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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 BDNF-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 BDNF (50 ng/ml) for 2 hr. Membrane fractions were separated and subjected to IB with antibodies against Rac and pan-cadherin. Total Rac or GAPDH was probed as loading controls. 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 after transfection with pSUPER or pSUPER-GGTβ-siRNA, without or with 50 ng/ml BDNF treatments. D) Quantification for total dendritic length between neighboring circles. E) Quantification for total dendritic length between the circle of 50 and 75 μm. F) Quantification for the number of crossings at indicated distances from soma. G) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. Data are shown as means ± SEM (n = 33 for control; n = 44 for GGT-siRNA; n = 23 for BDNF; n = 21 for BDNF 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 6: Effect of GGT-siRNA on BDNF-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 BDNF (50 ng/ml) for 2 hr. Membrane fractions were separated and subjected to IB with antibodies against Rac and pan-cadherin. Total Rac or GAPDH was probed as loading controls. 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 after transfection with pSUPER or pSUPER-GGTβ-siRNA, without or with 50 ng/ml BDNF treatments. D) Quantification for total dendritic length between neighboring circles. E) Quantification for total dendritic length between the circle of 50 and 75 μm. F) Quantification for the number of crossings at indicated distances from soma. G) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. Data are shown as means ± SEM (n = 33 for control; n = 44 for GGT-siRNA; n = 23 for BDNF; n = 21 for BDNF 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: Neurotrophins such as NGF, BDNF, NT-3, 4 are known to regulate dendritic growth [18-20]. Our previous report shows that GGT is activated by BDNF and mediates BDNF-induced dendrite growth [12]. In BDNF-knockout mice, growth of Purkinje cell dendrites is impeded [21]. Granule cell death was also observed in the BDNF-knockout mice, suggesting that the effect on Purkinje cell development could be indirect [21]. We determined the role of BDNF by treating cultured cerebellar slices with BDNF. We found that BDNF treatment caused an increase in the level of membrane-associated Rac, thereafter referred to as Rac (m) (Figure 6A and 6B), but had no effect on GGT expression (data not shown), suggesting the activation of GGT. This elevation is similar to that seen in cultured cortical neurons [12], and depended on the GGT activity, since the presence of GGTi-2147 prevented the increase in the level of Rac (m) induced by BDNF (Figure 6A and 6B). Furthermore, treatment with BDNF promoted dendritic growth, as reflected from increased total dendritic length between circles of 50 and 75 μm to the soma (Figure 6C-E). However, number of crossings was mildly, but not significantly, affected by the treatment with BDNF (Figure 6F and 6G). Thus, in our experimental conditions, BDNF stimulates Purkinje cell dendrite growth rather than arborization. The effect of BDNF on dendrite length was attenuated when endogenous GGT was down-regulated by GGTβ-siRNA (Figure 6D-G). This result suggests that GGT also participates in BDNF-mediated PC dendrite development.


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 BDNF-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 BDNF (50 ng/ml) for 2 hr. Membrane fractions were separated and subjected to IB with antibodies against Rac and pan-cadherin. Total Rac or GAPDH was probed as loading controls. 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 after transfection with pSUPER or pSUPER-GGTβ-siRNA, without or with 50 ng/ml BDNF treatments. D) Quantification for total dendritic length between neighboring circles. E) Quantification for total dendritic length between the circle of 50 and 75 μm. F) Quantification for the number of crossings at indicated distances from soma. G) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. Data are shown as means ± SEM (n = 33 for control; n = 44 for GGT-siRNA; n = 23 for BDNF; n = 21 for BDNF 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 6: Effect of GGT-siRNA on BDNF-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 BDNF (50 ng/ml) for 2 hr. Membrane fractions were separated and subjected to IB with antibodies against Rac and pan-cadherin. Total Rac or GAPDH was probed as loading controls. 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 after transfection with pSUPER or pSUPER-GGTβ-siRNA, without or with 50 ng/ml BDNF treatments. D) Quantification for total dendritic length between neighboring circles. E) Quantification for total dendritic length between the circle of 50 and 75 μm. F) Quantification for the number of crossings at indicated distances from soma. G) Number of crossings at the circle with radius of 75 μm was quantitatively analyzed. Data are shown as means ± SEM (n = 33 for control; n = 44 for GGT-siRNA; n = 23 for BDNF; n = 21 for BDNF 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: Neurotrophins such as NGF, BDNF, NT-3, 4 are known to regulate dendritic growth [18-20]. Our previous report shows that GGT is activated by BDNF and mediates BDNF-induced dendrite growth [12]. In BDNF-knockout mice, growth of Purkinje cell dendrites is impeded [21]. Granule cell death was also observed in the BDNF-knockout mice, suggesting that the effect on Purkinje cell development could be indirect [21]. We determined the role of BDNF by treating cultured cerebellar slices with BDNF. We found that BDNF treatment caused an increase in the level of membrane-associated Rac, thereafter referred to as Rac (m) (Figure 6A and 6B), but had no effect on GGT expression (data not shown), suggesting the activation of GGT. This elevation is similar to that seen in cultured cortical neurons [12], and depended on the GGT activity, since the presence of GGTi-2147 prevented the increase in the level of Rac (m) induced by BDNF (Figure 6A and 6B). Furthermore, treatment with BDNF promoted dendritic growth, as reflected from increased total dendritic length between circles of 50 and 75 μm to the soma (Figure 6C-E). However, number of crossings was mildly, but not significantly, affected by the treatment with BDNF (Figure 6F and 6G). Thus, in our experimental conditions, BDNF stimulates Purkinje cell dendrite growth rather than arborization. The effect of BDNF on dendrite length was attenuated when endogenous GGT was down-regulated by GGTβ-siRNA (Figure 6D-G). This result suggests that GGT also participates in BDNF-mediated PC dendrite 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