Geranylgeranyltransferase I is essential for dendritic development of cerebellar Purkinje cells
© Wu et al. 2010
Received: 9 December 2009
Accepted: 11 June 2010
Published: 11 June 2010
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.
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.
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.
Protein prenyltransferases, mainly farnesyl transferase (FT) and geranylgeranyl transferase I (GGT), are responsible for posttranslational lipidation of proteins with C-terminal "CAAX" motifs, where C is cysteine, A is often an aliphatic amino acid, and X at the C-terminus determines the specificity of protein prenylation [1, 2]. GGT catalyzes the transfer of a 20-carbon prenyl group from geranylgeranyl pyrophosphate (GGPP) to a cysteine residue of proteins usually with leucine or phenylalanine at their C-terminus [1, 3]. GGT substrates include K-Ras and Rho family small GTPases, such as Rac1, Cdc42 and RhoA, whose prenylation is essential for membrane localization, activation, and functions in various signaling pathways . Given that mutations of these small GTPases are oncogenic to cause malignant transformation, many studies have tried to use GGT inhibitors to suppress tumor growth [5–8]. A recent report shows that gene ablation of GGTase I-specific beta subunit (GGTβ) reduced lung tumor formation, probably by eliminating GGTase activity, disrupting the actin cytoskeleton, reducing cell migration, and/or blocking tumor cell proliferation . Thus GGT is a potential target for anti-cancer drug development.
Interestingly, the highest activity of GGT is often observed in the brain . Indeed, bovine brain has been used as a rich source for GGT purification . However, the role of GGT in neuronal system is poorly understood. Our previous study shows that GGT is localized at the neuromuscular junction and regulates agrin-induced clustering of acetylcholine receptors (AChR) by interacting with muscle specific receptor tyrosine kinase (MuSK) . Recently, we found that neuronal activity and BDNF activate GGT, which in turn promotes membrane recruitment of Rac1 and increases dendritic arborization of cultured hippocampal neurons . Furthermore, the activity of GGT in the rat hippocampus markedly increased when rats were put in a novel environment . Thus, GGT plays an important role in neuronal development. Nevertheless, the function of GGT in regulating neural development needs further investigation, especially in more intact systems.
During postnatal cerebellar development, Purkinje cells (PCs) form the most elaborate dendritic trees among neurons in the brain, however the mechanism governing dendrite development of PCs has not been completely understood . Here we demonstrate a role of GGT in the morphogenesis of PCs in cultured cerebellar slices. We found that GGT is enriched in the molecular layer of developing cerebellum. Down-regulation of GGT markedly affected dendritic arborization of PCs. In agreement with the notion that BDNF or neuronal activity activates GGT , we found that the enhancement effect of BDNF or high K+ on dendrite development of PCs was dramatically impeded by down-regulation of GGT. Thus GGT plays an important role in cerebellar neuron development.
Expression of GGT in the rat cerebellum
Up-regulation of GGT promotes Purkinje cell arborization
GGT is required for dendritic growth and arborization of Purkinje cells in cultured cerebellar slices
GGT is required for BDNF-mediated dendritic growth of Purkinje cells
High K+-induced dendritic morphogenesis of Purkinje cells requires the function of GGT
During cerebellar development, Purkinje cells (PCs) undergo dendrite extension and become elaborately arborescent, followed by synapse formation between dendrites of PCs and climbing fibers (CF) of inferior olive neurons or parallel fibers (PF) of granue cells (GC) . Thus, dendritic arborization and branching of PCs are essential steps for establishing cerebellar neural circuits. Here we find that GGT is enriched in PCs and required for dendrite development of PCs. Furthermore, neuronal depolarization or BDNF is able to promote PC dendrite development, and these effects depend on GGT expression. Thus GGT plays an important role in cerebellum development.
Previous studies have identified a number of factors that regulate growth and branching of dendritic arbors, including external signals, such as neuronal activity and neurotrophins, and a variety of intracellular mediators, such as Rho family small GTPases [23, 24]. The combined actions of multiple factors lead to cytoskeletal reorganization or gene expression required for dendritic growth. Due to the large somata and extensive dendritic trees, cerebellar PC has been a good model to study neuronal morphogenesis. By using organotypic slice culture system, many studies have led to identification of a number of molecules that regulate PC dendrite development . These factors include neurotransmitters and their receptors [25, 26], neurotrophic factors , steroids , thyroid hormone [29, 30], neuropeptides , kinases [32, 33], cytoskeleton regulating proteins , and cell adhesion molecules .
Previously we have shown that GGT regulates dendritic development of cultured hippocampal neurons , where GGT activation by BDNF or depolarization increases membrane localization of Rac1 , a member of Rho family small GTPases which are important for distinct aspects of dendrite development by modulating actin cytoskeleton [35, 36]. It remains to be determined whether Rac1 or other members of Rho family small GTPase also regulate morphogenesis of PCs. In addition to Rho GTPases, dendrite development can be regulated by many other molecules; some of them need to be associated with the plasma membrane in order to be activated efficiently. For example, prenylation of Ca2+/calmodulin-dependent protein kinase CLICK-III/CaMKIγ is responsible for its association with the lipid raft and its role in dendritogenesis . In addition to CaMKI, CaMKII also plays important role in dendrite differentiation . Interestingly, inhibition of CaMKII reduced the number of primary dendrites and the total dendritic length of Purkinje cells . It would be of interest to determine the mechanism by which GGT regulates Purkinje cell dendrite development.
Like other cell types, dendrite development of PCs has also been shown to be regulated by neuronal activity , and extracellular factors, such as GDNF . In line with this notion, treatment with high K+, which is believed to induce depolarization, promoted dendrite development of PCs in cultured cerebellar slices (Figure 7). Consistent with the activation of GGT by high K+ shown in cultured hippocampal neurons, we found that high K+-induced dendrite development of PCs was prevented by down-regulation of GGT. A previous report shows that in BDNF-knock out mice, there is a stunted growth of Purkinje cell dendrites . Consistent with this, we found that treatment with BDNF indeed promoted dendrite growth of PCs, although the effect was not as dramatic as predicted (Figure 6). The mild effect of BDNF could be due to the presence of neurotrophic factors in slice culture environment, or the production of BDNF by granule cells .
Given that GGT regulate Rac1 activity, which is known to be important for other neural developmental events, such as spine formation , it would be of interest to determine the role of GGT in other aspects of cerebellar development. A number of proteins have been shown to be involved in PC dendrite development, including GGT described in this study. Further works are needed to identify the specific regulators for Purkinje cell dendrite development, in particular how elaborate arborization and overwhelmingly dentritic trees are formed.
GGT is newly identified regulator for Purkinje cell dendrite development and is required for spontaneous, BDNF- or depolarization-induced Purkinje cell dendrite growth and/or arborization. Along with the previous report that shows the role of GGT in dendrite development of cultured hippocampal neurons, the role of GGT can be generalized in various types of neurons.
Reagents, antibodies and plasmids
Recombinant human BDNF was from Peprotech. Spermidine was form sigma. All salts used were from Sigma. Millicells (PICM03050) were from Millipore. Antibodies used for immunostaining were from Santa Cruz Biotechnology (rabbit anti-GGTα), Millipore (rabbit anti-Calbindin), Invitrogen (rabbit anti-GFP, Alexa Flour488 goat anti-rabbit IgG, Alexa Flour 555 goat anti-mouse IgG), Sigma (monoclonal anti-MAP2). Rabbit GGTβ antibody was generated by AbMART using a synthetic peptide derived from GGTβ sequence and affinity purified. The constructs of pSUPER-GGTβ-siRNA, HA-GGTβ and HA-GGTβRes were described previously .
Rat cerebellar organotypic culture
Cerebellar slices were prepared from P11 SD rat pups. Animals were anesthetized and decapitated. The brains were dissected and sliced in ice-cold EBSS sagittally at the thickness of 400 μm with WPI vibratome. Slices were then transferred onto Millicell and cultured in 5% CO2 at 37°C. The culture medium was modified from that of described previously [14, 15]. For BDNF or high K+ treatment, 50 ng/ml BDNF or 10 mM KCl were added into the culture medium 24 h after transfections. The treated cerebellar slices were cultured 3 days in vitro before fixation and image processing.
Rat brains were homogenized in cold lysis buffer containing 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitors. After pretreatment with GGTi-2147 (2.5 μM) or DMSO for 45 min, cultured cerebellar slices were treated with KCl (10 mM) or BDNF (50 ng/ml) for 2 hr and lysed. Protein membrane fractionation was conducted by using plasma membrane protein extraction kit (Biovision). Immunoblot follows standard protocol with indicated antibodies.
Microcarrier preparation and biolistic transfection
Microcarrier preparation was performed according to the manufacturer's instructions (BioRad). Briefly, 75 μg of the plasmids mixture (0.75 μg/μl) and 25 mg of gold particles (1.0 μm in diameter) were mixed in 50 mM spermidine (100 μl). Plasmids-particle mixture was precipitated by adding 1 M CaCl2 (100 μl) gradually, followed by 3 × washes with ethanol. The plasmids-coated particles were suspended in 3 ml of 15 μg/ml polyvinylpyrrolidone and loaded into tefzel tubing, the tube was then dried and the microcarriers were ready for use. Cultured cerebellar slices were transfected using Helios Gene Gun system after 2 days in vitro. Testing plasmid was mixed with pCAG-EYFP at a ratio of 3:1.
Tissue processing and immunohistochemistry
P10 SD rats were anesthetized and perfused with 4% paraformaldehyde (PFA, pH7.4). The brains were then dissected and postfixed overnight. The fixed brains were cryoprotected in 30% sucrose solution and sagittally sectioned at the sickness of 30 μm with cryostat microtome. The sectioned brain slices were incubated in 0.3% Triton X-100 for 30 min at room temperature. After blocking with 10% goat serum in PBS at room temperature for 1 h, the slices were incubated in primary antibodies at 4°C overnight. After 3 × washes with PBS, the slices were incubated in corresponding secondary antibodies for 2 h at room temperature, then the slices were mounted.
Cultured cerebellar slices were washed with PBS, and fixed in 4% PFA at room temperature for 30 min. The procedure for the staining of cultured cerebellar slices was similar to that of cryostat microtome sectioned slices.
Confocal imaging and data analysis
Images were acquired by Zeiss LSM 510 laser scanning confocal microscopy with a 40 × oil immersion objective. The captured neurons were traced using neurolucida software. Sholl analysis was used to analyze the total dendritic length and dendritic arborization. Data analysis was performed using Student's t-test. Errors bars in graphs represent SEM.
We thank Dr. Q. Hu of ION Imaging Facility with microscope analysis. This work was supported by National Natural Science Foundation of China (30721004 and 30825013), National Basic Research Program (2006CB806600) and Key State Research Program of China (2006CB943900), Chinese Academy of Sciences Grant (KSCX2-YW-R-102), and Program of Shanghai Subject Chief Scientist (08XD14050).
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