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.
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