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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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Rescue effect of siRNA-resistant form of GGTβ. A) Representative images of Purkinje cells after transfection with pSUPER (control), pSUPER-GGTβ-siRNA, or GGTβ-siRNA together with HA-GGTβ or HA-GGTβRes. B) Quantification for the number of crossings. C) Quantification for total dendritic length between neighboring circles. D) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. E, F) Total dendritic length between neighboring circles at the interval of 25 μm (between 50 and 75 μm or between 75 and 100 μm). Data are shown as means ± SEM (n = 12 for control; n = 17 for GGT-siRNA; n = 15 for GGT-siRNA plus HA-GGTβ; n = 19 for GGT-siRNA plus HA-GGTβRes). N.S. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Student's t test. Scale bar = 20 μm.
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Figure 5: Rescue effect of siRNA-resistant form of GGTβ. A) Representative images of Purkinje cells after transfection with pSUPER (control), pSUPER-GGTβ-siRNA, or GGTβ-siRNA together with HA-GGTβ or HA-GGTβRes. B) Quantification for the number of crossings. C) Quantification for total dendritic length between neighboring circles. D) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. E, F) Total dendritic length between neighboring circles at the interval of 25 μm (between 50 and 75 μm or between 75 and 100 μm). Data are shown as means ± SEM (n = 12 for control; n = 17 for GGT-siRNA; n = 15 for GGT-siRNA plus HA-GGTβ; n = 19 for GGT-siRNA plus HA-GGTβRes). N.S. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Student's t test. Scale bar = 20 μm.

Mentions: To exclude possible off-target effect of GGTβ-siRNA, we performed rescue experiment with GGTβRes, the siRNA-resistant form of GGTβ, which has been described in our previous report [12]. Cerebellar slices were transfected with GGTβ-siRNA either alone or together with HA-GGTβ or HA-GGTβRes. We found that expression of HA-GGTβRes rescued dendritic development in GGTβ-siRNA-transfected neurons, whereas transfection with HA-GGTβ had no rescue effect (Figure 5A). The rescue effect could be reflected from the changes of the number of crossings (Figure 5B and 5D) and total dendritic length (Figure 5C, E, F) in indicated dendritic fields. These results suggest GGT effect on PC dendrtic development is specific.


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

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

Rescue effect of siRNA-resistant form of GGTβ. A) Representative images of Purkinje cells after transfection with pSUPER (control), pSUPER-GGTβ-siRNA, or GGTβ-siRNA together with HA-GGTβ or HA-GGTβRes. B) Quantification for the number of crossings. C) Quantification for total dendritic length between neighboring circles. D) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. E, F) Total dendritic length between neighboring circles at the interval of 25 μm (between 50 and 75 μm or between 75 and 100 μm). Data are shown as means ± SEM (n = 12 for control; n = 17 for GGT-siRNA; n = 15 for GGT-siRNA plus HA-GGTβ; n = 19 for GGT-siRNA plus HA-GGTβRes). N.S. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Student's t test. Scale bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Rescue effect of siRNA-resistant form of GGTβ. A) Representative images of Purkinje cells after transfection with pSUPER (control), pSUPER-GGTβ-siRNA, or GGTβ-siRNA together with HA-GGTβ or HA-GGTβRes. B) Quantification for the number of crossings. C) Quantification for total dendritic length between neighboring circles. D) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. E, F) Total dendritic length between neighboring circles at the interval of 25 μm (between 50 and 75 μm or between 75 and 100 μm). Data are shown as means ± SEM (n = 12 for control; n = 17 for GGT-siRNA; n = 15 for GGT-siRNA plus HA-GGTβ; n = 19 for GGT-siRNA plus HA-GGTβRes). N.S. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Student's t test. Scale bar = 20 μm.
Mentions: To exclude possible off-target effect of GGTβ-siRNA, we performed rescue experiment with GGTβRes, the siRNA-resistant form of GGTβ, which has been described in our previous report [12]. Cerebellar slices were transfected with GGTβ-siRNA either alone or together with HA-GGTβ or HA-GGTβRes. We found that expression of HA-GGTβRes rescued dendritic development in GGTβ-siRNA-transfected neurons, whereas transfection with HA-GGTβ had no rescue effect (Figure 5A). The rescue effect could be reflected from the changes of the number of crossings (Figure 5B and 5D) and total dendritic length (Figure 5C, E, F) in indicated dendritic fields. These results suggest GGT effect on PC dendrtic development is specific.

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