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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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Down regulation of GGTβ decreases dendrite growth and branching of PCs. A) HEK293 cells were co-transfected with Myc-GGTα and HA-GGTβ, together with pSUPER-GGTβ-siRNA or pSUPER. Cell lysates were subjected to IB with HA, Myc, GFP, or β-actin antibodies. B) Cultured cerebellar slices were transfected with pSUPER or pSUPER-GGTβ-siRNA, followed by staining with antibodies against GGTβ and Calbindin. Note the decrease of GGTβ signals in GGTβ-siRNA -transfected PCs. Scale bar = 20 μm. C) Cultured cerebellar slices at DIV2 were transfected with pSUPER-GGTβ-siRNA or pSUPER, together with pCAG-EYFP plasmid (3:1) to mark transfected cells. Shown are representative images at DIV5. Scale bar = 20 μm. D) Quantification for the number of crossings between dendrites and circles with a radius of indicated distance. E) Quantification for total dendritic length in indicated fields. Data shown are presented as means ± SEM (n = 17 for control, n = 23 for GGTβ-siRNA). *P < 0.05, Student's t test. Scale bar is 20 μm.
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Figure 4: Down regulation of GGTβ decreases dendrite growth and branching of PCs. A) HEK293 cells were co-transfected with Myc-GGTα and HA-GGTβ, together with pSUPER-GGTβ-siRNA or pSUPER. Cell lysates were subjected to IB with HA, Myc, GFP, or β-actin antibodies. B) Cultured cerebellar slices were transfected with pSUPER or pSUPER-GGTβ-siRNA, followed by staining with antibodies against GGTβ and Calbindin. Note the decrease of GGTβ signals in GGTβ-siRNA -transfected PCs. Scale bar = 20 μm. C) Cultured cerebellar slices at DIV2 were transfected with pSUPER-GGTβ-siRNA or pSUPER, together with pCAG-EYFP plasmid (3:1) to mark transfected cells. Shown are representative images at DIV5. Scale bar = 20 μm. D) Quantification for the number of crossings between dendrites and circles with a radius of indicated distance. E) Quantification for total dendritic length in indicated fields. Data shown are presented as means ± SEM (n = 17 for control, n = 23 for GGTβ-siRNA). *P < 0.05, Student's t test. Scale bar is 20 μm.

Mentions: Given the potential side effects of synthetic inhibitors, we took advantage of vector-based small interference RNA (siRNA) against GGTβ that has been shown to be able to down-regulate expression of endogenous GGTβ in primary neurons [12]. As shown in Figure 4A, this GGTβ-siRNA suppressed the expression of ectopic GGTβ, but not GGTα, expressed in HEK293 cells. The effectiveness of GGTβ-siRNA in suppressing endogenous GGTβ expression was observed in PCs of cultured cerebellar slices (Figure 4B). We found that the Purkinje cells developed much simpler dendritic arbors when transfected with GGT-siRNA, in comparison to control (Figure 4B). Transfected Purkinje cells were classified into three groups based on the severity of the phenotypes (mild: total dendritic length: >1000 μm; severe: 500-1000 μm; extreme: <500 μm) [17]. Among 23 GGT-siRNA-transfected neurons, the distribution of the phenotypes were 2 mild, 13 severe and 8 extreme (Figure 4C). Quantitatively, the number of crossings between dendrites and a 75 μm radius circle was significantly reduced in GGTβ-siRNA-transfected neurons, compared to control cells (Figure 4D, n = 17 for control, n = 23 for GGT-siRNA, P < 0.05). In addition, the total dendritic length between 50 and 75 μm or 75 and 100 μm circles to the soma, was significantly reduced in GGTβ-siRNA-transfected neurons (Figure 4E, P < 0.05). These results indicate that GGT itself plays a critical role in dendritic morphogenesis of PCs.


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

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

Down regulation of GGTβ decreases dendrite growth and branching of PCs. A) HEK293 cells were co-transfected with Myc-GGTα and HA-GGTβ, together with pSUPER-GGTβ-siRNA or pSUPER. Cell lysates were subjected to IB with HA, Myc, GFP, or β-actin antibodies. B) Cultured cerebellar slices were transfected with pSUPER or pSUPER-GGTβ-siRNA, followed by staining with antibodies against GGTβ and Calbindin. Note the decrease of GGTβ signals in GGTβ-siRNA -transfected PCs. Scale bar = 20 μm. C) Cultured cerebellar slices at DIV2 were transfected with pSUPER-GGTβ-siRNA or pSUPER, together with pCAG-EYFP plasmid (3:1) to mark transfected cells. Shown are representative images at DIV5. Scale bar = 20 μm. D) Quantification for the number of crossings between dendrites and circles with a radius of indicated distance. E) Quantification for total dendritic length in indicated fields. Data shown are presented as means ± SEM (n = 17 for control, n = 23 for GGTβ-siRNA). *P < 0.05, Student's t test. Scale bar is 20 μm.
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Related In: Results  -  Collection

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Figure 4: Down regulation of GGTβ decreases dendrite growth and branching of PCs. A) HEK293 cells were co-transfected with Myc-GGTα and HA-GGTβ, together with pSUPER-GGTβ-siRNA or pSUPER. Cell lysates were subjected to IB with HA, Myc, GFP, or β-actin antibodies. B) Cultured cerebellar slices were transfected with pSUPER or pSUPER-GGTβ-siRNA, followed by staining with antibodies against GGTβ and Calbindin. Note the decrease of GGTβ signals in GGTβ-siRNA -transfected PCs. Scale bar = 20 μm. C) Cultured cerebellar slices at DIV2 were transfected with pSUPER-GGTβ-siRNA or pSUPER, together with pCAG-EYFP plasmid (3:1) to mark transfected cells. Shown are representative images at DIV5. Scale bar = 20 μm. D) Quantification for the number of crossings between dendrites and circles with a radius of indicated distance. E) Quantification for total dendritic length in indicated fields. Data shown are presented as means ± SEM (n = 17 for control, n = 23 for GGTβ-siRNA). *P < 0.05, Student's t test. Scale bar is 20 μm.
Mentions: Given the potential side effects of synthetic inhibitors, we took advantage of vector-based small interference RNA (siRNA) against GGTβ that has been shown to be able to down-regulate expression of endogenous GGTβ in primary neurons [12]. As shown in Figure 4A, this GGTβ-siRNA suppressed the expression of ectopic GGTβ, but not GGTα, expressed in HEK293 cells. The effectiveness of GGTβ-siRNA in suppressing endogenous GGTβ expression was observed in PCs of cultured cerebellar slices (Figure 4B). We found that the Purkinje cells developed much simpler dendritic arbors when transfected with GGT-siRNA, in comparison to control (Figure 4B). Transfected Purkinje cells were classified into three groups based on the severity of the phenotypes (mild: total dendritic length: >1000 μm; severe: 500-1000 μm; extreme: <500 μm) [17]. Among 23 GGT-siRNA-transfected neurons, the distribution of the phenotypes were 2 mild, 13 severe and 8 extreme (Figure 4C). Quantitatively, the number of crossings between dendrites and a 75 μm radius circle was significantly reduced in GGTβ-siRNA-transfected neurons, compared to control cells (Figure 4D, n = 17 for control, n = 23 for GGT-siRNA, P < 0.05). In addition, the total dendritic length between 50 and 75 μm or 75 and 100 μm circles to the soma, was significantly reduced in GGTβ-siRNA-transfected neurons (Figure 4E, P < 0.05). These results indicate that GGT itself plays a critical role in dendritic morphogenesis 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