Journal List > Lab Anim Res > v.26(3) > 1053624

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


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.


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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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