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Doublecortin-like kinase enhances dendritic remodelling and negatively regulates synapse maturation.

Shin E, Kashiwagi Y, Kuriu T, Iwasaki H, Tanaka T, Koizumi H, Gleeson JG, Okabe S - Nat Commun (2013)

Bottom Line: Here we report two distinct functions of doublecortin-like kinases, chimeric proteins containing both a microtubule-binding domain and a kinase domain in postmitotic neurons.First, doublecortin-like kinases localize to the distal dendrites and promote their growth by enhancing microtubule bundling.Thus, doublecortin-like kinases are critical regulators of dendritic development by means of their specific targeting to the distal dendrites, and their local control of dendritic growth and synapse maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Neurobiology, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Dendritic morphogenesis and formation of synapses at appropriate dendritic locations are essential for the establishment of proper neuronal connectivity. Recent imaging studies provide evidence for stabilization of dynamic distal branches of dendrites by the addition of new synapses. However, molecules involved in both dendritic growth and suppression of synapse maturation remain to be identified. Here we report two distinct functions of doublecortin-like kinases, chimeric proteins containing both a microtubule-binding domain and a kinase domain in postmitotic neurons. First, doublecortin-like kinases localize to the distal dendrites and promote their growth by enhancing microtubule bundling. Second, doublecortin-like kinases suppress maturation of synapses through multiple pathways, including reduction of PSD-95 by the kinase domain and suppression of spine structural maturation by the microtubule-binding domain. Thus, doublecortin-like kinases are critical regulators of dendritic development by means of their specific targeting to the distal dendrites, and their local control of dendritic growth and synapse maturation.

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Regulation of dendritic growth by DCLKs(a,b) Dissociated hippocampal neurons expressing GFP or DCLK1-GFP from 8 to 14 DIV. Note the more complex dendritic branching in neurons expressing DCLK1-GFP. (c,d) Sholl analysis and total dendritic lengths of neurons expressing GFP or DCLK1-GFP (number of cells analysed; GFP: 30 and DCLK1-GFP: 30; t-test: ***P<0.001). (e,f) Comparison of dendritic morphology before and after induction of DCLK1-GFP or GFP only, by Cre-dependent excision of loxp-stop-loxp sequences. The images before and 3 days after application of Cre-expressing recombinant adenoviruses were recorded (12 and 15 DIV). Prominent upregulation of dendritic growth was observed only in neurons expressing DCLK1-GFP (red dotted circles). (g) The total number of dendritic branches per neurons was significantly increased in neurons after induction of DCLK1-GFP expression. (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: ***P<0.001.) (h) Overexpression of DCLK1-GFP suppressed pruning of dendrites and increased the rate of appearance of new dendritic segments (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: *P<0.05). (i) The total length of dendrites per neurons was significantly increased in neurons overexpressing DCLK1-GFP. (Dendritic growth (%) = sum of dendritic length (after)/sum of dendritic length (before)) (Number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: **P<0.01). (j,k) Sholl analyses of neurons before and after induction of GFP (j) or DCLK1-GFP expression (k) (number of cells analysed; GFP: 9 and DCLK1-GFP: 13). (l) Anti-α-tubulin immunnostaining of growth-cone MTs in neurons transfected with plasmids expressing GFP or DCLK1-GFP. DCLK1-GFP expression induced tightly bundled MTs within growth cones. (m) Time-lapse imaging of both DCLK1-RFP and tubulin-GFP. DCLK1-positive segments show proximal to distal translocation in dendrites. Arrowheads indicate the distal end of the DCLK1-positive segment. All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, b, e and f; 5 μm for l and m.
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Figure 2: Regulation of dendritic growth by DCLKs(a,b) Dissociated hippocampal neurons expressing GFP or DCLK1-GFP from 8 to 14 DIV. Note the more complex dendritic branching in neurons expressing DCLK1-GFP. (c,d) Sholl analysis and total dendritic lengths of neurons expressing GFP or DCLK1-GFP (number of cells analysed; GFP: 30 and DCLK1-GFP: 30; t-test: ***P<0.001). (e,f) Comparison of dendritic morphology before and after induction of DCLK1-GFP or GFP only, by Cre-dependent excision of loxp-stop-loxp sequences. The images before and 3 days after application of Cre-expressing recombinant adenoviruses were recorded (12 and 15 DIV). Prominent upregulation of dendritic growth was observed only in neurons expressing DCLK1-GFP (red dotted circles). (g) The total number of dendritic branches per neurons was significantly increased in neurons after induction of DCLK1-GFP expression. (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: ***P<0.001.) (h) Overexpression of DCLK1-GFP suppressed pruning of dendrites and increased the rate of appearance of new dendritic segments (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: *P<0.05). (i) The total length of dendrites per neurons was significantly increased in neurons overexpressing DCLK1-GFP. (Dendritic growth (%) = sum of dendritic length (after)/sum of dendritic length (before)) (Number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: **P<0.01). (j,k) Sholl analyses of neurons before and after induction of GFP (j) or DCLK1-GFP expression (k) (number of cells analysed; GFP: 9 and DCLK1-GFP: 13). (l) Anti-α-tubulin immunnostaining of growth-cone MTs in neurons transfected with plasmids expressing GFP or DCLK1-GFP. DCLK1-GFP expression induced tightly bundled MTs within growth cones. (m) Time-lapse imaging of both DCLK1-RFP and tubulin-GFP. DCLK1-positive segments show proximal to distal translocation in dendrites. Arrowheads indicate the distal end of the DCLK1-positive segment. All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, b, e and f; 5 μm for l and m.

Mentions: The distal-to-proximal concentration gradient of DCLK in dendrites at 7 DIV suggests active roles for DCLK proteins in dendritic growth. To analyse this, we first adopted a gain-of-function strategy and evaluated the effects of DCLK1 (long form) overexpression by transfection of DCLK1-GFP plus lacZ at 8 DIV and subsequent fixation at 14 DIV (Fig. 2a,b). The morphology of dendrites was visualized by anti-β-galactosidase immunostaining and was evaluated by Sholl analysis. Neurons overexpressing DCLK1-GFP showed a significant increase in dendritic complexity (Fig. 2c) and the total dendritic length (Fig. 2d).


Doublecortin-like kinase enhances dendritic remodelling and negatively regulates synapse maturation.

Shin E, Kashiwagi Y, Kuriu T, Iwasaki H, Tanaka T, Koizumi H, Gleeson JG, Okabe S - Nat Commun (2013)

Regulation of dendritic growth by DCLKs(a,b) Dissociated hippocampal neurons expressing GFP or DCLK1-GFP from 8 to 14 DIV. Note the more complex dendritic branching in neurons expressing DCLK1-GFP. (c,d) Sholl analysis and total dendritic lengths of neurons expressing GFP or DCLK1-GFP (number of cells analysed; GFP: 30 and DCLK1-GFP: 30; t-test: ***P<0.001). (e,f) Comparison of dendritic morphology before and after induction of DCLK1-GFP or GFP only, by Cre-dependent excision of loxp-stop-loxp sequences. The images before and 3 days after application of Cre-expressing recombinant adenoviruses were recorded (12 and 15 DIV). Prominent upregulation of dendritic growth was observed only in neurons expressing DCLK1-GFP (red dotted circles). (g) The total number of dendritic branches per neurons was significantly increased in neurons after induction of DCLK1-GFP expression. (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: ***P<0.001.) (h) Overexpression of DCLK1-GFP suppressed pruning of dendrites and increased the rate of appearance of new dendritic segments (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: *P<0.05). (i) The total length of dendrites per neurons was significantly increased in neurons overexpressing DCLK1-GFP. (Dendritic growth (%) = sum of dendritic length (after)/sum of dendritic length (before)) (Number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: **P<0.01). (j,k) Sholl analyses of neurons before and after induction of GFP (j) or DCLK1-GFP expression (k) (number of cells analysed; GFP: 9 and DCLK1-GFP: 13). (l) Anti-α-tubulin immunnostaining of growth-cone MTs in neurons transfected with plasmids expressing GFP or DCLK1-GFP. DCLK1-GFP expression induced tightly bundled MTs within growth cones. (m) Time-lapse imaging of both DCLK1-RFP and tubulin-GFP. DCLK1-positive segments show proximal to distal translocation in dendrites. Arrowheads indicate the distal end of the DCLK1-positive segment. All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, b, e and f; 5 μm for l and m.
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Figure 2: Regulation of dendritic growth by DCLKs(a,b) Dissociated hippocampal neurons expressing GFP or DCLK1-GFP from 8 to 14 DIV. Note the more complex dendritic branching in neurons expressing DCLK1-GFP. (c,d) Sholl analysis and total dendritic lengths of neurons expressing GFP or DCLK1-GFP (number of cells analysed; GFP: 30 and DCLK1-GFP: 30; t-test: ***P<0.001). (e,f) Comparison of dendritic morphology before and after induction of DCLK1-GFP or GFP only, by Cre-dependent excision of loxp-stop-loxp sequences. The images before and 3 days after application of Cre-expressing recombinant adenoviruses were recorded (12 and 15 DIV). Prominent upregulation of dendritic growth was observed only in neurons expressing DCLK1-GFP (red dotted circles). (g) The total number of dendritic branches per neurons was significantly increased in neurons after induction of DCLK1-GFP expression. (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: ***P<0.001.) (h) Overexpression of DCLK1-GFP suppressed pruning of dendrites and increased the rate of appearance of new dendritic segments (number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: *P<0.05). (i) The total length of dendrites per neurons was significantly increased in neurons overexpressing DCLK1-GFP. (Dendritic growth (%) = sum of dendritic length (after)/sum of dendritic length (before)) (Number of cells analysed; GFP: 9 and DCLK1-GFP: 13; t-test: **P<0.01). (j,k) Sholl analyses of neurons before and after induction of GFP (j) or DCLK1-GFP expression (k) (number of cells analysed; GFP: 9 and DCLK1-GFP: 13). (l) Anti-α-tubulin immunnostaining of growth-cone MTs in neurons transfected with plasmids expressing GFP or DCLK1-GFP. DCLK1-GFP expression induced tightly bundled MTs within growth cones. (m) Time-lapse imaging of both DCLK1-RFP and tubulin-GFP. DCLK1-positive segments show proximal to distal translocation in dendrites. Arrowheads indicate the distal end of the DCLK1-positive segment. All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, b, e and f; 5 μm for l and m.
Mentions: The distal-to-proximal concentration gradient of DCLK in dendrites at 7 DIV suggests active roles for DCLK proteins in dendritic growth. To analyse this, we first adopted a gain-of-function strategy and evaluated the effects of DCLK1 (long form) overexpression by transfection of DCLK1-GFP plus lacZ at 8 DIV and subsequent fixation at 14 DIV (Fig. 2a,b). The morphology of dendrites was visualized by anti-β-galactosidase immunostaining and was evaluated by Sholl analysis. Neurons overexpressing DCLK1-GFP showed a significant increase in dendritic complexity (Fig. 2c) and the total dendritic length (Fig. 2d).

Bottom Line: Here we report two distinct functions of doublecortin-like kinases, chimeric proteins containing both a microtubule-binding domain and a kinase domain in postmitotic neurons.First, doublecortin-like kinases localize to the distal dendrites and promote their growth by enhancing microtubule bundling.Thus, doublecortin-like kinases are critical regulators of dendritic development by means of their specific targeting to the distal dendrites, and their local control of dendritic growth and synapse maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Neurobiology, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Dendritic morphogenesis and formation of synapses at appropriate dendritic locations are essential for the establishment of proper neuronal connectivity. Recent imaging studies provide evidence for stabilization of dynamic distal branches of dendrites by the addition of new synapses. However, molecules involved in both dendritic growth and suppression of synapse maturation remain to be identified. Here we report two distinct functions of doublecortin-like kinases, chimeric proteins containing both a microtubule-binding domain and a kinase domain in postmitotic neurons. First, doublecortin-like kinases localize to the distal dendrites and promote their growth by enhancing microtubule bundling. Second, doublecortin-like kinases suppress maturation of synapses through multiple pathways, including reduction of PSD-95 by the kinase domain and suppression of spine structural maturation by the microtubule-binding domain. Thus, doublecortin-like kinases are critical regulators of dendritic development by means of their specific targeting to the distal dendrites, and their local control of dendritic growth and synapse maturation.

Show MeSH
Related in: MedlinePlus