Abstract
Autophagy is an essential degradative pathway that maintains neuronal homeostasis and prevents axon degeneration. However, the mechanisms of autophagy in neurons are only beginning to be understood. To address this fundamental gap in knowledge, we have established several key methodologies for live-cell imaging and quantitative analysis of autophagy in primary hippocampal neurons. Using these methods, we have defined compartment-specific dynamics of autophagy in real-time under basal versus stress conditions. For example, we have characterized autophagosome biogenesis in the distal axon and subsequent retrograde transport to the soma for degradation. Autophagosomes are also generated locally within the soma. In contrast to the axon, the majority of autophagosomes in dendrites are stationary, while some exhibit bidirectional movement. These studies establish an initial road map for autophagosome dynamics in each compartment of the neuron and set the stage for a more detailed understanding of neuronal autophagy in stress and disease.
Audrey Dong and Vineet Vinay Kulkarni are co-first authors.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Maday S (2016) Mechanisms of neuronal homeostasis: autophagy in the axon. Brain Res 1649(Pt B):143–150. https://doi.org/10.1016/j.brainres.2016.03.047
Yamamoto A, Yue Z (2014) Autophagy and its normal and pathogenic states in the brain. Annu Rev Neurosci 37:55–78. https://doi.org/10.1146/annurev-neuro-071013-014149
Ariosa AR, Klionsky DJ (2016) Autophagy core machinery: overcoming spatial barriers in neurons. J Mol Med (Berl) 94(11):1217–1227. https://doi.org/10.1007/s00109-016-1461-9
Maday S, Wallace KE, Holzbaur EL (2012) Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons. J Cell Biol 196(4):407–417. https://doi.org/10.1083/jcb.201106120
Maday S, Holzbaur EL (2014) Autophagosome biogenesis in primary neurons follows an ordered and spatially regulated pathway. Dev Cell 30(1):71–85. https://doi.org/10.1016/j.devcel.2014.06.001
Maday S, Holzbaur EL (2016) Compartment-specific regulation of autophagy in primary neurons. J Neurosci 36(22):5933–5945. https://doi.org/10.1523/JNEUROSCI.4401-15.2016
Weidberg H, Shvets E, Elazar Z (2011) Biogenesis and cargo selectivity of autophagosomes. Annu Rev Biochem 80:125–156. https://doi.org/10.1146/annurev-biochem-052709-094552
Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132. https://doi.org/10.1146/annurev-cellbio-092910-154005
Spalding KL, Bhardwaj RD, Buchholz BA, Druid H, Frisen J (2005) Retrospective birth dating of cells in humans. Cell 122(1):133–143. https://doi.org/10.1016/j.cell.2005.04.028
Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441(7095):885–889. https://doi.org/10.1038/nature04724
Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E, Tanaka K (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441(7095):880–884. https://doi.org/10.1038/nature04723
Komatsu M, Wang QJ, Holstein GR, Friedrich VL Jr, Iwata J, Kominami E, Chait BT, Tanaka K, Yue Z (2007) Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci U S A 104(36):14489–14494. https://doi.org/10.1073/pnas.0701311104
Nishiyama J, Miura E, Mizushima N, Watanabe M, Yuzaki M (2007) Aberrant membranes and double-membrane structures accumulate in the axons of Atg5-null Purkinje cells before neuronal death. Autophagy 3(6):591–596
Kim M, Sandford E, Gatica D, Qiu Y, Liu X, Zheng Y, Schulman BA, Xu J, Semple I, Ro SH, Kim B, Mavioglu RN, Tolun A, Jipa A, Takats S, Karpati M, Li JZ, Yapici Z, Juhasz G, Lee JH, Klionsky DJ, Burmeister M (2016) Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay. Elife 5. https://doi.org/10.7554/eLife.12245
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728. https://doi.org/10.1093/emboj/19.21.5720
Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15(3):1101–1111. https://doi.org/10.1091/mbc.E03-09-0704
Baas PW, Deitch JS, Black MM, Banker GA (1988) Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite. Proc Natl Acad Sci U S A 85(21):8335–8339
Kleele T, Marinkovic P, Williams PR, Stern S, Weigand EE, Engerer P, Naumann R, Hartmann J, Karl RM, Bradke F, Bishop D, Herms J, Konnerth A, Kerschensteiner M, Godinho L, Misgeld T (2014) An assay to image neuronal microtubule dynamics in mice. Nat Commun 5:4827. https://doi.org/10.1038/ncomms5827
Gowrishankar S, Yuan P, Wu Y, Schrag M, Paradise S, Grutzendler J, De Camilli P, Ferguson SM (2015) Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques. Proc Natl Acad Sci U S A 112(28):E3699–E3708. https://doi.org/10.1073/pnas.1510329112
Lee S, Sato Y, Nixon RA (2011) Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy. J Neurosci 31(21):7817–7830. https://doi.org/10.1523/JNEUROSCI.6412-10.2011
Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1(5):2406–2415. https://doi.org/10.1038/nprot.2006.356
Acknowledgments
This work was funded by NIH grants K99NS082619 and R00NS082619, the McCabe Fund Fellow Award, the University of Pennsylvania Alzheimer’s Disease Core Center, the Intellectual and Developmental Disabilities Research Center at the Children’s Hospital of Philadelphia and the University of Pennsylvania, and The Philadelphia Foundation to S.M.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dong, A., Kulkarni, V.V., Maday, S. (2019). Methods for Imaging Autophagosome Dynamics in Primary Neurons. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8873-0_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8872-3
Online ISBN: 978-1-4939-8873-0
eBook Packages: Springer Protocols