Journal: Nature neuroscience

Article Title: Reduced mitochondrial fusion and Huntingtin levels contribute to impaired dendritic maturation and behavioral deficits in Fmr1 mutant mice

doi: 10.1038/s41593-019-0338-y

Figure Lengend Snippet: a, Schematic illustration of dual retroviral vectors expressing Cre driven by DCX promoter (Retro-DCX-Cre) and expressing Cre-dependent GFP (Retro-Flip-GFP) to infect DCX+ cells. b, Schematic diagram showing selective deletion or restoration of FMRP using FMRP-cKO (left) and FMRP-cON (right) mice combined with Cre virus–mediated recombination. c, Experimental scheme for assessing the impact of specific deletion of FMRP from immature neurons on dendritic maturation. d, Representative confocal images (from 3 independently repeated experiments with similar results) and Neurolucida software-created traces of GFP+ neurons in WT (top), cKO (middle) and cON (bottom) mice at 28 d post-viral injection. Scale bar, 50 μm. e-f, Dendritic complexity analysis of GFP+ neurons in WT and cKO and cON mice. e, Scholl analysis (Multi-ANOVA, F( 2,72) = 16.024, P < 0.0001. WT vs. cKO: P < 0.0001, WT vs. cON: P = 0.772, cKO vs. cON: P < 0.0001. WT: n = 28 neurons in 5 mice, cKO: n = 27 neurons in 4 mice, cON: n = 18 neurons in 3 mice); f, Dendritic length (one-way ANOVA with Bonferroni post hoc test: F( 2,70) = 8.842, P = 0.0004; Bonferroni post hoc test, WT vs. cKO: P = 0.0018; WT vs. cON: P > 0.9999; cKO vs. cON: P = 0.0019. WT: 0.9240 ± 0.05876, n = 28 neurons in 5 mice, cKO: 0.6010 ± 0.07261, n = 27 neurons in 4 mice, cON: 0.9645 ± 0.06987, n = 18 neurons in 3 mice). g, Dendritic nodes (one-way ANOVA with Bonferroni post hoc test: F (2, 70) = 7.437, P = 0.0012; Bonferroni post hoc test, WT vs. cKO: P = 0.0123, WT vs. cON: P> 0.9999, cKO vs. cON: P = 0.0022. WT: 5.929 ± 0.3948, n = 28 neurons in 5 mice, cKO: 4.111 ± 0.4077, n = 27 neurons in 4 mice, cON: 6.556 ± 0.6428, n = 18 neurons in 3 mice). h, Dendritic ends (one-way ANOVA with two-sided Bonferroni post hoc test: F (2, 70) = 5.169, P = 0.0081; Bonferroni post hoc test, WT vs. cKO: P = 0.0902, WT vs. cON: P = 0.7935; cKO vs. cON: P = 0.0089. WT: 7.714 ± 0.3668, n = 28 neurons in 5 mice, cKO: 6.333 ± 0.4498, n = 27, n = 27 neurons in 4 mice, cON: 8.500 ± 0.6530, n = 18 neurons in 3 mice). i, Experimental scheme for assessing the impact of specific deletion of FMRP from immature neurons on synaptic transmission of DG newborn neurons. j, Representative traces of mEPSCs recorded from GFP+ DG neurons in acute hippocampal slices derived from WT and cKO mice 4 weeks after retroviral injection. (7 independently repeated experiments with similar results). k-l, Cumulative probability distribution (WT, n = 20 cells from 7 mice; cKO, n = 22 cells from 8 mice. P = 0.084, nonparametric Wilcoxon Rank test, two-sided) and average amplitude (Student’s t-test, two-sided, t(40) = 0.2630, P = 0.7936. WT: 7.154 ± 0.1483; cKO: 7.068 ± 0.2818) of mEPSC. m-n, Cumulative probability distribution (WT, n = 20 cells; cKO, n = 22 cells. P < 0.0001, nonparametric Wilcoxon Rank test, two-sided) and average frequency (Student’s t-test, two-sided, t(40) = 2.191, P = 0.0344. WT: 1.132 ± 0.08012; cKO: 0.8934 ± 0.07382) of mEPSC. * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars reflect the Mean ± S.E.M

Article Snippet: Z-stacks of eGFP+ dendrites were captured at 3 μm intervals and the dendrites and the cell body of single eGFP+ neurons were analyzed by Neurolucida software with 3D module plug-in (MicroBrightField, Inc. Williston, VI, http://www.mbfbioscience.com/ ).

Techniques: Expressing, Software, Injection, Transmission Assay, Derivative Assay

Journal: Nature neuroscience

Article Title: Reduced mitochondrial fusion and Huntingtin levels contribute to impaired dendritic maturation and behavioral deficits in Fmr1 mutant mice

doi: 10.1038/s41593-019-0338-y

Figure Lengend Snippet: a, Representative confocal images (from 3 independently repeated experiments with similar results) of HTT expression in the DCX+ immature neurons in the DG of cKO;Cre;tdT and Ctrl;Cre;tdT mice. Scale bars, 50 μm. b, The expression level of HTT in the tdT+ cells of the DG from cKO; Cre; tdT and Ctrl;Cre;tdT mice (Student’s t-test, two-sided, t(8) = 2.348, P = 0.0468. Ctrl: 1.000 ± 0.1304, n = 5 mice; cKO: 0.6139 ± 0.1002, n = 5 mice). c, Quantification of mRNA levels of Htt in 3-week-old WT and Fmr1 KO DG tissues. Gapdh was used as the internal control for quantitative PCR analysis. (Student’s t-test, two-sided, t(5) = 3.056, P = 0.0282. WT: 1.000 ± 0.06252, n = 4 mice; KO: 0.7457 ± 0.04554, n = 3 mice). d, Quantification of mRNA levels of Htt in DIV 7 WT and Fmr1 KO primary hippocampal cells. Gapdh was used as the internal control for quantitative PCR analysis. (Student’s t-test, two-sided, t(12) = 4.530, P = 0.0007. WT: 1.000 ± 1.000 ± 0.09355, n = 9; KO: 0.3492 ± 0.09103, n = 5 independent experiments). e, Schematic illustration of retroviral vectors expressing shNC and expressing shHtt . f, Experimental scheme for assessing the effect of down-regulation of Htt on mitochondrial fusion and fission genes and dendritic maturation of hippocampal neurons. g-l, Quantification of mRNA levels of Htt , mitochondrial fusion and fission genes in primary hippocampal cells infected by lentivirus- shHtt or lentivirus-shNC (above) or transfected with shHtt /shNC (below). (Student’s t-test, two-sided. Htt , shNC = 1.000 ± 0.1345, n = 4; shHtt = 0.1493 ± 0.02515, n = 4 biologically independent samples, t(6) = 6.291, P = 0.0008; Opa1 , shNC = 1.000 ± 0.08564, n = 4; shHtt = 0.6141 ± 0.09681, n = 4, t(6) = 2.985, P = 0.0245; Mfn1, shNC = 1.000 ± 0.07478, n = 4; shHtt = 0.8673 ± 0.04440, n = 4, t(6) = 1.526, P = 0.1778; Mfn2, shNC = 1.000 ± 0.04351, n = 4; shHtt = 0.5720 ± 0.09657, n = 4, t(6) = 4.041, P = 0.0068; Drp1, shNC = 1.000 ± 0.04058, n = 4; shHtt = 1.011 ± 0.1528, n = 4, t(6) = 0.06726, P = 0.9486; Fis1, shNC = 1.000 ± 0.2099, n = 4; shHtt = 0.5505 ± 0.1316, n = 4, t(6) = 1.814, P = 0.1196). m, Examples of confocal images and Neurolucida software-created traces of DIV7 primary hippocampal neurons transfected with shHtt or shNC at DIV4. 3 independently repeated experiments with similar results. Scale bar, 20 μm. m, Sholl analysis of dendritic complexity of hippocampal neurons transfected with shHtt or shNC (Multi-ANOVA, shNC vs. shHtt : F( 1,61) = 27.712, P < 0.0001. shNC, n = 22; shHtt, n = 40 cells from 3 independent experiments). o, Quantification of dendritic length of hippocampal neurons transfected with shHtt or shNC (Student’s t-test, t(60) = 6.454, P < 0.0001. shNC , 574.4 ± 31.77, n = 22; shHtt , 366.6 ± 16.34, n = 40 cells). p, Quantification of dendritic nodes of hippocampal neurons transfected with shHtt or shNC (Student’s t-test, two-sided. t(60) = 13.57, P < 0.0001. shNC , 6.909 ± 0.3598, n = 22; shHtt , 2.175 ± 0.1675, n = 40 cells). * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars reflect the Mean ± S.E.M.

Article Snippet: Z-stacks of eGFP+ dendrites were captured at 3 μm intervals and the dendrites and the cell body of single eGFP+ neurons were analyzed by Neurolucida software with 3D module plug-in (MicroBrightField, Inc. Williston, VI, http://www.mbfbioscience.com/ ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Infection, Transfection, Software