1. Volume 18, Issue 5, Pages 1118-1131 (January 2017)
Altered Tau Isoform Ratio Caused by Loss of FUS and SFPQ Function Leads to FTLD- like Phenotypes 
Shinsuke Ishigaki, Yusuke Fujioka, Yohei Okada, Yuichi Riku, Tsuyoshi Udagawa, Daiyu Honda, Satoshi Yokoi, Kuniyuki Endo, Kensuke Ikenaka, Shinnosuke Takagi, Yohei Iguchi, Naruhiko Sahara, Akihiko Takashima, Hideyuki Okano, Mari Yoshida, Hitoshi Warita, Masashi Aoki, Hirohisa Watanabe, Haruo Okado, Masahisa Katsuno, Gen Sobue 
Cell Reports 
Volume 18, Issue 5, Pages (January 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 1118-1131DOI: (10.1016/j.celrep.2017.01.013)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
FUS and SFPQ Comprise the Intranuclear Complex Required for Regulating Alternative Splicing of Mapt Exon 10 and Neurite Maintenance
(A) Fractionation of cytoplasmic and nuclear extracts of NSC-34 cells. Fractions 2–17 were immunoblotted using an anti-FUS antibody. Co-migrating molecular mass markers are indicated. A high-MW peak (fractions 12–14, red box) and a low-MW peak (fractions 4–6, blue box) are indicated (left panels). Multiple RNA-binding proteins that interact with FUS were identified (Table S1). SFPQ is enriched in the high-MW FUS immunoprecipitates (middle panel, silver staining; right panels, immunoblot). Other RNA-binding proteins were relatively enriched in the low-MW FUS immunoprecipitates (right panels).
(B) Immunoblots of nuclear extracts from SFPQ-silenced (shSFPQ) and control shRNA (shCont)-infected cells (left panels). Anti-FUS immunoblot of fractionated nuclear extracts (right panels).
(C) Lentivirus-mediated shRNAs targeting FUS (shFUS1 and shFUS2), SFPQ (shSFPQ1 and shSFPQ2), and shCont in primary cortical neurons. The mouse FUS and SFPQ genes were measured by qPCR. Alternative splicing of Mapt exon 10 was measured by RT-PCR. Quantification is shown as the exon 10+/exon 10– ratio (n = 3 for FUS, n = 4 for SFPQ, one-way ANOVA).
(D) Flag-tagged human FUSs were overexpressed in NSC-34 cells, and nuclear extracts were immunoprecipitated with an anti-Flag antibody followed by immunoblotting. Input lysates were immunoblotted as shown.
(E) Lentivirus-mediated shRNA targeting endogenous mouse FUS (shFUS) or a scramble control shRNA (shCont) in primary cortical neurons using increasing concentrations of lentivirus expressing FUSWT (left panels) or FUSR521G (right panels). Expression of the endogenous mouse FUS gene (blue) and exogenous human FUS gene (red) was determined by qPCR (bottom graphs). Alternative splicing of Mapt exon 10 was measured by RT-PCR. Quantification is shown as the exon 10+/exon– ratio (top graphs) (n = 3, one-way ANOVA).
(F) Lentivirus-mediated shFUS and shCont in primary cortical neurons with lentivirus expressing FUSWT, FUSR521G, and FUSR495X. The protein extracts from each group were immunoblotted as shown (left panels). Quantification of the 4R-T/3R-T ratio is shown (right graph) (n = 3, one-way ANOVA).
(G) The neurite outlength was measured as in Figure S2A (one-way ANOVA).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N.S., not significant. Data are mean ± SD. See also Figures S1 and S2 and Table S1.
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4. Figure 2
Mice with Hippocampal-Specific Knockdown of FUS or SFPQ Exhibit FTLD-like Behavioral Impairments and Hippocampal Atrophy with Neuronal Loss
(A) An elevated-plus maze test of mice treated with shCont and shFUS (upper graphs) or shCont and shSFPQ (lower graphs). Time spent in the open (left graphs) and the closed (middle graphs) arm of an elevated-plus maze and the number of entries into the open arm (right graphs) were measured for shCont (n = 30) versus shFUS (n = 33) and shCont (n = 19) versus shSFPQ (n = 25) (Student’s t test).
(B) A social interaction defect was observed in both shFUS and shSFPQ mice. A resident-intruder test measuring the investigation time of the test “resident” mouse from the shCont (n = 25) and shFUS (n = 15) sample sets, or the shCont (n = 18) and shSFPQ (n = 21) sample sets on an “intruder” wild-type mouse in four consecutive sessions (upper panel for shFUS and lower panel for shSFPQ).
(C) An open-field test of mice treated with shCont and shFUS (left graphs, n = 13 for each) or Cont and shSFPQ (right graphs, n = 19 and 24, respectively). The distance moved in the indicated compartment of an open field during the 5-min test session is shown (Student’s t test).
(D) MRI-based determination of hippocampus volume in shFUS and shSFPQ-treated mice (left panels and graphs). Representative brain imaging of shCont and shFUS-treated mice at 12 and 18 months post-injection, and shCont and shSFPQ-treated mice at 6 months post-injection are shown. Yellow arrows and dashed lines indicate a hippocampus with normal volume, whereas red arrows and dashed lines indicate an atrophic hippocampus. Quantification of the volume ratio of the hippocampus relative to the whole brain is shown (n = 4, Student’s t test). Sections of the hippocampus from shCont and shFUS mice at 12 and 18 months post-injection, and shCont and shSFPQ mice at 6 months post-injection were stained with an anti-NeuN antibody (right images). Severe neuronal loss in the hippocampus was observed in shFUS mice at 18 months post-injection, but not at 12 months post-injection. Prominent neuronal loss in the hippocampus was observed in shSFPQ mice at 6 months post-injection.
Scale bars, 200 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N.S., not significant. Data are mean ± SD. See also Figure S3.
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5. Figure 3
Mice with Hippocampal-Specific Knockdown of FUS or SFPQ Exhibit Reduced Adult Neurogenesis
(A) The sections of the hippocampus from shCont and shFUS-treated mice were stained with an anti-3R-T antibody using a DAB peroxidase substrate. Scale bars, 50 μm.
(B) A BrdU incorporation assay was performed as indicated (upper panel). Sections of the hippocampus at 2 weeks post-BrdU administration were stained with anti-BrdU and anti-NeuN antibodies. Scale bar, 20 μm. Arrows indicate BrdU- and NeuN-positive cells in the dentate gyrus (DG) (lower images).
(C) BrdU-positive cells in the DG are shown for sections from shCont and shFUS, and shCont and shSFPQ. Scale bars, 100 μm. The number of BrdU-positive cells in the DG was determined in sections from shCont and shFUS mice (left graph), and shCont and shSFPQ mice (right graph, n = 4 for each, Student’s t test).
(D) Sections of the hippocampus from shCont and shFUS mice were stained with anti-3R-T, anti-DCX (a marker for neurogenesis), and anti-FUS antibodies (upper images). Scale bars, 50 μm. Sections of the hippocampus from shCont and shSFPQ mice were stained with anti-3R-T, anti-DCX, and anti-SFPQ antibodies (lower images).
Scale bars, 20 μm. ∗∗p < 0.01, ∗∗∗p < Data are mean ± SD. See also Figure S4.
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6. Figure 4
Aged Mice with Hippocampal-Specific Knockdown of FUS or SFPQ Exhibit Phosphorylated Tau Accumulation
(A) Sections of the hippocampus from shCont and shFUS mice at 12, 18, and 24 months post-injection (n = 5 and 3, 4 and 6, and 6 and 4, respectively), and shCont and shSFPQ mice at 6 months post-injection (n = 5 for each) were stained with an MC1 antibody.
(B) Sections of the hippocampus from shCont and shFUS mice at 12, 18, and 24 months post-injection (n = 6 for each, 4 and 8, and 4 for each, respectively), and shCont and shSFPQ mice at 6 months post-injection (n = 5 for each) were stained with an AT8 antibody. Scale bars, 20 μm. The signal positive area was quantified using Tissuemorph DP (Visiopharm). Statistical analysis via Student’s t test. Data are represented as mean ± SD.
(C) Protein extracts from the hippocampus of shCont and shFUS mice at 2 years post-injection were fractionated into TBS-soluble and sarcosyl-insoluble fractions as previously reported (Sahara et al., 2013). The TBS-soluble fractions were immunoblotted with anti-phosphorylated tau (PHF1), anti-total tau (an originally developed mouse monoclonal antibody, 2A1-2E1), anti-3R-T, anti-4R-T, and anti-actin antibodies (left upper panels). The sarcosyl-insoluble fractions were immunoblotted with the monoclonal 2A1-2E1 total tau antibody (left lower panel). The signal intensities were quantified and phosphorylated tau (PHF1)/actin and total tau/actin ratios were determined (right graphs, shown as the ratio relative to shCont). Statistical analysis via Student’s t test (n = 3 for each).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N.S., not significant. Data are mean ± SD. See also Figure S5.
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7. Figure 5
Mice with Hippocampal-Specific Knockdown of FUS and SFPQ Exhibit Increased 4R-T/3R-T Ratios, and Their Altered Phenotypes Are Rescued by Co-suppression of 4R-T
(A) Hippocampal immunoblots of mice treated with shCont, shFUS + shCont, and shFUS + sh4R-T (upper panels) or shCont, shSFPQ + shCont, and shSFPQ + sh4R-T (lower panels). The 4R-T/3R-T ratios were determined (n = 3 for each, one-way ANOVA).
(B) Elevated-plus maze test of shFUS + shCont versus shFUS + sh4R-T mice (n = 22 and 20, respectively, upper graphs) or shSFPQ + shCont versus shSFPQ + sh4R-T mice (n = 12 for each, lower graphs). Statistical analysis via Student’s t test.
(C) The social interaction defects of shFUS- or shSFPQ-treated mice were rescued by co-silencing 4R-T (upper graph, n = 12 for each; lower graph, n = 9 and 10, respectively) Statistical analysis via Student’s t test.
(D) Open-field test of shFUS + shCont and shFUS + sh4R-T mice (left graph, n = 5 and 4, respectively) or shSFPQ + shCont and shSFPQ + sh4R-T mice (right graph, n = 12 for each). Statistical analysis via Student’s t test.
(E) MRI-based determination of hippocampus volume in shCont, shFUS + shCont, and shFUS + sh4R-T-treated mice. Representative images at 15 months post-injection with normal (yellow arrows) and atrophic (red arrows) hippocampal volume (n = 5 for each, one-way ANOVA).
(F) BrdU-positive cells in the DG of shCont, shFUS, and shFUS + sh4R-T mice (left images) or shCont, shSFPQ, and shSFPQ + sh4R-T mice (right images). The shCont, shFUS, and shSFPQ sections are the same as in Figure 3C. Scale bars, 100 μm. BrdU-positive cells in the DG were determined for shCont, shFUS, and shFUS + sh4R-T-treated mice (n = 4 for each, one-way ANOVA).
(G) Hippocampal sections of shFUS + shCont-treated and shFUS + sh4R-T-treated mice at 15 months post-injection were stained with either an MC1 (n = 6 for each, Student’s t test) or an AT8 antibody (n = 4 for each, Student’s t test). Scale bars, 20 μm. The signal positive area was quantified using Tissuemorph DP (Visiopharm).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N.S., not significant. Data are mean ± SD. See also Figures S6 and S7.
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8. Figure 6
FUSWT Overexpression Rescued Reduced Adult Neurogenesis at the SGZ in the Hippocampus Caused by Loss of FUS, but FUSR521G Overexpression Did Not
(A) Protein extracts from shCont, shFUS + GFP (Cont), and shFUS + FUSWT-treated mice were immunoblotted with the indicated antibodies (left panels). The signal intensities were quantified and the 4R-T/3R-T ratio determined (left graph, n = 3, Student’s t test, shFUS + Cont versus shFUS + FUSWT). The protein extracts from shCont, shFUS + GFP (Cont), and shFUS + FUSR521G-treated mice were immunoblotted with the indicated antibodies (right panels). The signal intensities were quantified and the 4R-T/3R-T ratio determined (right graph, n = 3, Student’s t test, shFUS + Cont vs. shFUS + FUSR521G).
(B) The elevated-plus maze test was performed using mice treated with shFUS + GFP (Cont) (n = 20), shFUS + FUSWT (n = 21), and shFUS + FUSR521G (n = 13). The time spent in the open (left graph) and the closed (middle graph) arm of an elevated-plus maze and the number of entries into the open arm (right graph) were measured (one-way ANOVA).
(C) A social interaction defect observed in shFUS mice was rescued by overexpressing FUSWT but not FUSR521G. A resident-intruder test of shFUS + GFP (Cont) (n = 8), shFUS + FUSWT (n = 7), and shFUS + FUSR521G -treated mice (n = 13) on an “intruder” mouse in four consecutive sessions was performed (one-way ANOVA).
(D) Sections of the hippocampus from mice treated with shFUS + control and shFUS + FUSWT were stained with anti-DCX and anti-FUS antibodies (left images). The number of BrdU-positive cells in the DG was determined (left graph; n = 4 for each; Student’s t test). Hippocampal sections from mice treated with shFUS + control and shFUS + FUSR521G were stained with anti-DCX and anti-FUS antibodies (right images). The number of BrdU-positive cells in the DG were determined (right graph; n = 4 for each; Student’s t test). Scale bars, 20 μm.
(E) Hippocampal protein extracts from shCont and shSFPQ2 mice were immunoblotted with the indicated antibodies.
(F) An elevated-plus maze test was performed using mice treated with shCont versus shSFPQ2 (n = 18 for each). Time spent in the open (left graph) and the closed (middle graph) arm of an elevated-plus maze, and the number of entries into the open arm (right graph) were measured (Student’s t test).
(G) Sections of the hippocampus from shCont and shSFPQ2 mice at 6 weeks post-injection were stained with anti-3R-T, anti-DCX (a marker for neurogenesis), and anti-SFPQ antibodies. Scale bars, 50 μm. The 3R-T-positive and DCX-positive neurons at the DG are decreased in shSFPQ2 mice.
(H) A BrdU incorporation assay was performed. The number of BrdU-positive cells in the DG was determined in sections from shCont and shSFPQ2 mice (n = 4 for each, Student’s t test).
∗p < 0.05, ∗∗∗p < 0.001, N.S., not significant. Data are shown as mean ± SD.
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9. Figure 7
Both FUS and SFPQ Regulate Alternative Splicing of MAPT at Exon 10 in Human Tissue
(A) To confirm alternative splicing regulation of exon 10 in human MAPT by FUS and SFPQ, a human mini-gene, which is comprised of exons 9–11 and the adjacent introns, was transfected into neuro2a cells with siRNAs targeting endogenous FUS (siFUS1 and siFUS2), SFPQ (siSFPQ), and control scrambled siRNA (siCont). Subsequent RT-PCR analysis revealed a change in the human MAPT exon 10+/ exon 10– ratio (left panel). Expression of the endogenous mouse FUS and SFPQ genes was measured by qPCR (middle and right graphs).
(B) Lentivirus-mediated shRNA targeting human FUS (sh-hFUS1 and sh-hFUS2) and a scramble control shRNA (shCont) were introduced into human iPSC-derived neurons. Expression of the human FUS gene was measured by qPCR (left graph). Alternative splicing of exon 10 in MAPT was measured by qPCR with TaqMan probes (right graph, one-way ANOVA).
(C) Lentivirus-mediated shRNA targeting human SFPQ (sh-hSFPQ) and a shCont were introduced into human iPSC-derived neurons. Expression of the human SFPQ gene was measured by qPCR (left graph). Alternative splicing of exon 10 in MAPT was measured by qPCR with TaqMan probes (right graph, Student’s t test).
(D) Human iPSC-derived neurons with a homozygous H517D mutation in the FUS gene (FUSH517D/H517D) were established by gene-editing. Alternative splicing of MAPT exon 10 was measured by qPCR with TaqMan probes on FUSH517D/H517D neurons and the unedited control neurons (n = 3 iPSC-derived neuronal lines for each, Student’s t test).
∗p < 0.05, ∗∗p < 0.01, N.S., not significant. Data are shown as mean ± SD. All qPCR and RT-PCR quantifications were done in triplicate (mean ± SD).
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