Differential expression patterns of NDRG family proteins in the central nervous system

Abstract

The N-myc downstream-regulated gene (NDRG) family consists of four proteins: NDRG1, NDRG2, NDRG3, and NDRG4 in mammals. NDRG1 has been thoroughly studied as an intracellular protein associated with stress response, cell growth, and differentiation. A nonsense mutation in the NDRG1 gene causes hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D. We previously generated Ndrg1-deficient mice and found that they exhibited peripheral nerve degeneration caused by severe demyelination, but that the complicated motor abilities were retained. These results implied that other NDRG family proteins may compensate for the NDRG1 deficiency in the central nervous system. In this study we raised specific antibodies against each member of the NDRG protein family and examined their cellular expression patterns in the mouse brain. In the cerebrum, NDRG1 and NDRG2 were localized in oligodendrocytes and astrocytes, respectively, whereas NDRG3 and NDRG4 were ubiquitous. In the cerebellum, NDRG1 and NDRG4 were localized in Purkinje cells and NDRG2 in Bergmann glial cells. NDRG3 was detected in the nuclei in most cells. These expression patterns demonstrated the cell type-specific and ubiquitous localization of the NDRG family proteins. Each NDRG may play a partially redundant role in specific cells in the brain.

Figure 1

Antibody specificity against NDRG family proteins. Lysates of COS-1 cells transfected with green fluorescent protein (GFP) or mouse NDRG–GFP vectors were analyzed by Western blotting. Each antibody specifically reacted with respective NDRG–GFP fusion proteins (closed arrowheads). Bands indicated by open arrowheads were endogenous NDRG proteins. Each lane contained 5 μg of total protein.

Figure 2

Expression of NDRG family proteins in the mouse brain. Brain lysates were prepared from the wild-type and Ndrg1-deficient mice aged 4 or 12 weeks and analyzed by Western blotting. Each lane contained 5 μg of total protein. ^+/+, wild-type mice; ^−/−, homozygous Ndrg1-deficient mice; ^+/−, heterozygous Ndrg1-deficient mice.

Figure 3

Histological assessment of the brain of adult Ndrg1-deficient mice. Transverse sections of cerebral neocortex (A,B) and hippocampus (C,D) of adult wild-type (^+/+; A,C) and adult Ndrg1-deficient (^−/−; B,D) mice were compared. There were no significant differences in the structure of the brain regardless of genotype. Transverse sections of the forebrain at the level of the corpus callosum (E,F) and lateral ventricle (G,H) of adult wild-type (E,G) and adult Ndrg1-deficient (F,H) mice were compared. Normal myelination was observed in wild-type (E) and Ndrg1-deficient mice (F). NDRG1 was detected in the axon bundles of the corpus callosum and corpus striatum in wild-type mice (arrowheads in G). NDRG1 expression was not detected in the Ndrg1-deficient mice (H). HE, hematoxylin–eosin staining; myelin, luxol–fast blue staining; NDRG1, anti-NDRG1 immunostaining. Bar = 100 μm.

Figure 4

Localization of NDRG1 in the brain. NDRG1 was detected in the cytoplasm of the oligodendrocyte in the cerebrum (arrowhead in A) and in Purkinje cells in the cerebellum (open arrowhead in B). Expression of NDRG1 (C) was colocalized with an oligodendrocyte-specific marker CNPase (D). Merged image is shown in E. Bar = 10 μm.

Figure 5

Localization of NDRG2 in the brain. NDRG2 was detected in the astrocytes in the cerebrum (arrowhead in A) and in Bergmann glial cells in the cerebellum (open arrowhead in B). Expression of NDRG2 (C,F) was colocalized with an astrocyte-specific marker GFAP (D), but not with a neuron marker NeuN (G). Merged images are shown in E and H, respectively. Bar = 10 μm.

Figure 6

Localization of NDRG3 in the brain. NDRG3 was detected in the nucleus of most cells in the cerebrum, but especially strongly in the neurons (arrowhead in A) and nucleus of Purkinje cells in the cerebellum (open arrowhead in B). Expression of NDRG3 (C) was colocalized with a neuron marker NeuN (D). Merged images are shown in E. Bar = 10 μm.

Figure 7

Localization of NDRG4 in the brain. NDRG4 was detected in most cells in the cerebrum, but especially strongly in the neurons (arrowhead in A) and Purkinje cells in the cerebellum (open arrowhead in B). Strong expression of NDRG4 (C) was colocalized with a neuron marker NeuN (D). Merged images are shown in E. Bar = 10 μm.