Comparative Study
doi: 10.1523/JNEUROSCI.2284-05.2005.Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways
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- PMID:16339024
- PMCID: PMC6725910
- DOI: 10.1523/JNEUROSCI.2284-05.2005
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Comparative Study
Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways
Vikas Kumar et al. J Neurosci..
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Abstract
Dendritic arborization and spine formation are critical for the functioning of neurons. Although many proteins have been identified recently as regulators of dendritic morphogenesis, the intracellular signaling pathways that control these processes are not well understood. Here we report that the Ras-phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway plays pivotal roles in the regulation of many aspects of dendrite formation. Whereas the PI3K-Akt-mTOR pathway alone controlled soma and dendrite size, a coordinated activation together with the Ras-mitogen-activated protein kinase signaling pathway was required for increasing dendritic complexity. Chronic inhibition of PI3K or mTOR reduced soma and dendrite size and dendritic complexity, as well as density of dendritic filopodia and spines, whereas a short-term inhibition promoted the formation of mushroom-shaped spines on cells expressing constitutively active mutants of Ras, PI3K, or Akt, or treated with the upstream activator BDNF. Together, our data underscore the central role of a spatiotemporally regulated key cell survival and growth pathway on trophic regulation of the coordinated development of dendrite size and shape.
Figures
Constitutively active PI3K and Akt expression increases dendritic complexity and alters spine morphology.A–C depict overall and close-up view of hippocampal neurons double-transfected with EGFP, DsRed, and active mutants of Akt(CA Akt) and PI3K (CA PI3K). The empty vector (A), CA Akt (B), and CA PI3K (C) constructs were cotransfected with EGFP on 5–7 DIV, followed by DsRed transfection 12–24 h later. The graphs show mean ± SEM for TDBL (D), terminal tip number (E), primary-order dendrite number (F), dendritic caliber (G), and soma area (H). The bottom panels illustrate dendritic protrusions at 15 DIV in GFP (I), CA PI3K (J), and CA Akt (K) expressing cells.L illustrates spine and filopodia density of the representative groups (mean ± SEM).M shows cumulative distribution of dendritic protrusion length. Data are fromat least three independent experiments (n = 49 GFP, 58 CA PI3K, and 94 CA Akt cells;n = 600 GFP, 455 CA PI3K, and 540 CA Akt dendritic protrusions). There is no significant difference in width among the three groups (data not shown). *p < 0.05, **p < 0.005, and ***p < 0.0001 demonstrates statistical significance by one-way ANOVA (Tukey' stest). Scale bars:C, 100 μm;I, 20μm. Note that the fold overexpression for CA Akt was quantitated using immunostaining for Akt1 antibody and was found to be increased by fourfold to fivefold (n = 50 neurons from 3 independent experiments).
Inhibition of PI3K–Akt signaling reduces dendritic complexity, soma size, and dendritic protrusions.A, Overall view of 15 DIV neurons transfected with GFP, CA PI3K, and CA Akt cells on 6 DIV and treated with 50μm LY294002 (LY) starting on 6 DIV. Also shown are DNPI3K- and DNAkt-expressing cells. The bar graphs show mean ± SEM for TDBL (B), number of terminal tips (C), and soma area (D). The data were quantified from three independent experiments (n = 49GFP, 58CAPI3K, 94CAAkt, 35GFPplus LY294002, 40 CA PI3K plus LY294002, 58 CA Akt plus LY294002, 25 DN PI3K, and 35 DN Akt cells).E, The micrograph panel represents the dendritic protrusions in 15 DIV neurons transfected with GFP, CA PI3K, and CA Akt on 6 DIV and treated with LY294002 (50μm ) starting on 7 DIV and cells expressing DN PI3K and DN Akt. The bar graph (F) shows mean ± SEM for spine and filopodia densities. Quantifications were performed from at least three independent experiments (n = 600 GFP, 455 CA PI3K, 540 CA Akt, 350 GFP plus LY294002, 350 CA PI3K plus LY294002, 300 CA Akt plus LY294002, 400 DN PI3K, and 390 DN Akt dendritic protrusions). Statistical difference within group: *p < 0.05, **p < 0.01, ***p < 0.001. Difference with control group:##p < 0.001, one-way ANOVA (Dunnett's test). Scale bars:A, 50μm;E, 10μm. Note that the control set used for this set of experiments was treated with DMSO (vehicle) but was not found to be statistically different compared with control data shown in Figure 1. Also, the dataset for CA PI3K and CA Akt from Figure 1 was used here with proper statisticalpost hoc test.
Increased dendritic complexity, soma size, and alteration of spine morphology in RasL61-overexpressing neurons. Overall views of 15 DIV neurons expressing GFP (A), RasL61 (B), and RasL61 cells treated with 50μm LY294002 (LY) (C) or 10μm U0126 (U0) (D). The neurons were transfected on 6 DIV, and the inhibitors were added on 7 DIV. U0126 were replaced every 2 d, whereas LY294002 was found to remain stable for the whole period.E–G represent mean ± SEM for TDBL, number of terminal tips, and soma area, respectively.H–K depict dendritic protrusions in the respective groups.L andM show quantification for spine and filopodia densities. Quantifications are from at least three independent experiments (n = 50 GFP, 35 U0126, 40 RasL61, 60 RasL61 plus LY294002, 46 RasL61 plus U0126 neurons; andn = 600 GFP, 350 U0126, 350 RasL61, 300 RasL61 plus LY294002, and 350 RasL61 plus U0126 dendritic protrusions). Statistical difference with RasL61 group: ***p < 0.0001. Difference with control group:###p < 0.0001, one-way ANOVA (Dunnett's test). Scale bars:D, 50 μm;K, 10 μm. Note that the fold overexpression for RasL61 was quantitated using pan-Ras antibody (n = 35 neurons from 2 independent experiments) and was found to be twofold increased. Similar expression level was also found for other RasL61 mutants. The same data for GFP controls with LY294002 as in Figure 2 are plotted here for easy comparison.
Regulation of dendritic size and shape by Ras–PI3K–Akt–mTOR signaling.A–F depict overall view of 15 DIV neurons transfected with GFP (A), RasL61 (B), RasL61S35 (C), RasL61C40 (D), CA PI3K (E), and CA Akt (F) cells on 6 DIV.G–L depict matching groups of neurons treated with rapamycin (Rap) (1μm ) starting on 6 DIV.M–O show mean ± SEM for TDBL, terminal tip number, and soma size, respectively, from more than two independent experiments (n = 24 GFP, 20 RasL61, 19 RasL61S35, 33 RasL61C40, 30 CA PI3K, 35 CA Akt, 26 GFP plus rapamycin, 22 RasL61 plus rapamycin, 26 RasL61S35 plus rapamycin, 21 RasL61C40 plus rapamycin, 20 CA PI3K plus rapamycin, and 25 CA Akt plus rapamycin cells). Statistical difference within group: *p < 0.05, **p < 0.01, ***p < 0.001. Difference with control group:###p < 0.001, one-way ANOVA (Dunnett's test). Scale bar, 50 μm.P, Sholl analysis of 15 DIV CA1/CA3 neurons to analyze detailed morphometric assessment shows that neurons overexpressing RasL61S35 have more profound effect on the distal dendrites without effects on primary dendrites, whereas RasL61C40-overexpressing neurons show a larger dendrite size and increases in both primary and distal dendrite branches. Application of rapamycin reduced the dendrite size in both the RasL61 mutants, but the increased distal dendritic branching persists in RasL61S35-expressing cells, whereas rapamycin completely blocked the primary and distal dendrite increases in RasL61C40-expressing cell. Concentric circles at 20μm spacing difference were drawn around the cell body, and the number of intersections of all dendritic branches with the circles were counted and plotted.n is same as above for all groups. Statistical difference shown is only with respect to control group: *p < 0.01, **p < 0.0001, one-way ANOVA (least significant difference).
Regulation of spine morphology by Ras–PI3K–Akt–mTOR signaling.A–F illustrate close-up views of dendritic protrusions from 15 DIV hippocampal neurons transfected with GFP (A), RasL61 (B), RasL61S35 (C), RasL61C40 (D), CA PI3K (E), and CA Akt (F) on 6 DIV.G–L show neurons from sister coverslips treated with rapamycin (1μm ) (Rap) starting on 6 DIV. The bar graphs indicate mean ± SEM for spine (M) and filopodia (N) densities. The densities were quantified from two independent set of experiments (n = 600 GFP, 350 RasL61, 375 RasL61S35, 200 RasL61C40, 450 CA PI3K, 500 CA Akt, 270 GFP plus rapamycin, 240 RasL61 plus rapamycin, 430 RasL61S35 plus rapamycin, 340 RasL61C40 plus rapamycin, 240 CA PI3K plus rapamycin, and 300 CA Akt plus rapamycin dendritic protrusions). Statistical difference within group: *p < 0.05, **p < 0.01, ***p < 0.001. Difference with control group:###p < 0.001, one-way ANOVA (Dunnett's test). Scale bar, 10 μm.
BDNF promotes growth of filopodia and destabilizes dendritic spines, and short-term inhibition of PI3K–Akt–mTOR promotes spine development. The micrographs depicts close-up views of 15 DIV dendritic protrusions from hippocampal neurons transfected with GFP on 6 DIV and treated with BDNF and BDNF plus drugs for 24 h starting on 14 DIV. GFP control (A), BDNF (20 ng/ml) (B), and coapplication of BDNF with K252a (200 nm )(C), LY294002 (50μm )(D; LY), wortmannin (1μm )(E; Wort), U0126 (10μm )(F; U0), LY294002 (50μm ) plus U0126 (10μm )(G), and rapamycin (1μm )(H; Rap).I andJ show mean ± SEM for spine and filopodia densities, respectively.K–L, Western blot analysis to ascertain the efficacy and specificity of treatment with BDNF and pharmacological inhibitors for above morphological experiments. Sister coverslips were harvested and probed for pAkt, pMAPK, and pS6. A representative experiment is shown inK.L, Quantifications of the Western blots from three independent experiments. The quantifications for spine density were from at least three independent experiments (n = 600 GFP, 350 BDNF, 470 BDNF plus K252a, 458 BDNF plus LY294002, 487 BDNF plus wortmannin, 463 BDNF plus U0126, 460 266 BDNF plus LY294002 plus U0126, and 300 BDNF plus rapamycin dendritic protrusions). ***p < 0.0001, statistical difference with BDNF.##p < 0.001, ###p < 0.0001, difference with respect to GFP control group, one-way ANOVA (Dunnett's test). Scale bar, 10 μm.
Short-term manipulation of PI3K–Akt–mTOR signaling promotes mushroom-shaped spines.A–D depict dendritic protrusions in 15 DIV cells transfected with GFP, RasL61, CAPI3K, and CA Akt on 6 DIV. The middle and right panels shows cells from sister coverslips treated with LY294002 (50 μm )(E–H; LY) and rapamycin (1 μm )(I–L; Rap) for 24 h starting on 14 DIV. The bar graphs show mean ± SEM for spine (M) and filopodia (N) densities. Quantification for the data were done from at least three experiments (n = 600 GFP, 350 RasL61, 450 CA PI3K, 500 CA Akt, 325 GFP plus LY294002, 250 GFP plus rapamycin, 275 RasL61 plus LY294002, 275 RasL61 plus rapamycin, 450 CA PI3K plus LY294002, 350 CA PI3K plus rapamycin, 400 Ca Akt plus LY294002, and 315 CA Akt plus rapamycin dendritic protrusions).###p < 0.0001, statistical difference with respect to GFP control. *p < 0.05, ***p < 0.0001, difference within respective groups, one-way ANOVA (Dunnett's test). Scale bar, 10 μm.
PI3K–Akt–mTOR signaling regulates dendritic complexity, soma size, and spine morphology in slice culture. The top micrograph panels depicts neurons from 10 DIV hippocampal slice culture, transfected with GFP (A), CA Akt (B), and CA Akt (C) on 2 DIV and treated with rapamycin (1μm ) for 24 h starting at 9 DIV.D–F illustrates the mean ± SEM for TDBL, primaryorder dendrite, and soma area, respectively.G depicts dendritic protrusions and their quantification (H). Quantification of the data were done from two independent experiments (n > 25 cells for each group and >1200 μm dendritic segments for quantifying protrusion density). Statistical difference with respect to control:#p < 0.005,###p < 0.0001. Difference with respect to CA Akt: *p < 0.05, **p < 0.005, ***p < 0.0001, one-way ANOVA (Tukey's test). Scale bars:C, 50 μm;G, 10 μm.
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