Dissecting early steps in the authophagy machinery to understand the molecular basis of neurodegeneration

Paola Lenzi, Alessandra Falleni, Gianfranco Natale, Alessia Bartalucci, Paola Soldani, Gabriele Siciliano, Antonio Paparelli


Autophagy is involved in degrading long-lived proteins such as aged/oxidized cell components including alpha-synuclein, SOD1 huntingtin in addition to organelles, such as mitochondria. Autophagy is involved in a variety of neurodegenerative disorders and the ability to detect autophagy alterations represents a seminal point in clinical anatomy. Despite such a critical role it remains unclear how autophagy starts at sub-cellular level. In detail, the ability to dissect the fine morphological correlates of the autophagy machinery remains a critical point to unravel the early molecular and morphological events in neurodegeneration. In the present study we used transmission electron microscopy both in baseline condition and following neurodegeneration-related autophagy-dependent triggers at various time intervals. Plain transmission electron microscopy was paralleled by immunocytochemistry for LC3, beclin-1 and Rab24 to localize early autophagy structures. The present data provide morphological evidence for the occurrence of the so-called phagophore during early steps in autophagy induction. The phagophore appears on nuclear and endoplasmic membranes as a beclin1 positive membrane bulging. We found a correlation between the number of altered mitochondria and phagophore-like labelled membranes. These data suggest that immunogold-based TEM provides a useful tool to detect early autophagy activation during neurodegeneration allowing to dissect early vs late autophagy compartments such as autophagolysosomes and stagnant autophagy vacuoles. This work was supported by research grant: PRIN 2010-2011 F.F. This work was supported by an educational grant from CUCCS.


autophagy; PC12 cell line; immunoelectron microscopy

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DOI: http://dx.doi.org/10.13128/IJAE-14867

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