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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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Effect of GGT-siRNA on high K+-induced Purkinje cell dendrite development. A) Rat cerebellar slices at DIV2 were pre-treated with GGTi-2147 (2.5 μM) or vehicle DMSO for 45 min, followed by the treatment with KCl (10 mM) for 2 hr. Membrane fractions were separated and subjected to IB with indicated antibodies. B) Quantification for the levels of membrane Rac, Rac (m). Data were shown as means ± SEM from three independent experiments. *P < 0.05. Student's t test. C) Representative images of Purkinje cells transfected with pSUPER or pSUPER-GGTβ-siRNA, without or with 10 mM KCl treatments. D, E) Quantification for the number of crossings at indicated distances from soma. F) Quantification for total dendritic length between neighboring circles. G) Total dendritic length between the circle of 50 and 75 μm. Data are shown as means ± SEM (n = 39 for control; n = 47 for GGT-siRNA; n = 36 for KCl; n = 34 for KCl with GGT-siRNA). 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 7: Effect of GGT-siRNA on high K+-induced Purkinje cell dendrite development. A) Rat cerebellar slices at DIV2 were pre-treated with GGTi-2147 (2.5 μM) or vehicle DMSO for 45 min, followed by the treatment with KCl (10 mM) for 2 hr. Membrane fractions were separated and subjected to IB with indicated antibodies. B) Quantification for the levels of membrane Rac, Rac (m). Data were shown as means ± SEM from three independent experiments. *P < 0.05. Student's t test. C) Representative images of Purkinje cells transfected with pSUPER or pSUPER-GGTβ-siRNA, without or with 10 mM KCl treatments. D, E) Quantification for the number of crossings at indicated distances from soma. F) Quantification for total dendritic length between neighboring circles. G) Total dendritic length between the circle of 50 and 75 μm. Data are shown as means ± SEM (n = 39 for control; n = 47 for GGT-siRNA; n = 36 for KCl; n = 34 for KCl with GGT-siRNA). N.S. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Student's t test. Scale bar = 20 μm.

Mentions: Like other types of neurons, dendritic differentiation of PCs is also shown to be regulated by neuronal activity [22]. Our previous study shows that high K+-induced depolarization promotes dendrite development of cultured hippocampal neurons, and this process requires TrkB-mediated activation of GGT [12]. In line with this notion, exposure of cultured cerebellar slices to high K+ resulted in an increase in the level of Rac (m), but not total Rac or GGT (Figure 7A, 7B and data not shown). The high K+-induced Rac membrane association was prevented by inhibiting GGT with GGTi-2147 (Figure 7A, 7B). This result suggests neuronal depolarization may also activate GGT in cerebeller neurons. Next, we determined the effect of high K+ on dendritic development of PCs. We found that high K+ treatment caused a remarkably increase in the number of dendritic branches indicated by number of crossings (Figure 7C-E), as well as dendrite length (Figure 7F and 7G). However, the effect of high K+ was attenuated in neurons transfected with GGTβ-siRNA (Figure 7C-G). The reduction of high K+-induced dendrite development caused by GGT-siRNA was exemplified by the decreased number of crossings at the 75 μm circle (Figure 7E) and total dendritic length between 50 and 75 μm circles to the soma (Figure 7G). These results suggest that GGT is required for depolarization-induced dendrite development of PCs.


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

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

Effect of GGT-siRNA on high K+-induced Purkinje cell dendrite development. A) Rat cerebellar slices at DIV2 were pre-treated with GGTi-2147 (2.5 μM) or vehicle DMSO for 45 min, followed by the treatment with KCl (10 mM) for 2 hr. Membrane fractions were separated and subjected to IB with indicated antibodies. B) Quantification for the levels of membrane Rac, Rac (m). Data were shown as means ± SEM from three independent experiments. *P < 0.05. Student's t test. C) Representative images of Purkinje cells transfected with pSUPER or pSUPER-GGTβ-siRNA, without or with 10 mM KCl treatments. D, E) Quantification for the number of crossings at indicated distances from soma. F) Quantification for total dendritic length between neighboring circles. G) Total dendritic length between the circle of 50 and 75 μm. Data are shown as means ± SEM (n = 39 for control; n = 47 for GGT-siRNA; n = 36 for KCl; n = 34 for KCl with GGT-siRNA). 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 7: Effect of GGT-siRNA on high K+-induced Purkinje cell dendrite development. A) Rat cerebellar slices at DIV2 were pre-treated with GGTi-2147 (2.5 μM) or vehicle DMSO for 45 min, followed by the treatment with KCl (10 mM) for 2 hr. Membrane fractions were separated and subjected to IB with indicated antibodies. B) Quantification for the levels of membrane Rac, Rac (m). Data were shown as means ± SEM from three independent experiments. *P < 0.05. Student's t test. C) Representative images of Purkinje cells transfected with pSUPER or pSUPER-GGTβ-siRNA, without or with 10 mM KCl treatments. D, E) Quantification for the number of crossings at indicated distances from soma. F) Quantification for total dendritic length between neighboring circles. G) Total dendritic length between the circle of 50 and 75 μm. Data are shown as means ± SEM (n = 39 for control; n = 47 for GGT-siRNA; n = 36 for KCl; n = 34 for KCl with GGT-siRNA). N.S. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Student's t test. Scale bar = 20 μm.
Mentions: Like other types of neurons, dendritic differentiation of PCs is also shown to be regulated by neuronal activity [22]. Our previous study shows that high K+-induced depolarization promotes dendrite development of cultured hippocampal neurons, and this process requires TrkB-mediated activation of GGT [12]. In line with this notion, exposure of cultured cerebellar slices to high K+ resulted in an increase in the level of Rac (m), but not total Rac or GGT (Figure 7A, 7B and data not shown). The high K+-induced Rac membrane association was prevented by inhibiting GGT with GGTi-2147 (Figure 7A, 7B). This result suggests neuronal depolarization may also activate GGT in cerebeller neurons. Next, we determined the effect of high K+ on dendritic development of PCs. We found that high K+ treatment caused a remarkably increase in the number of dendritic branches indicated by number of crossings (Figure 7C-E), as well as dendrite length (Figure 7F and 7G). However, the effect of high K+ was attenuated in neurons transfected with GGTβ-siRNA (Figure 7C-G). The reduction of high K+-induced dendrite development caused by GGT-siRNA was exemplified by the decreased number of crossings at the 75 μm circle (Figure 7E) and total dendritic length between 50 and 75 μm circles to the soma (Figure 7G). These results suggest that GGT is required for depolarization-induced dendrite development of PCs.

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