Seiji Miyake1, Yuji Takihara1,2, Satoshi Yokota1,3, Yoshihiro Takamura1, Masaru Inatani1. 1. a Department of Ophthalmology, Faculty of Medical Sciences , University of Fukui , Fukui , Japan. 2. b Cancer Science Institute of Singapore , National University of Singapore , Medical Drive , Singapore. 3. c Department of Ophthalmology and Visual Sciences , Kyoto University Graduate School of Medicine , Kyoto , Japan.
Abstract
PURPOSE: Axonal transport is fundamental to autophagy in neuronal cells. To understand its biological significance in various conditions, it is necessary to monitor the process of autophagy. However, monitoring methods are often limited to static analyses, such as protein expression and histological observations. Autophagy has multistep process and is highly dynamic; therefore, additional techniques are necessary to study autophagy. In this study, we quantified the dynamics of autophagy-related organelle transport under conditions of dynamic instability and catastrophic disruption of microtubules using in vitro live imaging. MATERIALS AND METHODS: Retinal ganglion cells (RGCs) were isolated from postnatal day 3 Sprague-Dawley rats by immunopanning. After 7 days of culture, acidic organelles were stained by LysoTracker. Dynamics of acidic organelles was quantified using kymographs. Colchicine was used to induce microtubule disruption. Movement of acidic organelles was observed at five time points: before, and at 6, 24, 72, and 120 h after colchicine stimulation. Ethidium homodimer-1 (EthD-1) was used to determine cell viability. RESULTS: The status of axonal transport of acidic organelles (n = 363) from 27 RGCs was classified into four categories: anterograde (1.4%), retrograde (90%), stationary (8.0%), and fluttering (0.28%). Six hours after the induction of microtubule disruption in 14 of 27 RGCs, almost all acidic organelles (n = 236) were stationary. All acidic components had completely stopped moving 24 h later. At 72 h after stimulation, axonal fragmentation, and shrinking and disappearance of soma were observed in 71% of RGCs. Finally, the remaining RGCs became positive for EthD-1. In the control (13 of 27 RGCs), axonal transport was maintained for 120 h and EthD-1-positive RGCs were not observed. CONCLUSION: Almost all acidic organelles were transported retrogradely along the axon, which was inhibited by colchicine. Understanding the dynamics of acidic organelles may provide useful parameters for characterizing autophagy of neuronal cells in pathophysiological conditions.
PURPOSE: Axonal transport is fundamental to autophagy in neuronal cells. To understand its biological significance in various conditions, it is necessary to monitor the process of autophagy. However, monitoring methods are often limited to static analyses, such as protein expression and histological observations. Autophagy has multistep process and is highly dynamic; therefore, additional techniques are necessary to study autophagy. In this study, we quantified the dynamics of autophagy-related organelle transport under conditions of dynamic instability and catastrophic disruption of microtubules using in vitro live imaging. MATERIALS AND METHODS: Retinal ganglion cells (RGCs) were isolated from postnatal day 3 Sprague-Dawley rats by immunopanning. After 7 days of culture, acidic organelles were stained by LysoTracker. Dynamics of acidic organelles was quantified using kymographs. Colchicine was used to induce microtubule disruption. Movement of acidic organelles was observed at five time points: before, and at 6, 24, 72, and 120 h after colchicine stimulation. Ethidium homodimer-1 (EthD-1) was used to determine cell viability. RESULTS: The status of axonal transport of acidic organelles (n = 363) from 27 RGCs was classified into four categories: anterograde (1.4%), retrograde (90%), stationary (8.0%), and fluttering (0.28%). Six hours after the induction of microtubule disruption in 14 of 27 RGCs, almost all acidic organelles (n = 236) were stationary. All acidic components had completely stopped moving 24 h later. At 72 h after stimulation, axonal fragmentation, and shrinking and disappearance of soma were observed in 71% of RGCs. Finally, the remaining RGCs became positive for EthD-1. In the control (13 of 27 RGCs), axonal transport was maintained for 120 h and EthD-1-positive RGCs were not observed. CONCLUSION: Almost all acidic organelles were transported retrogradely along the axon, which was inhibited by colchicine. Understanding the dynamics of acidic organelles may provide useful parameters for characterizing autophagy of neuronal cells in pathophysiological conditions.
Authors: Barry Boland; Wai Haung Yu; Olga Corti; Bertrand Mollereau; Alexandre Henriques; Erwan Bezard; Greg M Pastores; David C Rubinsztein; Ralph A Nixon; Michael R Duchen; Giovanna R Mallucci; Guido Kroemer; Beth Levine; Eeva-Liisa Eskelinen; Fanny Mochel; Michael Spedding; Caroline Louis; Olivier R Martin; Mark J Millan Journal: Nat Rev Drug Discov Date: 2018-08-17 Impact factor: 84.694
Authors: R G Valencia; E Mihailovska; L Winter; K Bauer; I Fischer; G Walko; J Jorgacevski; M Potokar; R Zorec; G Wiche Journal: Neuropathol Appl Neurobiol Date: 2020-06-25 Impact factor: 8.090