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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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Postnatal expression and subcellular localization of DCLKs(a) Domain organizations of DCX, DCLK1 isoforms and DCLK2. A black line indicates the DCLK1 fragment used for antibody generation. S/P rich, serine/proline-rich domain. (b,c) Western blotting of DCLK protein. The immunoreactive bands of 85 kDa corresponding to the size of the full-length DCLK1 and DCLK2 proteins were detected in the extracts prepared from multiple brain regions (b) or dissociated hippocampal neurons at various days after plating (c). (d,e) Immunocytochemistry of dissociated hippocampal neurons at 7 DIV (d) and 24 DIV (e) using anti-DCLK and anti-MAP2 antibodies. DCLK proteins were preferentially concentrated in the distal part of dendrites at 7 DIV (arrows). (f) Immunocytochemistry of GFP-expressing hippocampal neurons with anti-DCLK antibody at 7 DIV. DCLK proteins were preferentially concentrated in the distal part of dendrites (arrows). (g) Transfection of DCLK1-GFP together with a lacZ reporter plasmid and subsequent immunocytochemistry with an anti-β-galactosidase antibody. (h) Relative intensity of DCLK1-GFP and anti-β-galactosidase immunoreactive signals at 7 DIV and 25 DIV. (DIV 7: n = 13 cells, DIV 25: n =15 cells.) (i,j) Presence of DCLK immunoreactivity and DCLK1-GFP fluorescence in PSD-95-positive spines (arrows). (k) Presence of DCLK immunoreactivity in PSD-1T and PSD-2T fractions, which contain purified PSDs (P2: crude synaptosomal pellet, S3: crude synaptic vesicle fraction, P3: lysed synaptosomal membrane fraction, SV: synaptic vesicle fraction, SPM: synaptic plasma membrane fraction, PSD-1T,PSD-2T: purified PSD fractions, PSD3S: PSD fraction after sarkosyl treatment). (l) Immunoprecipitation of PSD-95 by anti-DCLK antibody in extracts from the adult brain (rabbit IgG (Rb-IgG) as a control antibody of immunoprecipitation). All numeric data are given as mean ± s.e.m. Bar, 20 μm for d, 50 and 20 μm for e, 10 μm for f and g and 5 μm for i and j.
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Figure 1: Postnatal expression and subcellular localization of DCLKs(a) Domain organizations of DCX, DCLK1 isoforms and DCLK2. A black line indicates the DCLK1 fragment used for antibody generation. S/P rich, serine/proline-rich domain. (b,c) Western blotting of DCLK protein. The immunoreactive bands of 85 kDa corresponding to the size of the full-length DCLK1 and DCLK2 proteins were detected in the extracts prepared from multiple brain regions (b) or dissociated hippocampal neurons at various days after plating (c). (d,e) Immunocytochemistry of dissociated hippocampal neurons at 7 DIV (d) and 24 DIV (e) using anti-DCLK and anti-MAP2 antibodies. DCLK proteins were preferentially concentrated in the distal part of dendrites at 7 DIV (arrows). (f) Immunocytochemistry of GFP-expressing hippocampal neurons with anti-DCLK antibody at 7 DIV. DCLK proteins were preferentially concentrated in the distal part of dendrites (arrows). (g) Transfection of DCLK1-GFP together with a lacZ reporter plasmid and subsequent immunocytochemistry with an anti-β-galactosidase antibody. (h) Relative intensity of DCLK1-GFP and anti-β-galactosidase immunoreactive signals at 7 DIV and 25 DIV. (DIV 7: n = 13 cells, DIV 25: n =15 cells.) (i,j) Presence of DCLK immunoreactivity and DCLK1-GFP fluorescence in PSD-95-positive spines (arrows). (k) Presence of DCLK immunoreactivity in PSD-1T and PSD-2T fractions, which contain purified PSDs (P2: crude synaptosomal pellet, S3: crude synaptic vesicle fraction, P3: lysed synaptosomal membrane fraction, SV: synaptic vesicle fraction, SPM: synaptic plasma membrane fraction, PSD-1T,PSD-2T: purified PSD fractions, PSD3S: PSD fraction after sarkosyl treatment). (l) Immunoprecipitation of PSD-95 by anti-DCLK antibody in extracts from the adult brain (rabbit IgG (Rb-IgG) as a control antibody of immunoprecipitation). All numeric data are given as mean ± s.e.m. Bar, 20 μm for d, 50 and 20 μm for e, 10 μm for f and g and 5 μm for i and j.

Mentions: DCLK1 gives rise to multiple transcripts, and three major splice variants exist in the brain (Fig. 1a): a full-length isoform (long form), a short isoform with a MT-binding domain (DCX-like; DCL) and another short isoform with a kinase domain (short form)11-14. Furthermore, two major splice variants of DCLK2 exist; both of these contain the DCX domain and the kinase domain15. We generated a polyclonal pan-DCLK-antibody by using the DCLK1 DCX domain as an antigen (Supplementary Fig. S1). This region shows high homology between DCLK1 and DCLK2, and our antibody showed comparable reactivity with both DCLK1 (long form) and DCLK2. Western blotting with this antibody revealed immunoreactive bands of 85 kDa, corresponding to the size of DCLK1 (long form) and DCLK2 in extracts of the cerebrum, cerebellum and hippocampus (Fig. 1b). The additional immunoreactivity of 43 kDa was likely to correspond to DCL and disappeared in samples taken from DCLK1−/− mice (Supplementary Fig. S1C). The level of DCL was much lower than the level of DCLK1 (long form) and DCLK2 in the postnatal brain. DCLK expression was highest at postnatal day 7 and gradually decreased in the cerebrum and cerebellum (Fig. 1b). In the hippocampus, DCLK expression peaked around postnatal day 14 and started to decline thereafter. The immunoblots also showed weak nonspecific bands of 70 kDa, which did not disappear in samples taken from DCLK1−/− and DCLK2−/− mice (Supplementary Fig. S1C).


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)

Postnatal expression and subcellular localization of DCLKs(a) Domain organizations of DCX, DCLK1 isoforms and DCLK2. A black line indicates the DCLK1 fragment used for antibody generation. S/P rich, serine/proline-rich domain. (b,c) Western blotting of DCLK protein. The immunoreactive bands of 85 kDa corresponding to the size of the full-length DCLK1 and DCLK2 proteins were detected in the extracts prepared from multiple brain regions (b) or dissociated hippocampal neurons at various days after plating (c). (d,e) Immunocytochemistry of dissociated hippocampal neurons at 7 DIV (d) and 24 DIV (e) using anti-DCLK and anti-MAP2 antibodies. DCLK proteins were preferentially concentrated in the distal part of dendrites at 7 DIV (arrows). (f) Immunocytochemistry of GFP-expressing hippocampal neurons with anti-DCLK antibody at 7 DIV. DCLK proteins were preferentially concentrated in the distal part of dendrites (arrows). (g) Transfection of DCLK1-GFP together with a lacZ reporter plasmid and subsequent immunocytochemistry with an anti-β-galactosidase antibody. (h) Relative intensity of DCLK1-GFP and anti-β-galactosidase immunoreactive signals at 7 DIV and 25 DIV. (DIV 7: n = 13 cells, DIV 25: n =15 cells.) (i,j) Presence of DCLK immunoreactivity and DCLK1-GFP fluorescence in PSD-95-positive spines (arrows). (k) Presence of DCLK immunoreactivity in PSD-1T and PSD-2T fractions, which contain purified PSDs (P2: crude synaptosomal pellet, S3: crude synaptic vesicle fraction, P3: lysed synaptosomal membrane fraction, SV: synaptic vesicle fraction, SPM: synaptic plasma membrane fraction, PSD-1T,PSD-2T: purified PSD fractions, PSD3S: PSD fraction after sarkosyl treatment). (l) Immunoprecipitation of PSD-95 by anti-DCLK antibody in extracts from the adult brain (rabbit IgG (Rb-IgG) as a control antibody of immunoprecipitation). All numeric data are given as mean ± s.e.m. Bar, 20 μm for d, 50 and 20 μm for e, 10 μm for f and g and 5 μm for i and j.
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Figure 1: Postnatal expression and subcellular localization of DCLKs(a) Domain organizations of DCX, DCLK1 isoforms and DCLK2. A black line indicates the DCLK1 fragment used for antibody generation. S/P rich, serine/proline-rich domain. (b,c) Western blotting of DCLK protein. The immunoreactive bands of 85 kDa corresponding to the size of the full-length DCLK1 and DCLK2 proteins were detected in the extracts prepared from multiple brain regions (b) or dissociated hippocampal neurons at various days after plating (c). (d,e) Immunocytochemistry of dissociated hippocampal neurons at 7 DIV (d) and 24 DIV (e) using anti-DCLK and anti-MAP2 antibodies. DCLK proteins were preferentially concentrated in the distal part of dendrites at 7 DIV (arrows). (f) Immunocytochemistry of GFP-expressing hippocampal neurons with anti-DCLK antibody at 7 DIV. DCLK proteins were preferentially concentrated in the distal part of dendrites (arrows). (g) Transfection of DCLK1-GFP together with a lacZ reporter plasmid and subsequent immunocytochemistry with an anti-β-galactosidase antibody. (h) Relative intensity of DCLK1-GFP and anti-β-galactosidase immunoreactive signals at 7 DIV and 25 DIV. (DIV 7: n = 13 cells, DIV 25: n =15 cells.) (i,j) Presence of DCLK immunoreactivity and DCLK1-GFP fluorescence in PSD-95-positive spines (arrows). (k) Presence of DCLK immunoreactivity in PSD-1T and PSD-2T fractions, which contain purified PSDs (P2: crude synaptosomal pellet, S3: crude synaptic vesicle fraction, P3: lysed synaptosomal membrane fraction, SV: synaptic vesicle fraction, SPM: synaptic plasma membrane fraction, PSD-1T,PSD-2T: purified PSD fractions, PSD3S: PSD fraction after sarkosyl treatment). (l) Immunoprecipitation of PSD-95 by anti-DCLK antibody in extracts from the adult brain (rabbit IgG (Rb-IgG) as a control antibody of immunoprecipitation). All numeric data are given as mean ± s.e.m. Bar, 20 μm for d, 50 and 20 μm for e, 10 μm for f and g and 5 μm for i and j.
Mentions: DCLK1 gives rise to multiple transcripts, and three major splice variants exist in the brain (Fig. 1a): a full-length isoform (long form), a short isoform with a MT-binding domain (DCX-like; DCL) and another short isoform with a kinase domain (short form)11-14. Furthermore, two major splice variants of DCLK2 exist; both of these contain the DCX domain and the kinase domain15. We generated a polyclonal pan-DCLK-antibody by using the DCLK1 DCX domain as an antigen (Supplementary Fig. S1). This region shows high homology between DCLK1 and DCLK2, and our antibody showed comparable reactivity with both DCLK1 (long form) and DCLK2. Western blotting with this antibody revealed immunoreactive bands of 85 kDa, corresponding to the size of DCLK1 (long form) and DCLK2 in extracts of the cerebrum, cerebellum and hippocampus (Fig. 1b). The additional immunoreactivity of 43 kDa was likely to correspond to DCL and disappeared in samples taken from DCLK1−/− mice (Supplementary Fig. S1C). The level of DCL was much lower than the level of DCLK1 (long form) and DCLK2 in the postnatal brain. DCLK expression was highest at postnatal day 7 and gradually decreased in the cerebrum and cerebellum (Fig. 1b). In the hippocampus, DCLK expression peaked around postnatal day 14 and started to decline thereafter. The immunoblots also showed weak nonspecific bands of 70 kDa, which did not disappear in samples taken from DCLK1−/− and DCLK2−/− mice (Supplementary Fig. S1C).

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