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First published online 17 June 2008
doi: 10.1242/jcs.026906

Journal of Cell Science 121, 2301-2307 (2008)
Published by The Company of Biologists 2008
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TRPC6 channels promote dendritic growth via the CaMKIV-CREB pathway

Yilin Tai1, Shengjie Feng1, Ruiliang Ge2, Wanlu Du1, Xiaoxing Zhang1, Zhuohao He1 and Yizheng Wang1,*

1 Laboratory of Neural Signal Transduction, Institute of Neuroscience, Shanghai Institutes of Biological Sciences, State Key Laboratory of Neuroscience, The Graduate School, Chinese Academy of Science, 320 Yueyang Road, Shanghai 200031, People's Republic of China
2 Eastern Hepatobiliary Hospital, Shanghai, 200438, People's Republic of China


Figure 1
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  		Fig. 1. Expression pattern of TRPCs in hippocampal development. (A) Western blot analysis of rat hippocampi at the indicated ages with the indicated antibodies. Ad, adult. Positions of molecular size markers (in kDa) are indicated on right. (B) In situ hybridization of P14 brain sections shows that TRPC6 is expressed in all regions of the hippocampus. (C) Immunohistochemical analysis of rat hippocampi at the indicated ages with TRPC6 antibody. (D) Confocal laser-scanning microscopy images of TRPC6 (green) and MAP2 (red) immunolabeling of P14 hippocampal CA1 neurons. Scale bar: upper left, 50 µm; enlargements of boxed area, 10 µm.

 

Figure 2
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  		Fig. 2. Overexpression of TRPC6 promotes dendritic growth. (A) Representative images of hippocampal neurons transfected at 5 DIV for 2 days with pcDNA or wild-type TRPC6 (WTC6), plus GFP to visualize the cell morphology. Scale bar: 20 µm. Quantification of the number of primary dendrites (B), total dendritic tips (C) and total dendritic length (D) of the neurons transfected with the indicated constructs. **P<0.01 versus control (ctrl).

 

Figure 3
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  		Fig. 3. Knockdown of TRPC6 impairs dendritic growth. (A) Effectiveness and specificity of TRPC6-shRNAi. The two upper blots are total lysates of hippocampal cultures transfected with the shRNAi constructs targeting rat TRPC6 (shRNA6i-1 or shRNA6i-2) and control shRNAi construct targeting firefly luciferase (pPRIME-FF3) 96 hours after transfection using antibodies against TRPC6 and tubulin. The three lower blots show total lysates of HEK293 cells transfected with rat TRPC5 or human TRPC6 and shTRPC6i-1 (targeting both rat and human TRPC6) or shTRPC6i-2 (targeting only rat TRPC6) 48 hours after transfection detected using the indicated antibodies. (B) Representative images of the neurons transfected at 3 DIV for 4 days with control vector, shTRPC6i-2, shTRPC6i-1 plus human TRPC6 or shTRPC6i-2 plus human TRPC6. Scale bar: 20 µm. (C,D) Quantification of total dendritic tips (C) and total dendritic length (D) of the neurons shown in B. **P<0.01 versus control (ctrl) or shTRPC6i-2 (shC6i-2) plus hTRPC6.

 

Figure 4
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  		Fig. 4. The Ca2+ influx through TRPC6 is important for dendritic development. (A,B) Quantification of total dendritic tips and total dendritic length of hippocampal neurons transfected with the indicated constructs at 5 DIV for 2 days. Vehicle, 2 mM EGTA or 10 µM Cd2+ was applied for 24 hours before neurons were fixed. Data are the mean ± s.e.m. n.s., not significant. (C) Effect of downregulation of TRPC6 on OAG-induced [Ca2+]i elevation. Intracellular Ca2+ levels in the neurons transfected with pPRIME-FF3 or shTRPC6 RNAi were determined by the F340/F380 ratio. The relative change in [Ca2+]i was depicted by 340/380 ratio normalized to the baseline. Data are mean ± s.e.m. of 45 cells from three independent experiments. (D,E) Quantification of total dendritic tips (D) and total dendritic length (E) of the neurons transfected with the indicated constructs at 3 DIV for 4 days. DMSO or 100 mM OAG was applied for 24 hours before the neurons were fixed. **P<0.01 versus control.

 

Figure 5
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  		Fig. 5. TRPC6-induced dendritic growth depends on CaMKIV activity. (A) Total lysates of cells treated as indicated blotted with antibodies to phosphorylated CaMKIV, total CaMKIV, phosphorylated CaMKII{alpha} or total CaMKII{alpha}. (B) Quantification of CaMKIV phosphorylation in the neurons transfected with the indicated constructs. *P<0.05 versus ctrl. (C) Representative images of the neurons transfected at 3 DIV for 4 days with wild-type TRPC6 (WTC6) and the dominant-negative CaMKIV mutant, or shTRPC6 RNAi construct and the constitutive active CaMKIV mutant. Scale bar: 20 µm. (D,E) Quantification of total dendritic tips (D) and total dendritic length (E) of the neurons shown in C. **P<0.01 versus ctrl. All data are the means ± s.e.m. n.s., not significant.

 

Figure 6
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  		Fig. 6. TRPC6-induced dendritic growth depends on CREB activity. (A) The total lysates of the cells transfected with the indicated plasmids were blotted with antibodies to phosphorylated CREB or total CREB. (B) Quantification of CREB phosphorylation in the neurons transfected with the indicated constructs. *P<0.05 or **P<0.01 versus control. (C) Representative images of the neurons transfected at 3 DIV for 4 days with wild-type TRPC6 (WTC6) and the dominant-negative CREB mutant (KCREB). Scale bar: 20 µm. (D,E) Quantification of total dendritic tips (D) and total dendritic length (E) of the neurons shown in C. **P<0.01 versus ctrl. All data are the means ± s.e.m. n.s., not significant.

 

Figure 7
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  		Fig. 7. TRPC6 promotes dendritic growth in vivo. (A) The total lysates of P14 mouse hippocampi from wild-type or TRPC6 transgenic mice blotted with the antibodies to TRPC6, {alpha}-tubulin, phosphorylated CaMKIV, total CaMKIV, phosphorylated CREB and total CREB. (B) Quantification of TRPC6, phosphorylated CaMKIV (p-CaMKIV) and phophorylated CREB (p-CREB) in the hippocampi of wild-type and TRPC6 transgenic mice. *P<0.05 versus wild-type. (C,D) Representative projections of CA1 hippocampal pyramidal neurons from wild-type (WT) and TRPC6 transgenic (TG) mice. (E,F) Quantification of dendritic tips (E) and dendritic length (F) of the neurons shown in C and D. Data are the means ± s.e.m. **P<0.01 versus wild type.

 

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