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LIS1-dependent retrograde translocation of excitatory synapses in developing interneuron dendrites.

Kawabata I, Kashiwagi Y, Obashi K, Ohkura M, Nakai J, Wynshaw-Boris A, Yanagawa Y, Okabe S - Nat Commun (2012)

Bottom Line: This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility.Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites.Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Synaptic remodelling coordinated with dendritic growth is essential for proper development of neural connections. After establishment of synaptic contacts, synaptic junctions are thought to become stationary and provide fixed anchoring points for further dendritic growth. However, the possibility of active translocation of synapses along dendritic protrusions, to guide the proper arrangement of synaptic distribution, has not yet been fully investigated. Here we show that immature dendrites of γ-aminobutyric acid-positive interneurons form long protrusions and that these protrusions serve as conduits for retrograde translocation of synaptic contacts to the parental dendrites. This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility. Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites. Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

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Effect of manipulating LIS1/NDEL1/dynein activity in interneurons on PSD mobility.(a) Fraction of mobile PSD puncta in interneurons transfected with PSD-95-GFP and RFP (control) or PSD-95-GFP, RFP and respective short hairpin RNA (shRNA) constructs. Two additional methods for inhibiting dynein-based mobility were also included (treatment with 1 mM erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) and dynamitin transfection). PSD mobility was suppressed by LIS1 or NDEL1 shRNA and rescued by overexpression of RNAi-insensitive LIS1 and NDEL1 mutants ('LIS1 RNAi+mLIS1' and 'NDEL1 RNAi+mNDEL1'). Mutant LIS1 and NDEL1 RNAi constructs containing three mismatched nucleotides ('mLIS1 RNAi' and 'mNDEL1 RNAi') did not suppress PSD mobility. Both EHNA treatment and expression of dynamitin suppressed PSD mobility. (n=18 cells for control, n=6 cells for LIS1 RNAi, n=7 cells for mLIS1 RNAi, n=6 cells for LIS1 RNAi+mLIS1, n=8 cells for NDEL1 RNAi, n=5 cells for mNDEL1 RNAi, n=5 cells for NDEL1 RNAi+mNDEL1, n=5 cells for EHNA treatment, n=5 cells for dynamitin transfection.) Significant differences (one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests): *P<0.05. (b) Time-lapse imaging of an interneuron expressing PSD-95-GFP with NDEL1-KillerRed before and after CALI (within 60 min after irradiation). Arrowheads indicate the position of a PSD-95-GFP punctum. Scale bar, 5 μm. (c) Suppression of PSD mobility by CALI of NDEL1-KillerRed. The effect of CALI on the mobile fraction was significant at 5–50 min after CALI (53±13.4%, n=6 cells). The fraction of motile PSD-95-GFP puncta returned to the control level at 60–105 min after CALI (91±15.6%, n=6 cells). The mean velocity of mobile PSD puncta also decreased after CALI and this effect was sustained for more than 60 min (2.8±0.30 μm h−1 before CALI, 1.1±0.17 μm h−1 at 5–50 min after CALI, 1.9±0.24 μm h−1 at 60–105 min after CALI, n=6 cells). Significant differences (one-way ANOVA followed by Tukey–Kramer multiple comparison tests): *P<0.05. All numeric data are mean±s.e.m.
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f6: Effect of manipulating LIS1/NDEL1/dynein activity in interneurons on PSD mobility.(a) Fraction of mobile PSD puncta in interneurons transfected with PSD-95-GFP and RFP (control) or PSD-95-GFP, RFP and respective short hairpin RNA (shRNA) constructs. Two additional methods for inhibiting dynein-based mobility were also included (treatment with 1 mM erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) and dynamitin transfection). PSD mobility was suppressed by LIS1 or NDEL1 shRNA and rescued by overexpression of RNAi-insensitive LIS1 and NDEL1 mutants ('LIS1 RNAi+mLIS1' and 'NDEL1 RNAi+mNDEL1'). Mutant LIS1 and NDEL1 RNAi constructs containing three mismatched nucleotides ('mLIS1 RNAi' and 'mNDEL1 RNAi') did not suppress PSD mobility. Both EHNA treatment and expression of dynamitin suppressed PSD mobility. (n=18 cells for control, n=6 cells for LIS1 RNAi, n=7 cells for mLIS1 RNAi, n=6 cells for LIS1 RNAi+mLIS1, n=8 cells for NDEL1 RNAi, n=5 cells for mNDEL1 RNAi, n=5 cells for NDEL1 RNAi+mNDEL1, n=5 cells for EHNA treatment, n=5 cells for dynamitin transfection.) Significant differences (one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests): *P<0.05. (b) Time-lapse imaging of an interneuron expressing PSD-95-GFP with NDEL1-KillerRed before and after CALI (within 60 min after irradiation). Arrowheads indicate the position of a PSD-95-GFP punctum. Scale bar, 5 μm. (c) Suppression of PSD mobility by CALI of NDEL1-KillerRed. The effect of CALI on the mobile fraction was significant at 5–50 min after CALI (53±13.4%, n=6 cells). The fraction of motile PSD-95-GFP puncta returned to the control level at 60–105 min after CALI (91±15.6%, n=6 cells). The mean velocity of mobile PSD puncta also decreased after CALI and this effect was sustained for more than 60 min (2.8±0.30 μm h−1 before CALI, 1.1±0.17 μm h−1 at 5–50 min after CALI, 1.9±0.24 μm h−1 at 60–105 min after CALI, n=6 cells). Significant differences (one-way ANOVA followed by Tukey–Kramer multiple comparison tests): *P<0.05. All numeric data are mean±s.e.m.

Mentions: Our results in dissociated neurons indicated the presence of growing microtubules with plus ends distal in dendritic protrusions. It is thus likely that PSD translocation is regulated by minus-end-directed motors, such as dynein molecules. Therefore, we evaluated the role of LIS1, a known regulator of the dynein motor complex1516. Both LIS1 and its interacting protein NDEL1 are expressed in the postnatal brain and enriched in the purified PSD fraction2122. First, we assessed the effect of LIS1 and NDEL1 knockdown using targeted short hairpin RNAs in dissociated hippocampal neurons at 9 DIV. LIS1 and NDEL1 knockdown did not impair the normal development of dendrites, but significantly reduced the fraction of mobile PSD-95-GFP puncta (Fig. 6a). Impaired PSD-95 mobility was rescued by overexpression of interfering RNA (RNAi)-insensitive LIS1 and NDEL1 mutants, confirming the specificity of the knockdown.


LIS1-dependent retrograde translocation of excitatory synapses in developing interneuron dendrites.

Kawabata I, Kashiwagi Y, Obashi K, Ohkura M, Nakai J, Wynshaw-Boris A, Yanagawa Y, Okabe S - Nat Commun (2012)

Effect of manipulating LIS1/NDEL1/dynein activity in interneurons on PSD mobility.(a) Fraction of mobile PSD puncta in interneurons transfected with PSD-95-GFP and RFP (control) or PSD-95-GFP, RFP and respective short hairpin RNA (shRNA) constructs. Two additional methods for inhibiting dynein-based mobility were also included (treatment with 1 mM erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) and dynamitin transfection). PSD mobility was suppressed by LIS1 or NDEL1 shRNA and rescued by overexpression of RNAi-insensitive LIS1 and NDEL1 mutants ('LIS1 RNAi+mLIS1' and 'NDEL1 RNAi+mNDEL1'). Mutant LIS1 and NDEL1 RNAi constructs containing three mismatched nucleotides ('mLIS1 RNAi' and 'mNDEL1 RNAi') did not suppress PSD mobility. Both EHNA treatment and expression of dynamitin suppressed PSD mobility. (n=18 cells for control, n=6 cells for LIS1 RNAi, n=7 cells for mLIS1 RNAi, n=6 cells for LIS1 RNAi+mLIS1, n=8 cells for NDEL1 RNAi, n=5 cells for mNDEL1 RNAi, n=5 cells for NDEL1 RNAi+mNDEL1, n=5 cells for EHNA treatment, n=5 cells for dynamitin transfection.) Significant differences (one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests): *P<0.05. (b) Time-lapse imaging of an interneuron expressing PSD-95-GFP with NDEL1-KillerRed before and after CALI (within 60 min after irradiation). Arrowheads indicate the position of a PSD-95-GFP punctum. Scale bar, 5 μm. (c) Suppression of PSD mobility by CALI of NDEL1-KillerRed. The effect of CALI on the mobile fraction was significant at 5–50 min after CALI (53±13.4%, n=6 cells). The fraction of motile PSD-95-GFP puncta returned to the control level at 60–105 min after CALI (91±15.6%, n=6 cells). The mean velocity of mobile PSD puncta also decreased after CALI and this effect was sustained for more than 60 min (2.8±0.30 μm h−1 before CALI, 1.1±0.17 μm h−1 at 5–50 min after CALI, 1.9±0.24 μm h−1 at 60–105 min after CALI, n=6 cells). Significant differences (one-way ANOVA followed by Tukey–Kramer multiple comparison tests): *P<0.05. All numeric data are mean±s.e.m.
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f6: Effect of manipulating LIS1/NDEL1/dynein activity in interneurons on PSD mobility.(a) Fraction of mobile PSD puncta in interneurons transfected with PSD-95-GFP and RFP (control) or PSD-95-GFP, RFP and respective short hairpin RNA (shRNA) constructs. Two additional methods for inhibiting dynein-based mobility were also included (treatment with 1 mM erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) and dynamitin transfection). PSD mobility was suppressed by LIS1 or NDEL1 shRNA and rescued by overexpression of RNAi-insensitive LIS1 and NDEL1 mutants ('LIS1 RNAi+mLIS1' and 'NDEL1 RNAi+mNDEL1'). Mutant LIS1 and NDEL1 RNAi constructs containing three mismatched nucleotides ('mLIS1 RNAi' and 'mNDEL1 RNAi') did not suppress PSD mobility. Both EHNA treatment and expression of dynamitin suppressed PSD mobility. (n=18 cells for control, n=6 cells for LIS1 RNAi, n=7 cells for mLIS1 RNAi, n=6 cells for LIS1 RNAi+mLIS1, n=8 cells for NDEL1 RNAi, n=5 cells for mNDEL1 RNAi, n=5 cells for NDEL1 RNAi+mNDEL1, n=5 cells for EHNA treatment, n=5 cells for dynamitin transfection.) Significant differences (one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison tests): *P<0.05. (b) Time-lapse imaging of an interneuron expressing PSD-95-GFP with NDEL1-KillerRed before and after CALI (within 60 min after irradiation). Arrowheads indicate the position of a PSD-95-GFP punctum. Scale bar, 5 μm. (c) Suppression of PSD mobility by CALI of NDEL1-KillerRed. The effect of CALI on the mobile fraction was significant at 5–50 min after CALI (53±13.4%, n=6 cells). The fraction of motile PSD-95-GFP puncta returned to the control level at 60–105 min after CALI (91±15.6%, n=6 cells). The mean velocity of mobile PSD puncta also decreased after CALI and this effect was sustained for more than 60 min (2.8±0.30 μm h−1 before CALI, 1.1±0.17 μm h−1 at 5–50 min after CALI, 1.9±0.24 μm h−1 at 60–105 min after CALI, n=6 cells). Significant differences (one-way ANOVA followed by Tukey–Kramer multiple comparison tests): *P<0.05. All numeric data are mean±s.e.m.
Mentions: Our results in dissociated neurons indicated the presence of growing microtubules with plus ends distal in dendritic protrusions. It is thus likely that PSD translocation is regulated by minus-end-directed motors, such as dynein molecules. Therefore, we evaluated the role of LIS1, a known regulator of the dynein motor complex1516. Both LIS1 and its interacting protein NDEL1 are expressed in the postnatal brain and enriched in the purified PSD fraction2122. First, we assessed the effect of LIS1 and NDEL1 knockdown using targeted short hairpin RNAs in dissociated hippocampal neurons at 9 DIV. LIS1 and NDEL1 knockdown did not impair the normal development of dendrites, but significantly reduced the fraction of mobile PSD-95-GFP puncta (Fig. 6a). Impaired PSD-95 mobility was rescued by overexpression of interfering RNA (RNAi)-insensitive LIS1 and NDEL1 mutants, confirming the specificity of the knockdown.

Bottom Line: This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility.Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites.Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Synaptic remodelling coordinated with dendritic growth is essential for proper development of neural connections. After establishment of synaptic contacts, synaptic junctions are thought to become stationary and provide fixed anchoring points for further dendritic growth. However, the possibility of active translocation of synapses along dendritic protrusions, to guide the proper arrangement of synaptic distribution, has not yet been fully investigated. Here we show that immature dendrites of γ-aminobutyric acid-positive interneurons form long protrusions and that these protrusions serve as conduits for retrograde translocation of synaptic contacts to the parental dendrites. This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility. Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites. Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

Show MeSH
Related in: MedlinePlus