Laser Capture Microdissection Reveals Specific Genes Related to Purkinje Cell Death in the Leaner Mice

Sang-Soep Nahm; Ji Eun Yoo; Louise C. Abbott

doi:10.5625/lar.2010.26.3.301

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Nahm, Yoo, and Abbott: Laser Capture Microdissection Reveals Specific Genes Related to Purkinje Cell Death in the Leaner Mice

Original Article

Lab. Anim. Res. 2010;26(3):301-305.

Published online: 17 September 2010

DOI: https://doi.org/10.5625/lar.2010.26.3.301

Laser Capture Microdissection Reveals Specific Genes Related to Purkinje Cell Death in the Leaner Mice

Sang-Soep Nahm^1,^∗, Ji Eun Yoo¹, Louise C. Abbott²

¹Department of Veterinary Medicine, College of Veterinary Medicine, Konkuk University, Seoul, Korea

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, USA

^∗Corresponding author: Sang-Soep Nahm, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea Tel: +82-2-450-3705 Fax: +82-2-450-3037 E-mail: ssnahm@konkuk.ac.kr

Received 14 August 2010 Revised 15 September 2010 Accepted 18 September 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The leaner mouse carries a mutation in the gene encoding the α1A subunit of P/Q-type calcium channels. Leaner mice exhibit extensive cerebellar granule and Purkinje cell loss that results in cerebellar dysfunction. A previous study suggested that a small population of leaner Purkinje cells undergo apoptosis, however the cell death mode of the rest of degenerating Purkinje cells has not been identified. In order to investigate the mechanisms underlying leaner Purkinje cell death, gene arrays that contain 243 cell death related genes were carried out. To increase the chance of detecting Purkinje cell specific genes, laser capture microdissection was employed to obtain Purkinje cell enriched samples. The gene array analysis revealed several potential genes that are involved in autophagic cell death pathway including cathepsin D, a key lysosomal protease that triggers autophagic degradation. Further analysis on LC3, which is a hallmark for autophagic cell death showed that leaner Purkinje cells are degenerating via autophagic process. The present study provides evidence that calcium channel defects trigger different modes of neurodegeneration in the cerebellum.

Keywords: Cerebellum, autophagy, calcium channel mutation, cathepsin D, LC3

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Figure 1.

Photomicrographs show dissections of cerebellar Purkinje cells (white arrows) using laser capture microdissection. Pictures show before (A) and after (B) laser capture microdissection on an unstained cerebellar section and procured Purkinje cells on the transfer film (C). GCL, granule cell layer; WM, white matter.

Figure 2.

Western blot analysis of cathepsin D expression in wild type and leaner mice (A). Note that higher cathepsin D expression near 30 kDa in the leaner cerebellum. Densitometric analysis of cathepsin D normalized by b-actin. Leaner mice showed a significant increase in cathepsin D expression (B). Data are presented as mean±SEM. ∗P<0.05.

Figure 3.

Photomicrographs of cathepsin D immunohistochemistry of wild type (A) and leaner mouse (B). Note that the leaner cerebellum shows increased cathepsin D immunoreactivity in the Purkinje cell bodies. ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bar=20 µm.

Figure 4.

Photomicrographs of LC3 immunohistochemistry of wild type (A) and leaner mouse (B). Note that the leaner cerebellum shows increased LC3 immunoreactivity in the Purkinje cell bodies. ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bar=20 µm.

Table 1.

Up-regulated genes in leaner Purkinje cells∗

Group	Gene Name	Gene Description	Accession #	Ratio
ARF	Dap1	death-associated protein 1	AI196645	1.5
ARF	Ctsd	cathepsin D	NM_009983	1.7
ARF	MT-2	metallothionein 2	K02236	2.5
ARF	DAD-1	defender against cell death 1	NM_010015	3.6
MIT	Bak	Bcl2 homologous antagonist	NM_007523	1.3
STP	14-3-3 eta	tyrosine 3-monooxygenase	NM_011738	1.6
STP	TRANK	peroxiredoxin 4	NM_016764	2.4
CCR	PCNA	proliferating cell nuclear antigen	NM_011045	1.4
TNS	TALL-1	B-cell activating factor (Baff)	AF119383	2.0

Genes identified by confidence analysis (P<0.05). ARF, apoptosis-related factors; MIT, mitochondrial associated genes; STP, signal transduction genes; CCR, cell cycle regulators; TNS, tumor necrosis factor superfamily.

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