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AD and Autophagy—A Problem of Supply and Demand
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9 July 2008. Overproduction seems to be a major problem for neurons. In Alzheimer (AD), and other neurodegenerative diseases, toxic proteins such as amyloid-β are produced faster than they can be cleared. In AD, the abnormally high accumulation of autophagic vacuoles (AVs) in dystrophic neurites suggests that autophagy, too, has run amok. But is overzealous autophagy really the problem? In the July 2 Journal of Neuroscience, researchers led by Ralph Nixon, Nathan Kline Institute, Orangeburg, New York, show that primary cortical neurons have an extremely high capacity for disposing of autophagic vesicles. Even after autophagy is experimentally ramped up, neurons seem to have no problem mopping up these vacuoles. But when the researchers inhibited lysosomal proteolysis, it was a different story. Then, they found that AD-like AVs did accumulate. The results suggest that it is not autophagy per se but clearance of autophagic vacuoles that is detrimental in AD.
“The results were surprising to us in the sense that inducing autophagy as robustly as one could, that you rarely accumulate the kind of structures found in the AD brain,” said Nixon in an interview with ARF. The problem in AD dystrophic neurons, it seems, is that lysosomes are unable to keep up with the production of autophagic vacuoles. This suggestion has implications not only for AD, but for other neurodegenerative diseases such as Huntington disease. Boosting autophagy may be one strategy for dealing with unwanted proteins, but it may only work if lysosomes can keep up with the process. “If you induce autophagy and it is not an efficient process, you may actually compound the problem,” said Nixon.
The realization that AVs accumulate in AD and other disorders led to the notion that autophagy might be upregulated in certain diseases, suggested Nixon. But an equally reasonable explanation might be that clearance of these vacuoles is impaired. “The purpose of this study was to see what happens to neurons if you induce very strongly the autophagy pathway, or alternatively block the end steps,” said Nixon. Lead author Barry Boland and colleagues first tried boosting the process. Autophagy can be induced by rapamycin, an inhibitor of mTOR (mammalian target of rapamycin). When the researchers added rapamycin to primary cortical cultures, they found that LC3-II, a marker of autophagosomes, was increased. However, the researchers found that this marker turned up predominantly in autolysosomes, structures that contain lysosomal cathepsin-D protease. Autolysosomes—fusions of autophagosomes and lysosomes—are the end of the road for the autophagic pathway, and the findings suggest that although autophagy was induced, the vacuoles were rapidly cleared.
What would happen in a neuron if lysosomal clearance of autophagic vacuoles was impaired? To address this, the researchers tried two different experiments. They challenged neurons with lysosomal protease inhibitors and they also treated them with vinblastine, an inhibitor of the microtubule transport system that autophagosomes depend on to rendezvous with lysosomes. Inhibiting lysosomal proteolysis with leupeptin and pepstatin caused accumulation of autophagic vacuoles in both cell bodies and also in some neurites. Interestingly, these AVs contained amorphous, electron-dense material, just like the AVs found in AD dystrophic neurites. Vinblastine also induced a rapid accumulation of autophagic vacuoles and an elevation of LC3-II well above that seen with rapamycin. The vinblastine treatment resulted in an increase in mostly autophagosomes, while normal autolysosome levels were maintained. Here, too, the autophagic vacuoles that accumulated in cell bodies and neurites ultrastructurally resembled those found in AD.
“We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone,” write the authors.
This study adds to a growing literature indicating the importance of autophagy in neurons and in AD. Conditionally knocking out ATG5 and ATG7, key autophagy genes, in neurons causes neurodegeneration in mice (see ARF related news story), for example, and just recently researchers led by Tony Wyss-Coray at Stanford University, Palo Alto, California, reported that levels of beclin, another key autophagic regulator, are reduced in AD (see ARF related news story). Nixon’s new study also points the finger more squarely at the lysosomes, which, in addition to their role in lysosomal storage diseases, have been implicated in AD pathology. Aβ can accumulate and destabilize lysosomes (see Yang et al., 1998), an effect that is exacerbated by ApoE4 (see Ji et al., 2006), the strongest genetic risk factor identified to date for late-onset AD. “Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway,” conclude the authors.—Tom Fagan.
Reference:
Boland B, Kumar A, Lee S, Platt FM, Wegiel J, Yu WH, Nixon RA. Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer’s disease. J. Neurosci. 2008, July 2; 28:6926-6937. Abstract
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Primary Papers: Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease.
Comment by: Leonidas Stefanis
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Submitted 24 July 2008
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Posted 24 July 2008
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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...
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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 |
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