Anat Cell Biol.  2011 Jun;44(2):128-134. 10.5115/acb.2011.44.2.128.

ID4 mediates proliferation of astrocytes after excitotoxic damage in the mouse hippocampus

• Lee YS
• Kang JW
• Lee YH
• Kim DW

• KMID: 1447422
• DOI: http://doi.org/10.5115/acb.2011.44.2.128

Abstract

Inhibitor of DNA binding (ID) proteins bind to and inhibit the function of basic helix-loop-helix transcription factors, including those that regulate proliferation and differentiation during development. However, little is known about the role of ID proteins in glial activation under neuropathological conditions. In this study, we evaluated the expression of ID4 following induction of excitotoxic lesions in mouse brain by kainic acid injection. The number of ID4-expressing astrocytes increased in the CA1 layer of the injured hippocampus until 3 days post-lesion. To analyze the effects of ID4 on cell proliferation, primary astrocytes were transduced with recombinant adenovirus expressing GFP-ID4. Overexpression of ID4 led to increased proliferation of astrocytes. These results suggest that ID4 plays a proliferative role in astrocyte activation after excitotoxin-induced hippocampal neuronal death.

Keyword

ID4; Astrocytes; Excitotoxicity; Kainic acid; Proliferation

MeSH Terms

Adenoviridae
Animals
Astrocytes
Basic Helix-Loop-Helix Transcription Factors
Brain
Cell Proliferation
DNA
Hippocampus
Kainic Acid
Mice
Neurons
Proteins
Basic Helix-Loop-Helix Transcription Factors
DNA
Kainic Acid
Proteins

Figure

• 

  Fig. 1 Cresyl violet staining (A) and inhibitor of DNA binding 4 (ID4) immunoreactivity (IR) (B) in control (A1, A4, B1, B4) and kainic acid (KA)-treated mice on days 1 (A2, A5, B2, B5) and 3 (A3, A6, B3, B6). (A) In contrast to normal mice tissue, pyramidal degeneration was apparent in the CA3 region on days 1 and 3 post-lesion (arrows in A2, A3). In addition to pyramidal cell loss, small glial cell reactivities were evident (arrowheads in A5, A6). (B) In the control, ID4 IR was not found in the hippocampus (B1). In the KA-injured hippocampus, strong ID4 IR was observed on day 1 post-lesion (B2) and became maximal on day 3 (B3). Higher magnification of rectangular areas (B1-3) in the hippocampus shows sequential changes in ID4 IR cells. Note that ID4 IR cells exhibited the characteristic star-shaped morphology of astrocytes (B6). Cont, control; SO, stratum oriens; P, pyramidal cell; SR, stratum radiatum. Scale bars=200 µm (A1-3, B1-3), 20 µm (A4-6, B4-6).

• 

  Fig. 2 Double immunofluorscence staining for identification of inhibitor of DNA binding 4 (ID4)-positive cells in kainic acid (KA)-treated mice. ID4 (A, D) and GFAP (B, E) co-localized well in the CA1 region of the KA-injected hippocampus on day 3 (arrowheads in D, E). GFAP, glial fibrillar acidic protein; SO, stratum oriens; P, pyramidal cell; SR, stratum radiatum. Scale bars=100 µm (A-C), 20 µm (D-F).

• 

  Fig. 3 Western blot analysis showing the temporal pattern of inhibitor of DNA binding 4 (ID4) expression in the hippocampus following kainic acid treatment. The amount of ID4 began to increase significantly on day 1 post-lesion and reached a maximum on day 3. Subsequently, the amount of ID4 decreased on day 7. Cont, control; d, day.

• 

  Fig. 4 Expression of inhibitor of DNA binding 4 (ID4) by adenoviral transduction in astrocytes (A), and the effect of ID4 on proliferation of astrocytes (B). (A) Cells were transduced with adenovirus expressing GFP-ID4 (Ad/ID4) at a multiplicity of infection of 10 for 6 h. After washing twice with phosphate-buffered saline, cells were incubated in growth medium for 24 h. Expression of ID4 was verified by Western blotting using anti-ID4 antibody. Adenovirus expressing GFP (Ad/GFP) was used as a negative control. (B) Cellular proteins were separated on ployacrylamide gels, transferred onto a nitrocellulose membrane, and then reacted with anti-Ki-67 antibody. Anti-actin antibody was used as a loading control.

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