Supplementary Information
JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent
neuronal degeneration
Tokiharu Sato, Momoe Ishikawa, Miki Mochizuki, Masaki Ohta, Masamichi Ohkura,
Junichi Nakai, Nobuhiko Takamatsu and Katsuji Yoshioka
Supplementary Information includes:
Supplementary Figures S1-S7 (submitted as separate files)
Supplementary Movies S1-S27 (submitted as separate files)
Supplementary Figure legends
Supplementary Movie legends
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Supplementary Figure Legends
Figure S1. Phenotypic and immunohistological analysis of Jsap1:Jlp cdKO mice. (a)
Generation of a floxed Jlp allele. Schematic illustration of the targeting of the Jlp gene. The
wild-type allele (Jlp+) and targeted allele (JlploxP-frt) are shown with the targeting construct.
B, BamHI; E, EcoRI; N, NotI; neo, neomycin-resistance cassette; DT-A, diphtheria toxin-A
gene; blue circles, loxP sites; red triangles, frt sites. (b) Southern blot analysis of Jlp+/+ (+/+)
and JlploxP-frt/+ (loxP-frt/+) mice. Genomic DNAs isolated from the mice were double
digested with BamHI and EcoRI, and subjected to Southern blotting with the probe shown
in a. (c) Control (Jsap1f/f:Jlpf/f) and Jsap1:Jlp cdKO male mice at P21. Jsap1:Jlp cdKO
mice were smaller than control mice. (d) Nissl staining of coronal brain sections of Jsap1
cKO and Jlp cKO mice at P21. Ventricular enlargement was not observed in the mice. (e)
Defective telencephalic commissure formation in Jsap1 cKO and Jsap1:Jlp cdKO mice.
Coronal brain sections of control, Jsap1 cKO, Jlp cKO, and Jsap1:Jlp cdKO mice at P21
were stained with Nissl. Jsap1 cKO and Jsap1:Jlp cdKO mice, but not control or Jlp cKO
mice, lacked the telencephalic commissure. Lower panels show higher magnifications of
the boxed areas in the upper panels. (f,g) Control and Jsap1:Jlp cdKO mice at P21 were
subjected to TUNEL (f) and GFAP (g) staining. In addition to the color TUNEL images,
black-and-white binary images, created using ImageJ, are included for better visualization
(f). Scale bars, 1 mm (d,e); 100 µm (f,g).
Figure S2. Immunofluorescence microscopy of hippocampal neurons infected with
AxCANCre. Uninfected Jsap1f/f:Jlpf/f, single Jsap1 or Jlp KO, and Jsap1:Jlp dKO
hippocampal neurons infected with AxCANCre at 7 dpi (a) and 10 dpi (b) were stained
with antibodies against MAP2 (a dendritic marker) and Tau (an axonal marker).
MAP2-positive/Tau-negative and MAP2-negative/Tau-positive immunosignals were
detected in the confocal images. Right panels show higher magnifications of the boxed
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areas in the left panels. The numbers in the right panels correspond to the box numbers in
the left panels. Scale bars, 50 µm.
Figure S3. Distribution of Mito-, APP-, SYP-, and SYT1-eGFP in the axons of primary
neurons. These eGFP fusion proteins were lentivirally expressed in Jsap1f/f:Jlpf/f
hippocampal neurons, and were stained with MitoTracker and the indicated antibodies.
Lower panels show higher magnifications of the boxed areas in upper panels. Tau staining
was included as an axonal marker. Mito-eGFP (a) and APP-eGFP (b) colocalized with
MitoTracker-stained mitochondria and with APP stained with an anti-APP antibody,
respectively, in the axons. The eGFP signals of SYP (c) and SYT1 (d) mostly colocalized
with VAMP2, indicating that a large proportion of the SYP- and SYT1-eGFPs were
expressed in synaptic vesicle precursors. Scale bars, 50 µm.
Figure S4. Distribution of exogenously expressed wild-type and mutant JSAP1s in
Jsap1:Jlp dKO neurons. Jsap1f/f:Jlpf/f hippocampal neurons infected with AxCANCre and
HA-JSAP1-WT, -LZ, or -KBD lentiviruses were stained with DAPI and antibodies against
HA and Tau as indicated. The HA-JSAP1s were widely and similarly distributed along the
axons in AxCANCre-infected Jsap1:Jlp dKO neurons at 3 dpi. Lower panels show higher
magnifications of the boxed areas in the upper panels. Scale bars, 50 µm.
Figure S5. JLP-WT but not JLP-KBD rescued defective anterograde movement of
mitochondria and APP, and the axonal degeneration and the subsequent neuronal loss, in
Jsap1:Jlp dKO hippocampal neurons. (a) The distribution of lentivirally expressed
HA-JLP-WT and HA-JLP-KBD in Jsap1f/f:Jlpf/f hippocampal neurons stained with
anti-HA and anti-Tau antibodies. Lower panels show magnified views of the boxed areas in
the upper panels. HA-JLP-WT and -KBD were widely distributed in the neurons. Scale
bars, 50 µm. (b) Time-lapse studies and quantitative analysis of mitochondria and APP
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movement were performed as in Figure 4a-d using AxCANCre-infected Jsap1f/f:Jlpf/f
hippocampal neurons, and HA-JLP and APP-eGFP lentiviruses as indicated. Mitochondria
were stained with MitoTracker. See also Supplementary Movies 21, 22, 26, and 27. The
defective anterograde movement of mitochondria and APP, along with an increase in
stationary mitochondria and APP, were observed in the Jsap1:Jlp dKO neurons (Figure
4a-d); these defects were reversed by HA-JLP-WT, but not -KBD. (c) HA-JLP-WT, but
not -KBD rescued axonal degeneration (left) and neuron loss (right) in the Jsap1:Jlp
hippocampal dKO neurons. Immunofluorescence microscopy and quantitative analysis
were performed as in Figures 2c-f, using Jsap1f/f:Jlpf/f hippocampal neurons that were
infected or uninfected with AxCANCre or HA-JLP lentiviruses, as indicated.
**
P<0.01;
***
P<0.001; n.s., not significant.
Figure S6. JLP-WT, but not -KBD, rescues reduced kinesin-1 binding to microtubules
(MTs). (a) An MT binding assay was performed at 3 dpi as in Figure 6a, using Jsap1f/f:Jlpf/f
hippocampal neurons uninfected or infected with AxCANCre and HA-JLP lentiviruses as
indicated. The reduced KHC binding to MTs was reversed by the lentivirus expression of
HA-JLP-WT, but not -KBD. It should be noted there was very little JLP in the MT
pellets, from Jsap1:Jlp dKO neurons that expressed HA-JLP-KBD or from those not
expressing an HA-JLP fusion protein. (b) Quantification of MT-bound KHC in a.
***
P<0.001.
Figure S7. Reduced intra-axonal ATP levels in Jsap1:Jlp dKO neurons. (a,b) Jsap1f/f:Jlpf/f
hippocampal neurons were uninfected or infected with AxCANCre and AT1.03 lentiviruses,
as indicated. AT1.03-YEMK is an enhanced derivative of a FRET-based ATP indicator
ATeam, and AT1.03-R122K/R126K is a negative mutant of ATeam29. At 5 dpi with
AxCANCre, the neurons were fixed with 4% PFA for 30 min at room temperature, and
subjected to FRET analysis. These samples were also subjected to YFP direct excitation to
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examine the expression levels of AT1.03s in the neurons. Confocal images of FRET and
CFP (a) and YFP (b) were acquired as described in Materials and Methods. Lower panels
show higher magnifications of the boxed areas in the upper panels. (c) Quantification of the
ratio of the mean FRET intensity to the mean CFP intensity in a. (d) Ratio of the mean YFP
intensity in Jsap1f/f:Jlpf/f neurons uninfected or infected with AxCANCre and AT1.03
lentiviruses, as indicated, to that in AxCANCre-uninfected Jsap1f/f:Jlpf/f neurons expressing
AT1.03-YEMK in b. **P<0.01; n.s., not significant. Scale bars, 50 µm; enlarged images, 10
µm.
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Supplementary Movie Legends
Movie S1: Time-lapse movie of Mito-eGFP expressed in control (Jsap1f/f:Jlpf/f)
hippocampal neurons.
Mito-eGFP movement to the left represents anterograde axonal transport.
Movie S2: Time-lapse movie of Mito-eGFP expressed in AxCANCre-infected Jsap1
single-knockout (KO) hippocampal neurons at 3 dpi.
Mito-eGFP movement to the left represents anterograde axonal transport.
Movie S3: Time-lapse movie of Mito-eGFP expressed in AxCANCre-infected Jlp
single-knockout (KO) hippocampal neurons at 3 dpi.
Mito-eGFP movement to the right represents anterograde axonal transport.
Movie S4: Time-lapse movie of Mito-eGFP expressed in AxCANCre-infected Jsap1:Jlp
dKO hippocampal neurons at 3 dpi.
Mito-eGFP movement to the left represents anterograde axonal transport.
Movie S5: Time-lapse movie of APP-eGFP expressed in control (Jsap1f/f:Jlpf/f)
hippocampal neurons.
APP-eGFP movement to the right represents anterograde axonal transport.
Movie S6: Time-lapse movie of APP-eGFP expressed in AxCANCre-infected Jsap1 KO
hippocampal neurons at 3 dpi.
APP-eGFP movement to the right represents anterograde axonal transport.
Movie S7: Time-lapse movie of APP-eGFP expressed in AxCANCre-infected Jlp KO
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hippocampal neurons at 3 dpi.
APP-eGFP movement to the left represents anterograde axonal transport.
Movie S8: Time-lapse movie of APP-eGFP expressed in AxCANCre-infected Jsap1:Jlp
dKO hippocampal neurons at 3 dpi.
APP-eGFP movement to the right represents anterograde axonal transport.
Movie S9: Time-lapse movie of SYP-eGFP in control (Jsap1f/f:Jlpf/f) hippocampal neurons.
SYP-eGFP movement to the right represents anterograde axonal transport.
Movie S10: Time-lapse movie of SYP-eGFP in AxCANCre-infected Jsap1 KO
hippocampal neurons at 3 dpi.
SYP-eGFP movement to the left represents anterograde axonal transport.
Movie S11: Time-lapse movie of SYP-eGFP in AxCANCre-infected Jlp KO hippocampal
neurons at 3 dpi.
SYP-eGFP movement to the left represents anterograde axonal transport.
Movie S12: Time-lapse movie of SYP-eGFP in AxCANCre-infected Jsap1:Jlp dKO
hippocampal neurons at 3 dpi.
SYP-eGFP movement to the left represents anterograde axonal transport.
Movie S13: Time-lapse movie of SYT1-eGFP in control (Jsap1f/f:Jlpf/f) hippocampal
neurons.
SYT1-eGFP movement to the left represents anterograde axonal transport.
Movie S14: Time-lapse movie of SYT1-eGFP in AxCANCre-infected Jsap1 KO
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hippocampal neurons at 3 dpi.
SYT1-eGFP movement to the right represents anterograde axonal transport.
Movie S15: Time-lapse movie of SYT1-eGFP in AxCANCre-infected Jlp KO
hippocampal neurons at 3 dpi.
SYT1-eGFP movement to the left represents anterograde axonal transport.
Movie S16: Time-lapse movie of SYT1-eGFP in AxCANCre-infected Jsap1:Jlp dKO
hippocampal neurons at 3 dpi.
SYT1-eGFP movement to the left represents anterograde axonal transport.
Movie S17: Time-lapse movie of mitochondria stained with MitoTracker in
AxCANCre-infected Jsap1:Jlp dKO hippocampal neurons at 3 dpi.
Mitochondrial movement to the right represents anterograde axonal transport.
Movie S18: Time-lapse movie of mitochondria stained with MitoTracker in
AxCANCre-infected Jsap1:Jlp dKO hippocampal neurons expressing HA-JSAP1-WT at 3
dpi.
Mitochondrial movement to the left represents anterograde axonal transport.
Movie S19: Time-lapse movie of mitochondria stained with MitoTracker in
AxCANCre-infected Jsap1:Jlp dKO hippocampal neurons expressing HA-JSAP1-LZ at 3
dpi.
Mitochondrial movement to the left represents anterograde axonal transport.
Movie S20: Time-lapse movie of mitochondria stained with MitoTracker in
AxCANCre-infected Jsap1:Jlp dKO hippocampal neurons expressing HA-JSAP1-KBD
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at 3 dpi.
Mitochondrial movement to the left represents anterograde axonal transport.
Movie S21: Time-lapse movie of mitochondria stained with MitoTracker in
AxCANCre-infected Jsap1:Jlp dKO hippocampal neurons expressing HA-JLP-WT at 3
dpi.
Mitochondrial movement to the left represents anterograde axonal transport.
Movie S22: Time-lapse movie of mitochondria stained with MitoTracker in
AxCANCre-infected Jsap1:Jlp dKO hippocampal neurons expressing HA-JLP-KBD at 3
dpi.
Mitochondrial movement to the left represents anterograde axonal transport.
Movie S23: A time-lapse movie of APP-eGFP in AxCANCre-infected Jsap1:Jlp dKO
hippocampal neurons expressing HA-JSAP1-WT at 3 dpi.
APP-eGFP movement to the left represents anterograde axonal transport.
Movie S24: Time-lapse movie of APP-eGFP in AxCANCre-infected Jsap1:Jlp dKO
hippocampal neurons expressing HA-JSAP1-LZ at 3 dpi.
APP-eGFP movement to the left represents anterograde axonal transport.
Movie S25: Time-lapse movie of APP-eGFP in AxCANCre-infected Jsap1:Jlp dKO
hippocampal neurons expressing HA-JSAP1-KBD at 3 dpi.
APP-eGFP movement to the left represents anterograde axonal transport.
Movie S26: Time-lapse movie of APP-eGFP in AxCANCre-infected Jsap1:Jlp dKO
hippocampal neurons expressing HA-JLP-WT at 3 dpi.
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APP-eGFP movement to the left represents anterograde axonal transport.
Movie S27: Time-lapse movie of APP-eGFP in AxCANCre-infected Jsap1:Jlp dKO
hippocampal neurons expressing HA-JLP-KBD at 3 dpi.
APP-eGFP movement to the right represents anterograde axonal transport.
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