AD—thus extending the striking HSV1-amyloid connection that we recently discovered (2). HSV1 infects, and then resides lifelong, in the peripheral nervous system (PNS) of most humans in a latent state and is reactivated periodically by events such as stress; it then causes damage—cold sores—in some of those infected.

We detected HSV1 DNA some 18 years ago in the brain of many elderly humans (3), and subsequently showed that in brain, as in the PNS, it reactivates from latency (4), possibly recurrently, triggered presumably by stress, systemic infection, etc. Further, we found that HSV1 in ApoE-ε4 carriers’ brains conferred a strong risk of AD (5), and we suggested that brain damage caused on viral reactivation was greater in ApoE-ε4... Read more

Another Herpes Virus-Alzheimer’s Disease Connection: Beclin Beckons
This study not only strengthens the link between AD and autophagy by relating it to a reduced beclin 1 activity in the diseased brain. It also strengthens, indirectly, another link which we proposed (1), namely, among herpes simplex virus type 1 (HSV1), autophagy, and AD—thus extending the striking HSV1-amyloid connection that we recently discovered (2). HSV1 infects, and then resides lifelong, in the peripheral nervous system (PNS) of most humans in a latent state and is reactivated periodically by events such as stress; it then causes damage—cold sores—in some of those infected.

We detected HSV1 DNA some 18 years ago in the brain of many elderly humans (3), and subsequently showed that in brain, as in the PNS, it reactivates from latency (4), possibly recurrently, triggered presumably by stress, systemic infection, etc. Further, we found that HSV1 in ApoE-ε4 carriers’ brains conferred a strong risk of AD (5), and we suggested that brain damage caused on viral reactivation was greater in ApoE-ε4 carriers, leading to the development of AD (5,6). Since then, several studies by others have linked HSV1 to AD, detecting a close homology between a sequence in Aβ and one of the viral glycoproteins (7), and showing that APP associates with HSV1 during axonal transport (8). Also, recent studies have indicated that ApoE affects HSV1 (9-12), determining its transport in tissues and its expression. (Indeed, we have found that ApoE influences outcome of infection by several other viruses, including occurrence of cold sores in ApoE-ε4 carriers—paralleling the CNS situation—see, e.g., [6].)

Our recent data reveal that HSV1 infection of cultured cells causes a large accumulation of Aβ (2) and also AD-like tau phosphorylation, and infection of mice causes Aβ accumulation in brain.

Our recent hypothesis proposes that excess Aβ produced by HSV1 action is inadequately removed by autophagy because of viral hindrance, thus allowing plaque formation to occur (1). This was based on the fact that cells infected by HSV1 attempt to demolish the intruder by an autophagic process called xenophagy, but to evade this, the viral-encoded neurovirulence protein, ICP34.5, binds to beclin and inhibits its autophagic function (13). This ability to overcome xenophagy is shared by various other infectious agents, e.g., HIV (14) (and some microbes even use autophagy for their own advantage [15,16]), but HSV1 is the only pathogen (or indeed inflammation-producing agent) detected so far in many normal elderly human brains, and is therefore the only agent in a position to cause AD-like damage—as indeed we have shown it to do in cells and mice. In fact, one of the authors of the present study, too, has discussed the possibility that viral inhibition of autophagy might contribute to “non-infectious” neurodegenerative diseases such as AD (17).

References:
1. Itzhaki RF, Cosby SL, Wozniak MA. Herpes simplex virus type 1 and Alzheimer's disease: the autophagy connection. J Neurovirol. 2008;14:1-4. Abstract

2. Wozniak MA, Itzhaki RF, Shipley SJ, Dobson CB. Herpes simplex virus infection causes cellular beta-amyloid accumulation and secretase upregulation. Neurosci Lett. 2007;429:95-100. Abstract

3. Jamieson GA, Maitland NJ, Wilcock GK, Craske J, Itzhaki RF. Latent herpes simplex virus type 1 in normal and Alzheimer's disease brains. J Med Virol. 1991;33:224-7. Abstract

4. Wozniak MA, Shipley SJ, Combrinck M, Wilcock GK, Itzhaki RF. Productive herpes simplex virus in brain of elderly normal subjects and Alzheimer's disease patients. J Med Virol. 2005;75:300-6. Abstract

5. Itzhaki RF, Lin WR, Shang D, Wilcock GK, Faragher B, Jamieson GA. Herpes simplex virus type 1 in brain and risk of Alzheimer's disease. Lancet. 1997;349:241-4. Abstract

6. Itzhaki RF, Wozniak MA. Herpes Simplex Virus Type 1 in Alzheimer's disease: The Enemy Within. J Alzheimers Dis. 2008;13:393-405. Abstract

7. Cribbs DH, Azizeh BY, Cotman CW, LaFerla FM. Fibril formation and neurotoxicity by a herpes simplex virus glycoprotein B fragment with homology to the Alzheimer's A beta peptide. Biochemistry. 2000;39:5988-94. Abstract

8. Satpute-Krishnan P, DeGiorgis JA, Bearer EL. Fast anterograde transport of herpes simplex virus: role for the amyloid precursor protein of Alzheimer's disease. Aging Cell. 2003;2:305-18. Abstract

9. Burgos JS, Ramirez C, Sastre I, Bullido MJ, Valdivieso F. ApoE4 is more efficient than E3 in brain access by herpes simplex virus type 1. Neuroreport. 2003;14:1825-7. Abstract

10. Burgos JS, Ramirez C, Sastre I, Valdivieso F. Effect of apolipoprotein E on the cerebral load of latent herpes simplex virus type 1 DNA. J Virol. 2006;80:5383-7. Abstract

11. Bhattacharjee PS, Neumann DM, Stark D, Thompson HW, Hill JM. Apolipoprotein E modulates establishment of HSV-1 latency and survival in a mouse ocular model. Curr Eye Res. 2006;31:703-8. Abstract

12. Miller RM, Federoff HJ. Isoform-specific effects of ApoE on HSV immediate early gene expression and establishment of latency. Neurobiol Aging. 2008;29:71-7. Abstract

13. Orvedahl A, Alexander D, Tallóczy Z, Sun Q, Wei Y, Zhang W, Burns D, Leib DA, Levine B. HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe. 2007;1:23-35. Abstract

14. Zhou D, Spector S. Human immunodeficiency virus type-1 infection inhibits autophagy. AIDS. 2008;22:695-9. Abstract

15. Colombo MI. Pathogens and autophagy: subverting to survive. Cell Death Differ. 2005;12 Suppl 2:1481-3. Abstract

16. Wileman T. Aggresomes and autophagy generate sites for virus replication. Science. 2006;312:875-8. Abstract

17. Orvedahl A, Levine B. Autophagy and viral neurovirulence. Cell Microbiol. 2008 May 22. [Epub ahead of print] Abstract

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I recommend this paper

Macroautophagy is a major mechanism for intracellular protein degradation. It begins with the engulfment of part of the cytoplasm, including intracellular organelles, by double membrane vacuolar structures, the autophagosomes; these structures then fuse with lysosomes, thus creating the autolysosomes in which the intracellular constituents are degraded. Observations in a number of neurodegenerative diseases, including Alzheimer disease, indicate that there is an extensive accumulation of autophagic vacuoles in affected tissues. Whether this represents an induction of the process of macroautophagy or an inhibition of the conversion of autophagosomes to mature lysosomes has been unclear.

Boland et al. now report, using a number of careful imaging and biochemical tools, that in cultured primary cortical neurons induction of macroautophagy through inhibition of mTOR leads to little accumulation of double-membrane LC3-II-positive autophagosomes, as these are rapidly converted to autolysosomes. In contrast, inhibition of lysosomal proteolysis or disruption of... Read more

Macroautophagy is a major mechanism for intracellular protein degradation. It begins with the engulfment of part of the cytoplasm, including intracellular organelles, by double membrane vacuolar structures, the autophagosomes; these structures then fuse with lysosomes, thus creating the autolysosomes in which the intracellular constituents are degraded. Observations in a number of neurodegenerative diseases, including Alzheimer disease, indicate that there is an extensive accumulation of autophagic vacuoles in affected tissues. Whether this represents an induction of the process of macroautophagy or an inhibition of the conversion of autophagosomes to mature lysosomes has been unclear.

Boland et al. now report, using a number of careful imaging and biochemical tools, that in cultured primary cortical neurons induction of macroautophagy through inhibition of mTOR leads to little accumulation of double-membrane LC3-II-positive autophagosomes, as these are rapidly converted to autolysosomes. In contrast, inhibition of lysosomal proteolysis or disruption of autophagosome-lysosome fusion led to abundant accumulation of double-membrane structures, most of them positive for LC3-II, indicating inhibition of macroautophagy-dependent degradation. These latter structures resemble those seen in Alzheimer disease brains, as well as in the brains of APP/PS1 Tg mice. The authors conclude that disruption of the process downstream of autophagosome formation, rather than induction of macroautophagy, is likely to be the main determinant leading to the marked vacuolar accumulation seen in AD.

The findings raise a number of questions:

First, what is the nature of the presumed defect in the “maturation” of the autophagic pathway? One possibility is that there is a defect in the fusion of autophagosomes into autolysosomes. This could be due to a general defect in axonal transport or to yet unknown properties specific to the budding vacuoles. Alternatively, the problem could lie within the lysosomes. Lysosomal dysfunction in cultured neurons, as shown convincingly in this study, but also in experimental animals or in humans, leads to accumulation of autophagic vacuoles. Lysosomal dysfunction as a primary event could also, in part, explain the defects in endocytosis observed early in the course of AD.

A second question, perhaps more important from the point of view of potential therapies, is whether this accumulation of autophagic vacuoles is beneficial or detrimental, and whether preventing or inducing it would be a good therapeutic strategy against neurodegeneration. The data from this paper indicate that the presence of autophagosomes in AD is a readout for a dysfunction of the downstream protein degradation pathway within lysosomes. Therefore, it would make little sense to further induce the macroautophagy pathway in this setting as a therapeutic strategy, especially since autophagic vacuoles aid Aβ production (Yu et al., 2005). It would be better, in fact, to find ways to boost either the fusion event or lysosomal function in general.

Of course, the AD disease process could have additional effects on the autophagic/lysosomal pathway, as the authors themselves recognize. In fact, a recent manuscript (Pickford et al., 2008) has suggested that an early event in AD is the reduction of beclin-1, a protein that promotes macroautophagy; modulation of beclin-1 influenced the phenotype of APP transgenic mice, suggesting that the reduction of beclin-1, and the resultant reduction in macroautophagic degradation, observed in AD could have detrimental effects. Although seemingly contradictory, these reports both highlight the fact that there is dysfunction of the autophagic/lysosomal pathway in AD, perhaps at various levels. Lysosomal dysfunction with marked induction of autophagic vacuoles has been reported previously by us in the setting of aberrant α-synuclein expression in a PC12 cell model (Stefanis et al., 2001). Therefore, alterations of this pathway may play a significant role in various neurodegenerative conditions and could represent therapeutic targets.

View all comments by Leonidas Stefanis

The results in Jaeger et al. reinforce previous work from their lab (Pickford et al., 2008) showing that autophagy is a significant APP turnover pathway that can be strongly upregulated in cells. During autophagy induction, APP levels decreased somewhat more than Aβ levels, consistent with earlier data that Aβ is generated during autophagy and that efficient lysosomal proteolysis is needed to prevent the intracellular buildup of Aβ in autophagic vacuoles and lysosomes seen in AD. The authors also show that the block in autophagosome clearance in AD and AD mouse models can be exacerbated by beclin deficiency. This may be due to the impairment of early autophagy steps of autophagosome formation, which require beclin, as proposed in the paper, and it could also reflect interference with beclin-dependent late endosome functions, disturbances of which are known to disrupt amphisome formation and clearance through autophagy (1). Interestingly, the resultant buildup of APP and β-CTF in endosomal-related compartments is reminiscent of the... Read more

The results in Jaeger et al. reinforce previous work from their lab (Pickford et al., 2008) showing that autophagy is a significant APP turnover pathway that can be strongly upregulated in cells. During autophagy induction, APP levels decreased somewhat more than Aβ levels, consistent with earlier data that Aβ is generated during autophagy and that efficient lysosomal proteolysis is needed to prevent the intracellular buildup of Aβ in autophagic vacuoles and lysosomes seen in AD. The authors also show that the block in autophagosome clearance in AD and AD mouse models can be exacerbated by beclin deficiency. This may be due to the impairment of early autophagy steps of autophagosome formation, which require beclin, as proposed in the paper, and it could also reflect interference with beclin-dependent late endosome functions, disturbances of which are known to disrupt amphisome formation and clearance through autophagy (1). Interestingly, the resultant buildup of APP and β-CTF in endosomal-related compartments is reminiscent of the conditions that develop in Down syndrome, where it has been shown that APP duplication, through elevations of β-CTF, accelerates endocytosis (2). This creates more substrate traffic to lysosomes, interferes with endosome functions (2,3), and we suspect, also impairs autophagic turnover. These new investigations add to mounting evidence that disruption of lysosomal clearance mechanisms may be a critical factor in AD pathogenesis.

References:
1. Filimonenko M, Stuffers S, Raiborg C, Yamamoto A, Malerød L, Fisher EM, Isaacs A, Brech A, Stenmark H, Simonsen A. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. J Cell Biol. 2007 Nov 5;179(3):485-500. Abstract

2. Jiang Y, Mullaney KA, Peterhoff CM, Che S, Schmidt SD, Boyer-Boiteau A, Ginsberg SD, Cataldo AM, Mathews PM, Nixon RA. Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition. Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1630-5. Abstract

3. Cataldo AM, Mathews PM, Boiteau AB, Hassinger LC, Peterhoff CM, Jiang Y, Mullaney K, Neve RL, Gruenberg J, Nixon RA. Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects. Am J Pathol. 2008 Aug;173(2):370-84. Abstract

View all comments by Ralph Nixon

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