Effect of Microtubule Disruption on Dynamics of Acidic Organelles in the Axons of Primary Cultured Retinal Ganglion Cells

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.

KEYWORDS:

• Acidic organelle
• axonal transport
• microtubule disruption
• retinal ganglion cell

Acknowledgment

The authors thank Junko Yamamoto of Life Science Research Laboratory, Division of Bioresearch, University of Fukui, for excellent technical advice regarding the confocal laser scanning microscopy.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of paper.

Funding

This work was supported by JSPS KAKENHI Grant Numbers 26893100 and 16K20311.

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Additional information

Funding

This work was supported by JSPS KAKENHI Grant Numbers 26893100 and 16K20311.