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Geranylgeranyltransferase I is essential for dendritic development of cerebellar Purkinje cells.

Wu KY, Zhou XP, Luo ZG - Mol Brain (2010)

Bottom Line: We found that GGT was abundantly expressed in the developing rat cerebellum, in particular molecular layer (ML), the region enriched with PC dendrites.The effect of BDNF or high K+ was inhibited by inhibition or down-regulation of GGT.Our results indicate that GGT plays an important role in Purkinje cell development, and suggest a novel role of GGT in neuronal morphogenesis in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT

Background: During cerebellar development, Purkinje cells (PCs) form the most elaborate dendritic trees among neurons in the brain, but the mechanism regulating PC arborization remains largely unknown. Geranylgeranyltransferase I (GGT) is a prenyltransferase that is responsible for lipid modification of several signaling proteins, such as Rho family small GTPase Rac1, which has been shown to be involved in neuronal morphogenesis. Here we show that GGT plays an important role in dendritic development of PCs.

Results: We found that GGT was abundantly expressed in the developing rat cerebellum, in particular molecular layer (ML), the region enriched with PC dendrites. Inhibition or down-regulation of GGT using small interference RNA (siRNA) inhibited dendritic development of PCs. In contrast, up-regulation of GGT promoted dendritic arborization of PCs. Furthermore, neuronal depolarization induced by high K+ or treatment with brain-derived neurotrophic factor (BDNF) promoted membrane association of Rac1 and dendritic development of PCs in cultured cerebellar slices. The effect of BDNF or high K+ was inhibited by inhibition or down-regulation of GGT.

Conclusion: Our results indicate that GGT plays an important role in Purkinje cell development, and suggest a novel role of GGT in neuronal morphogenesis in vivo.

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Related in: MedlinePlus

Expression and localization of GGT in the rat cerebellum. A) Lysates of HEK293 cells transfected with HA-GGTβ or vehicle plasmid were subjected to immunoblotting (IB) with anti-HA or -GGTβ antibody at indicated dilution. B) Homogenates of cerebellum from rats at indicated ages were subjected to IB with indicated antibodies. C) P10 rat cerebellar sections were stained with antibody against GGTα or control antibody, together with MAP2. Hoechst was used to mark cell layers. ML: molecular layer; WM: white matter. Scale bar is 200 μm (left and right lanes) or 20 μm (middle lane). D) P15 rat cerebellar sections were stained with antibody against GGTβ or control antibody, together with Calbindin. DAPI was used to mark cell layers. Scale bar is 20 μm.
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Figure 1: Expression and localization of GGT in the rat cerebellum. A) Lysates of HEK293 cells transfected with HA-GGTβ or vehicle plasmid were subjected to immunoblotting (IB) with anti-HA or -GGTβ antibody at indicated dilution. B) Homogenates of cerebellum from rats at indicated ages were subjected to IB with indicated antibodies. C) P10 rat cerebellar sections were stained with antibody against GGTα or control antibody, together with MAP2. Hoechst was used to mark cell layers. ML: molecular layer; WM: white matter. Scale bar is 200 μm (left and right lanes) or 20 μm (middle lane). D) P15 rat cerebellar sections were stained with antibody against GGTβ or control antibody, together with Calbindin. DAPI was used to mark cell layers. Scale bar is 20 μm.

Mentions: Given that GGTα is shared between GGT and FT [1], to determine the expression of GGT in the brain, we generated an antibody, which was raised against synthetic GGTβ peptide. As shown in Figure 1A, this antibody recognized bands corresponding to exogenous HA-rGGTβ (rat GGTβ), as well as endogenous hGGTβ (human GGTβ) expressed in HEK293 cells, respectively. Next, we determined the levels of GGT in the rat cerebellum at different stages. We found that cerebellar GGTβ progressively increased after birth, and this pattern is similar to that of Calbindin, an intracellular calcium binding protein which is often used as a marker for cerebellum Purkinje cells. However, levels of GGTα remained unchanged (Figure 1B). Next, we examined regional distribution of GGT in the rat cerebellum. We found that GGTα was highly expressed in the cerebellar molecular layer, the region enriched with Purkinje cell dendritic trees (Figure 1C, left lane). Particularly, GGTα was enriched in the cell bodies and dendritic arbors, which were stained positive for MAP2 (Figure 1C, middle lane). GGTβ was also found to be expressed in Purkinje cells (PCs) that were positively labeled with Calbindin antibody (Figure 1D). These results suggest that GGT might be involved in Purkinje cell development.


Geranylgeranyltransferase I is essential for dendritic development of cerebellar Purkinje cells.

Wu KY, Zhou XP, Luo ZG - Mol Brain (2010)

Expression and localization of GGT in the rat cerebellum. A) Lysates of HEK293 cells transfected with HA-GGTβ or vehicle plasmid were subjected to immunoblotting (IB) with anti-HA or -GGTβ antibody at indicated dilution. B) Homogenates of cerebellum from rats at indicated ages were subjected to IB with indicated antibodies. C) P10 rat cerebellar sections were stained with antibody against GGTα or control antibody, together with MAP2. Hoechst was used to mark cell layers. ML: molecular layer; WM: white matter. Scale bar is 200 μm (left and right lanes) or 20 μm (middle lane). D) P15 rat cerebellar sections were stained with antibody against GGTβ or control antibody, together with Calbindin. DAPI was used to mark cell layers. Scale bar is 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902468&req=5

Figure 1: Expression and localization of GGT in the rat cerebellum. A) Lysates of HEK293 cells transfected with HA-GGTβ or vehicle plasmid were subjected to immunoblotting (IB) with anti-HA or -GGTβ antibody at indicated dilution. B) Homogenates of cerebellum from rats at indicated ages were subjected to IB with indicated antibodies. C) P10 rat cerebellar sections were stained with antibody against GGTα or control antibody, together with MAP2. Hoechst was used to mark cell layers. ML: molecular layer; WM: white matter. Scale bar is 200 μm (left and right lanes) or 20 μm (middle lane). D) P15 rat cerebellar sections were stained with antibody against GGTβ or control antibody, together with Calbindin. DAPI was used to mark cell layers. Scale bar is 20 μm.
Mentions: Given that GGTα is shared between GGT and FT [1], to determine the expression of GGT in the brain, we generated an antibody, which was raised against synthetic GGTβ peptide. As shown in Figure 1A, this antibody recognized bands corresponding to exogenous HA-rGGTβ (rat GGTβ), as well as endogenous hGGTβ (human GGTβ) expressed in HEK293 cells, respectively. Next, we determined the levels of GGT in the rat cerebellum at different stages. We found that cerebellar GGTβ progressively increased after birth, and this pattern is similar to that of Calbindin, an intracellular calcium binding protein which is often used as a marker for cerebellum Purkinje cells. However, levels of GGTα remained unchanged (Figure 1B). Next, we examined regional distribution of GGT in the rat cerebellum. We found that GGTα was highly expressed in the cerebellar molecular layer, the region enriched with Purkinje cell dendritic trees (Figure 1C, left lane). Particularly, GGTα was enriched in the cell bodies and dendritic arbors, which were stained positive for MAP2 (Figure 1C, middle lane). GGTβ was also found to be expressed in Purkinje cells (PCs) that were positively labeled with Calbindin antibody (Figure 1D). These results suggest that GGT might be involved in Purkinje cell development.

Bottom Line: We found that GGT was abundantly expressed in the developing rat cerebellum, in particular molecular layer (ML), the region enriched with PC dendrites.The effect of BDNF or high K+ was inhibited by inhibition or down-regulation of GGT.Our results indicate that GGT plays an important role in Purkinje cell development, and suggest a novel role of GGT in neuronal morphogenesis in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT

Background: During cerebellar development, Purkinje cells (PCs) form the most elaborate dendritic trees among neurons in the brain, but the mechanism regulating PC arborization remains largely unknown. Geranylgeranyltransferase I (GGT) is a prenyltransferase that is responsible for lipid modification of several signaling proteins, such as Rho family small GTPase Rac1, which has been shown to be involved in neuronal morphogenesis. Here we show that GGT plays an important role in dendritic development of PCs.

Results: We found that GGT was abundantly expressed in the developing rat cerebellum, in particular molecular layer (ML), the region enriched with PC dendrites. Inhibition or down-regulation of GGT using small interference RNA (siRNA) inhibited dendritic development of PCs. In contrast, up-regulation of GGT promoted dendritic arborization of PCs. Furthermore, neuronal depolarization induced by high K+ or treatment with brain-derived neurotrophic factor (BDNF) promoted membrane association of Rac1 and dendritic development of PCs in cultured cerebellar slices. The effect of BDNF or high K+ was inhibited by inhibition or down-regulation of GGT.

Conclusion: Our results indicate that GGT plays an important role in Purkinje cell development, and suggest a novel role of GGT in neuronal morphogenesis in vivo.

Show MeSH
Related in: MedlinePlus