Characterization of starvation-induced autophagy in cerebellar Purkinje cells of pHluorin-mKate2-human LC3B transgenic mice

Juan Alejandro Oliva Trejo,Chigure Suzuki,Norihiro Tada,Soichiro Kakuta,Isei Tanida,Yasuo Uchiyama

doi:10.1038/s41598-020-66370-6

Juan Alejandro Oliva Trejo, Chigure Suzuki + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-66370-6

Copy DOI

Journal: Scientific Reports	Publication Date: Jun 15, 2020
Citations: 9	License type: open-access

Affiliation: Juntendo University

Abstract

We generated a new transgenic mouse model that expresses a pHluorin-mKate2 fluorescent protein fused with human LC3B (PK-LC3 mice) for monitoring autophagy activity in neurons of the central nervous system. Histological analysis revealed fluorescent puncta in neurons of the cerebral cortex, hippocampus, cerebellar Purkinje cells, and anterior spinal regions. Using CLEM analysis, we confirmed that PK-LC3-positive puncta in the perikarya of Purkinje cells correspond to autophagic structures. To validate the usability of PK-LC3 mice, we quantified PK-LC3 puncta in Purkinje cells of mice kept in normal feeding conditions and of mice starved for 24 hours. Our results showed a significant increase in autophagosome number and in individual puncta areal size following starvation. To confirm these results, we used morphometry at the electron microscopic level to analyze the volume densities of autophagosomes and lysosomes/autolysosomes in Purkinje cells of PK-LC3 mice. The results revealed that the volume densities of autophagic structures increase significantly after starvation. Together, our data show that PK-LC3 mice are suitable for monitoring autophagy flux in Purkinje cells of the cerebellum, and potentially other areas in the central nervous system.

Highlights

IntroductionIn neurons of the central nervous system (CNS), macroautophagy (henceforward, autophagy) has long been established as an essential process for preserving homeostasis and preventing cell death
In neurons of the central nervous system (CNS), macroautophagy has long been established as an essential process for preserving homeostasis and preventing cell death
Our goal was to develop a transgenic mouse model that could enable in vivo monitoring of autophagy and autolysosome activity in CNS neurons

Summary

Introduction

In neurons of the central nervous system (CNS), macroautophagy (henceforward, autophagy) has long been established as an essential process for preserving homeostasis and preventing cell death. Intracellular changes in neurons feature ubiquitylated protein accumulation and cell death[5,6,7] These models have shown that the neuronal axon is vulnerable to autophagy deficiency, exhibiting dysregulated development[7] and pathological changes[5]. Tanida et al published a new method for monitoring autophagy flux that uses a new double fluorescent construct attached to human (h)LC3B23 Both fluorescent proteins were selected after a careful screening to ensure they could be used to monitor autophagosomes and autolysosomes. During protein degradation in autolysosomes, the internal pH is decreased due to the acidic milieu, and the green fluorescence emitted by pHluorin is rapidly diminished and the fluorescence from mKate[2] remains red Based on this principle, we constructed a new transgenic mouse model called pHluorin-mKate2-hLC3B (PK-LC3)

Objectives

Methods

Results

Conclusion