loHuntingtin recruits KIF1A to transport synaptic vesicle precursors along the mouse axon to support synaptic transmission and motor skill learning
- Univ. Grenoble Alpes, Inserm, U1216, CHU Grenoble Alpes, Grenoble Institut Neuroscience, France
- Center for Interdisciplinary Research in Biology, College de France, CNRS, INSERM, Université PSL, France
- Sorbonne Université, Institut du Cerveau, Paris Brain Institute, ICM, Inserm U1127, CNRS UMR7225, France
Figures
Figure 1 with 2 supplements
HTT phosphorylation at S421 increases synaptic vesicle precursor (SVP) anterograde axonal transport and SV exocytosis.
(A) Diagram of the microfluidic device for reconstituting a corticostriatal network compatible with live-cell imaging of axons. Cortical axons grow in the cortical chamber (yellow) and connect with …
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Figure 1—source data 1
Data analyzed for anterograde velocity.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-data1-v2.xlsx
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Figure 1—source data 2
Data analyzed for retrograde velocity.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-data2-v2.xlsx
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Figure 1—source data 3
Data analyzed for net flux.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-data3-v2.xlsx
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Figure 1—source data 4
Data analyzed for VGLUT1 pHluorin exocytosis number of events.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-data4-v2.xlsx
Figure 1—figure supplement 1
HTT phosphorylation at S421 increases synaptic vesicle precursor (SVP) anterograde axonal transport without affecting the quantity of SV released.
(A) Left: Number of anterograde (*p<0.05; N=117 WT axons, 156 HTT-SD axons, and 132 HTT-SA axons) and retrograde (*p<0.05; 118 WT axons, 159 HTT-SD axons, and 134 HTT-SA axons) VAMP2-mCherry …
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Figure 1—figure supplement 1—source data 1
Data analyzed for number of anterograde vesicles.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-figsupp1-data1-v2.xlsx
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Figure 1—figure supplement 1—source data 2
Data analyzed for number of retrograde vesicles.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-figsupp1-data2-v2.xlsx
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Figure 1—figure supplement 1—source data 3
Data analyzed for the linear flow rate.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-figsupp1-data3-v2.xlsx
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Figure 1—figure supplement 1—source data 4
Data analyzed for VGLUT1 pHluorin exocytosis amplitude of events.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig1-figsupp1-data4-v2.xlsx
Figure 1—video 1
Movie showing the glutamate release (VGLUT-pHluorin) in wild-type (WT) neurons after the stimulation.
Figure 2 with 1 supplement
Constitutive phosphorylation of HTT at S421 impairs motor skill learning in mice.
(A) Schematic of the accelerating rotarod protocol assessing motor skill learning over 8 days with 10 sessions per day. (B) Mean latency to fall each day for 8 days, for HTT-SD mice (*p<0.05: …
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Figure 2—source data 1
Data analyzed for accelerating rotarod over 8 days.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig2-data1-v2.xlsx
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Figure 2—source data 2
Data analyzed for accelerating rotarod during day 1.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig2-data2-v2.xlsx
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Figure 2—source data 3
Data analyzed for accelerating rotarod during day 8.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
HTT phosphorylation at S421 impairs motor skill learning without affecting motor performance.
(A) Results of the first rotarod trial of the first day for HTT-SD and HTT-SA mice (ns: non-significant, Mann-Whitney test). The box-whisker plots show the median, the 25th and the 75th percentiles, …
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Figure 2—figure supplement 1—source data 1
Data analyzed for accelerating rotarod first trial at day 1.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig2-figsupp1-data1-v2.xlsx
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Figure 2—figure supplement 1—source data 2
Data analyzed for accelerating rotarod during day 1 at 18 months.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig2-figsupp1-data2-v2.xlsx
Figure 3 with 1 supplement
HTT phosphorylation at S421 increases short-term plasticity in the corticostriatal network ex vivo.
(A) Schematic of medium-sized spiny neurons (MSNs) recording in the dorsolateral striatum (DLS) after paired-pulse stimulations in S2 cortex of mice at 2–3 months of age. (B) Representative traces …
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Figure 3—source data 1
Data analyzed for short-term plasticity.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
HTT phosphorylation does not regulate the spontaneous excitatory postsynaptic currents (sEPSCs) in the corticostriatal synapse.
(A) Schematic of the procedure for sEPSC recording in medium-sized spiny neurons (MSNs) within the dorsolateral striatum (DLS). (B) Representative traces, cumulative probability of the mean …
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Figure 3—figure supplement 1—source data 1
Data analyzed for mEPSC recordings.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
HTT phosphorylation increases the number of synaptic vesicles (SVs) distally to the presynaptic active zone.
(A) Representative images of SVs at the corticostriatal synapse, obtained by electronic microscopy, in dorsolateral striatum (DLS) slices from three wild-type (WT) and HTT-SD mice 3-month-old male. …
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Figure 4—source data 1
Data analyzed for the number of the corticostriatal synapses (i).
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-data1-v2.xlsx
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Figure 4—source data 2
Data analyzed for the number of the synaptic vesicles (ii).
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-data2-v2.xlsx
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Figure 4—source data 3
Data analyzed for the number of the axon terminal size in the presynaptic zone.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-data3-v2.xlsx
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Figure 4—source data 4
Data analyzed for the density of synaptic vesicles.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-data4-v2.xlsx
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Figure 4—source data 5
Data analyzed for the number of vesicles per zone.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-data5-v2.xlsx
Figure 4—figure supplement 1
Characterization of the three presynaptic zones of HTT-SD corticostriatal axon terminals and analysis of HTT-SA corticostriatal synapses.
(A) Area of the three zones in wild-type (WT) and HTT-SD axon terminals studied in 145±9 axon terminals from three WT and HTT-SD 3-month-old mice (ns: non-significant, two-way ANOVA followed by …
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Figure 4—figure supplement 1—source data 1
Data analyzed for the area of the presynaptic zones.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
Data analyzed for the the number of the corticostriatal synapses (i).
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-figsupp1-data2-v2.xlsx
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Figure 4—figure supplement 1—source data 3
Data analyzed for the number of the synaptic vesicles (ii).
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig4-figsupp1-data3-v2.xlsx
Figure 5 with 1 supplement
HTT phosphorylation recruits KIF1A on VAMP2-mCherry vesicles.
(A) Mass spectrometry analysis of vesicles purified from mouse brains identifies KIF1A (red) among HTT-associated vesicular proteins. (B) Confocal and two-dimensional stimulated emission depletion …
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Figure 5—source data 1
Data analyzed for HTT-KIF1A colocalization in axons.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-data1-v2.xlsx
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Figure 5—source data 2
Data analyzed for the proximity ligation assay performed between HTT and KIF1A.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-data2-v2.xlsx
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Figure 5—source data 3
Data analyzed for the protein content of KIF1A and VAMP2 levels in vesicular fractions.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-data3-v2.xlsx
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Figure 5—source data 4
Western blot scans for the data presented in Figure 5E (KIF1A and VAMP2 levels in brain vesicular fractions).
Shown in red are the cropped regions presented in Figure 5E. Films containing the second batch of samples (Gel 2) are shown.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-data4-v2.zip
Figure 5—figure supplement 1
HTT phosphorylation and subcellular localization and interaction of HTT and KIF1A with VAMP2.
(A) Representative immunostainings revealing HTT (cyan), KIF1A (green), and VAMP2-mCherry (magenta) within cortical axons localized in the long channels of the microfluidic devices. The images were …
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Figure 5—figure supplement 1—source data 1
Data analyzed for HTT-VAMP2 colocalization in axons.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-figsupp1-data1-v2.xlsx
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Figure 5—figure supplement 1—source data 2
Data analyzed for KIF1A-VAMP2 colocalization in axons.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-figsupp1-data2-v2.xlsx
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Figure 5—figure supplement 1—source data 3
Data analyzed for the proximity ligation assay performed between VAMP2/HTT.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-figsupp1-data3-v2.xlsx
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Figure 5—figure supplement 1—source data 4
Data analyzed for the proximity ligation assay performed between VAMP2/KIF1A.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-figsupp1-data4-v2.xlsx
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Figure 5—figure supplement 1—source data 5
Data analyzed for the protein content of KIF1A and VAMP2 levels in total fractions.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-figsupp1-data5-v2.xlsx
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Figure 5—figure supplement 1—source data 6
Western blot scans for the data presented in Figure 5—figure supplement 1D (KIF1A levels in whole brain lysates).
Shown in red are the cropped regions presented in Figure 5—figure supplement 1D. Films containing the first batch of samples (Gel 1) are shown.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig5-figsupp1-data6-v2.zip
Figure 6 with 2 supplements
HTT-dependent axonal transport of synaptic vesicle precursors (SVPs) is mediated by KIF1A.
(A) Diagram indicating lentiviral transduction of VAMP2-mCherry and sh-scramble (sh-Scr-GFP) or sh-Kif1a (sh-Kif1a-GFP) lentiviruses at day in vitro (DIV) 8 in the microfluidic device. On the right, …
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Figure 6—source data 1
Data analyzed for anterograde velocity.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-data1-v2.xlsx
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Figure 6—source data 2
Data analyzed for retrograde velocity.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-data2-v2.xlsx
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Figure 6—source data 3
Data analyzed for net flux.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-data3-v2.xlsx
Figure 6—figure supplement 1
KIF1A levels in HTT-SD neurons regulate VAMP2 axonal transport.
(A) Analysis of KIF1A levels by western blot in cortical neurons either not-transduced or transduced with sh-Kif1a or sh-Scr lentiviruses (*p<0.05, **p<0.01, ***p<0.001; one-way ANOVA followed by …
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Figure 6—figure supplement 1—source data 1
Western blot scans for the data presented in Figure 6—figure supplement 1A (KIF1A levels in cortical neurons).
Shown in red are the cropped regions presented in Figure 6—figure supplement 1A. Films containing the samples are shown.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp1-data1-v2.zip
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Figure 6—figure supplement 1—source data 2
Data analyzed for number of anterograde vesicles.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp1-data2-v2.xlsx
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Figure 6—figure supplement 1—source data 3
Data analyzed for number of retrograde vesicles.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp1-data3-v2.xlsx
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Figure 6—figure supplement 1—source data 4
Data analyzed for the linear flow rate.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp1-data4-v2.xlsx
Figure 6—figure supplement 2
KIF1A silencing doesn’t affect BDNF-mCherry transport.
(A) Diagram indicating transduction of BDNF-mCherry and sh-scramble (sh-scr-GFP) or sh-Kif1a (sh-Kif1a-GFP) lentiviruses (left). Representative kymographs of BDNF-mCherry vesicle transport within …
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Figure 6—figure supplement 2—source data 1
Data analyzed for for anterograde velocity.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp2-data1-v2.xlsx
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Figure 6—figure supplement 2—source data 2
Data analyzed for for retrograde velocity.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp2-data2-v2.xlsx
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Figure 6—figure supplement 2—source data 3
Data analyzed for the linear flow rate.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp2-data3-v2.xlsx
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Figure 6—figure supplement 2—source data 4
Data analyzed for the net flux.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig6-figsupp2-data4-v2.xlsx
Figure 7
In vivo KIF1A silencing in mice restores the synaptic vesicle (SV) synaptic pool.
(A) Immunolabeling of GFP within the injection site on a slice located at 1.5 mm before the bregma (left). Scale = 1 cm (insets, 100 µm). Immunolabeling of GFP within the projection site on a slice …
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Figure 7—source data 1
Data analyzed for the number of the synaptic vesicles.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig7-data1-v2.xlsx
Figure 8
Motor skill learning defects of S421D mice are rescued by KIF1A silencing in vivo.
(A) Schematic of the experimental procedure consisting in bilateral stereotaxic injections in the mouse brain followed 3 weeks later by the accelerating rotarod protocol over 8 days. (B) Mean time …
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Figure 8—source data 1
Data analyzed for accelerating rotarod over 8 days.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig8-data1-v2.xlsx
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Figure 8—source data 2
Data analyzed for accelerating rotarod during day 1.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig8-data2-v2.xlsx
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Figure 8—source data 3
Data analyzed for accelerating rotarod during day 8.
- https://cdn.elifesciences.org/articles/81011/elife-81011-fig8-data3-v2.xlsx
