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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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Dendritic growth was negatively regulated by DCLK shRNAs(a,b) Expression of DCLK1 and DCLK2 shRNA plasmids in dissociated hippocampal neurons from 4 to 9 DIV (a). A marked decrease of DCLK immunoreactivity was observed (b) (number of cells analysed; control: 5, DCLK1 shRNA: 6, DCLK2 shRNA: 5, DCLK1 and DCLK2 shRNA: 5; one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests: *P<0.05, ***P<0.001). (c) Neurons transfected with plasmids expressing DCLK1 and DCLK2 shRNAs showed severe impairment of dendritic growth (arrows), which could be rescued by expression of shRNA-resistant constructs for DCLK1 and 2. (d) Sholl analysis of dendritic complexity in neurons transfected with control, DCLK1 shRNA or DCLK2 shRNA plasmids, with or without rescue constructs (number of cells analysed; control: 31, DCLK1 shRNA: 29, DCLK2 shRNA: 30, DCLK1 shRNA plus DCLK1-GFP: 20, DCLK1 shRNA plus DCLK2-GFP: 19 and DCLK2 shRNA plus DCLK2-GFP: 20). (e) Total dendritic lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs, with or without rescue constructs (numbers of cells analysed were the same as in d; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). NS, no statistical difference. (f) Total axonal lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs. Total axonal length measured at 5 DIV showed a tendency for reduction without statistical significance (number of cells analysed; control: 30, DCLK1 shRNA: 29 and DCLK2 shRNA: 28). (g) Neurons transfected with DCLK1 shRNA plasmid and expression vectors for various mutants of GFP-tagged DCLK1. Dendritic morphology was visualized by anti-β-galactosidase immunostaining. (h,i) Sholl analyses of dendritic complexity (h) and evaluation of total dendritic length (i) in neurons expressing DCLK1 shRNA plasmid together with expression vectors for various mutants of GFP-tagged DCLK1 (number of cells analysed; control shRNA + GFP: 32, DCLK1 shRNA + GFP: 30, sh + DCLK1-GFP: 27, sh + DCLK1(K435R)-GFP: 27, sh + DCLK1(K435A)-GFP: 28, sh + DCLK1(ΔKD)-GFP: 30 and sh + DCLK1(MAP2 swap)-GFP: 32; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, c and g.
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Figure 3: Dendritic growth was negatively regulated by DCLK shRNAs(a,b) Expression of DCLK1 and DCLK2 shRNA plasmids in dissociated hippocampal neurons from 4 to 9 DIV (a). A marked decrease of DCLK immunoreactivity was observed (b) (number of cells analysed; control: 5, DCLK1 shRNA: 6, DCLK2 shRNA: 5, DCLK1 and DCLK2 shRNA: 5; one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests: *P<0.05, ***P<0.001). (c) Neurons transfected with plasmids expressing DCLK1 and DCLK2 shRNAs showed severe impairment of dendritic growth (arrows), which could be rescued by expression of shRNA-resistant constructs for DCLK1 and 2. (d) Sholl analysis of dendritic complexity in neurons transfected with control, DCLK1 shRNA or DCLK2 shRNA plasmids, with or without rescue constructs (number of cells analysed; control: 31, DCLK1 shRNA: 29, DCLK2 shRNA: 30, DCLK1 shRNA plus DCLK1-GFP: 20, DCLK1 shRNA plus DCLK2-GFP: 19 and DCLK2 shRNA plus DCLK2-GFP: 20). (e) Total dendritic lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs, with or without rescue constructs (numbers of cells analysed were the same as in d; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). NS, no statistical difference. (f) Total axonal lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs. Total axonal length measured at 5 DIV showed a tendency for reduction without statistical significance (number of cells analysed; control: 30, DCLK1 shRNA: 29 and DCLK2 shRNA: 28). (g) Neurons transfected with DCLK1 shRNA plasmid and expression vectors for various mutants of GFP-tagged DCLK1. Dendritic morphology was visualized by anti-β-galactosidase immunostaining. (h,i) Sholl analyses of dendritic complexity (h) and evaluation of total dendritic length (i) in neurons expressing DCLK1 shRNA plasmid together with expression vectors for various mutants of GFP-tagged DCLK1 (number of cells analysed; control shRNA + GFP: 32, DCLK1 shRNA + GFP: 30, sh + DCLK1-GFP: 27, sh + DCLK1(K435R)-GFP: 27, sh + DCLK1(K435A)-GFP: 28, sh + DCLK1(ΔKD)-GFP: 30 and sh + DCLK1(MAP2 swap)-GFP: 32; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, c and g.

Mentions: DCLK expression in cultured hippocampal neurons was also maintained throughout the culture period with the peak of expression at 14 days in vitro (DIV) (Fig. 1c). Because our hippocampal primary culture contains few glial cells, this result indicates continual expression of DCLK1 (long form) or DCLK2 in postmitotic neurons. We could also detect DCLK proteins in cortical and hippocampal pyramidal neurons by immunohistochemistry of adult brain sections (Supplementary Fig. S2). Specificity of our antibody was confirmed by significant reduction of immunoreactivity in primary neurons transfected with RNA interference (RNAi) constructs for both DCLK1 and DCLK2 (Fig. 3a). From these results, we concluded that DCLK protein expression is maintained in postnatal neurons, both in the cerebral cortex and hippocampus.


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)

Dendritic growth was negatively regulated by DCLK shRNAs(a,b) Expression of DCLK1 and DCLK2 shRNA plasmids in dissociated hippocampal neurons from 4 to 9 DIV (a). A marked decrease of DCLK immunoreactivity was observed (b) (number of cells analysed; control: 5, DCLK1 shRNA: 6, DCLK2 shRNA: 5, DCLK1 and DCLK2 shRNA: 5; one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests: *P<0.05, ***P<0.001). (c) Neurons transfected with plasmids expressing DCLK1 and DCLK2 shRNAs showed severe impairment of dendritic growth (arrows), which could be rescued by expression of shRNA-resistant constructs for DCLK1 and 2. (d) Sholl analysis of dendritic complexity in neurons transfected with control, DCLK1 shRNA or DCLK2 shRNA plasmids, with or without rescue constructs (number of cells analysed; control: 31, DCLK1 shRNA: 29, DCLK2 shRNA: 30, DCLK1 shRNA plus DCLK1-GFP: 20, DCLK1 shRNA plus DCLK2-GFP: 19 and DCLK2 shRNA plus DCLK2-GFP: 20). (e) Total dendritic lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs, with or without rescue constructs (numbers of cells analysed were the same as in d; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). NS, no statistical difference. (f) Total axonal lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs. Total axonal length measured at 5 DIV showed a tendency for reduction without statistical significance (number of cells analysed; control: 30, DCLK1 shRNA: 29 and DCLK2 shRNA: 28). (g) Neurons transfected with DCLK1 shRNA plasmid and expression vectors for various mutants of GFP-tagged DCLK1. Dendritic morphology was visualized by anti-β-galactosidase immunostaining. (h,i) Sholl analyses of dendritic complexity (h) and evaluation of total dendritic length (i) in neurons expressing DCLK1 shRNA plasmid together with expression vectors for various mutants of GFP-tagged DCLK1 (number of cells analysed; control shRNA + GFP: 32, DCLK1 shRNA + GFP: 30, sh + DCLK1-GFP: 27, sh + DCLK1(K435R)-GFP: 27, sh + DCLK1(K435A)-GFP: 28, sh + DCLK1(ΔKD)-GFP: 30 and sh + DCLK1(MAP2 swap)-GFP: 32; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, c and g.
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Figure 3: Dendritic growth was negatively regulated by DCLK shRNAs(a,b) Expression of DCLK1 and DCLK2 shRNA plasmids in dissociated hippocampal neurons from 4 to 9 DIV (a). A marked decrease of DCLK immunoreactivity was observed (b) (number of cells analysed; control: 5, DCLK1 shRNA: 6, DCLK2 shRNA: 5, DCLK1 and DCLK2 shRNA: 5; one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests: *P<0.05, ***P<0.001). (c) Neurons transfected with plasmids expressing DCLK1 and DCLK2 shRNAs showed severe impairment of dendritic growth (arrows), which could be rescued by expression of shRNA-resistant constructs for DCLK1 and 2. (d) Sholl analysis of dendritic complexity in neurons transfected with control, DCLK1 shRNA or DCLK2 shRNA plasmids, with or without rescue constructs (number of cells analysed; control: 31, DCLK1 shRNA: 29, DCLK2 shRNA: 30, DCLK1 shRNA plus DCLK1-GFP: 20, DCLK1 shRNA plus DCLK2-GFP: 19 and DCLK2 shRNA plus DCLK2-GFP: 20). (e) Total dendritic lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs, with or without rescue constructs (numbers of cells analysed were the same as in d; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). NS, no statistical difference. (f) Total axonal lengths of neurons expressing DCLK1 shRNAs or DCLK2 shRNAs. Total axonal length measured at 5 DIV showed a tendency for reduction without statistical significance (number of cells analysed; control: 30, DCLK1 shRNA: 29 and DCLK2 shRNA: 28). (g) Neurons transfected with DCLK1 shRNA plasmid and expression vectors for various mutants of GFP-tagged DCLK1. Dendritic morphology was visualized by anti-β-galactosidase immunostaining. (h,i) Sholl analyses of dendritic complexity (h) and evaluation of total dendritic length (i) in neurons expressing DCLK1 shRNA plasmid together with expression vectors for various mutants of GFP-tagged DCLK1 (number of cells analysed; control shRNA + GFP: 32, DCLK1 shRNA + GFP: 30, sh + DCLK1-GFP: 27, sh + DCLK1(K435R)-GFP: 27, sh + DCLK1(K435A)-GFP: 28, sh + DCLK1(ΔKD)-GFP: 30 and sh + DCLK1(MAP2 swap)-GFP: 32; one-way ANOVA followed by Tukey–Kramer multiple comparison tests: ***P<0.001). All numeric data are given as mean ± s.e.m. Bar, 50 μm for a, c and g.
Mentions: DCLK expression in cultured hippocampal neurons was also maintained throughout the culture period with the peak of expression at 14 days in vitro (DIV) (Fig. 1c). Because our hippocampal primary culture contains few glial cells, this result indicates continual expression of DCLK1 (long form) or DCLK2 in postmitotic neurons. We could also detect DCLK proteins in cortical and hippocampal pyramidal neurons by immunohistochemistry of adult brain sections (Supplementary Fig. S2). Specificity of our antibody was confirmed by significant reduction of immunoreactivity in primary neurons transfected with RNA interference (RNAi) constructs for both DCLK1 and DCLK2 (Fig. 3a). From these results, we concluded that DCLK protein expression is maintained in postnatal neurons, both in the cerebral cortex and hippocampus.

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