Figure 1A Immunohistochemical analysis of LONP1 expression in CKD children with mild ( n = 11 ) , moderate ( n = 10 ) , or severe fibrosis ( n = 9 ) . N = 4 in Normal group . D Western blot analysis for the expression of LONP1 in UUO models at different time points . F Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . G Sirius red staining in WT and cKI mice after UUO . Scale bar : 50μm . J , K Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and TGF - β1 ( monomer ) in WT and cKI mice after UUO ( n = 3 or 4 , biological replicates ) . M Succinate dehydrogenase ( SDH ) staining in WT and cKI mice after UUO . Scale bar : 50μm . N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO . Scale bar : 500nm .

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Figure 1A Immunohistochemical analysis of LONP1 expression in CKD children with mild (n=11), moderate (n=10), or severe fibrosis (n=9). N=4 in Normal group.D Western blot analysis for the expression of LONP1 in UUO models at different time points.F Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. G Sirius red staining in WT and cKI mice after UUO. Scale bar: 50μm.J, K Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and TGF-β1 (monomer) in WT and cKI mice after UUO (n=3 or 4, biological replicates).M Succinate dehydrogenase (SDH) staining in WT and cKI mice after UUO. Scale bar: 50μm.N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO. Scale bar: 500nm.


Figure 2A Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . B Sirius red staining in WT and cKO mice after UUO . Scale bar : 50μD , E Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after UUO ( n = 3 , biological replicates ) . I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO . Scale bar : 500nm .

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Figure 2A Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. B Sirius red staining in WT and cKO mice after UUO. Scale bar: 50μD, E Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after UUO (n=3, biological replicates).I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO. Scale bar: 500nm.


Figure 3C Masson ' s trichrome staining of WT and cKI mice after 5 / 6Nx . Scale bar : 100μm . D qRT - PCR analysis of FN1 , Collagen I and Collagen III in WT and cKI mice after 5 / 6Nx ( n = 6 in cKI + Sham group , n = 5 in the other three groups , biological replicates ) G Masson ' s trichrome staining of WT and cKO mice after 5 / 6Nx . Scale bar : 100μm . H qRT - PCR analysis of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 5 in WT + Sham group , n = 7 in cKO + Sham group , n = 6 in 5 / 6Nx groups , biological replicates ) . I , J Western blot and densitometric analysis for the expression of Collagen I and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 4 , biological replicates ) . L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5 / 6Nx . Scale bar : 500nm .

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Figure 3C Masson's trichrome staining of WT and cKI mice after 5/6Nx. Scale bar: 100μm.D qRT-PCR analysis of FN1, Collagen I and Collagen III in WT and cKI mice after 5/6Nx (n=6 in cKI+Sham group, n=5 in the other three groups, biological replicates)G Masson's trichrome staining of WT and cKO mice after 5/6Nx. Scale bar: 100μm.H qRT-PCR analysis of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after 5/6Nx (n=5 in WT+Sham group, n=7 in cKO+Sham group, n=6 in 5/6Nx groups, biological replicates).I, J Western blot and densitometric analysis for the expression of Collagen I and α-SMA in WT and cKO mice after 5/6Nx (n=4, biological replicates).L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5/6Nx. Scale bar: 500nm.


Figure 4A Quantification of mitochondrial ROS production ( n = 4 , biological replicates ) . D qRT - PCR analysis of FN1 , Collagen I , Collagen III , Collagen IV , α - SMA and Vimentin ( n = 3 , biological replicates ) . Measurement of OCR using an XF96 Extracellular Flux Analyzer ( n = 8 or 10 in each group , biological replicates ) . Western blot analysis for the expression of Collagen I , α - SMA , and Vimentin ( n = 3 , two independent experiments were carried out ) .

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Figure 4A Quantification of mitochondrial ROS production (n=4, biological replicates).D qRT-PCR analysis of FN1, Collagen I, Collagen III, Collagen IV, α-SMA and Vimentin (n=3, biological replicates).Measurement of OCR using an XF96 Extracellular Flux Analyzer (n=8 or 10 in each group, biological replicates).Western blot analysis for the expression of Collagen I, α-SMA, and Vimentin (n=3, two independent experiments were carried out).


Figure 5C Western blot validation of the up - regulated proteins in mitochondria ( n = 2 , biological replicates ) . E GST pull down analysis of interaction between LONP1 and HMGCS2 . G Immunofluorescence co - localization analysis of LONP1 , HMGCS2 and mitochondria in mPTC ( up , scale bar : 20μm ) and human kidney tissue ( down , scale bar : 200μm ) .

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Figure 5C Western blot validation of the up-regulated proteins in mitochondria (n=2, biological replicates).E GST pull down analysis of interaction between LONP1 and HMGCS2.G Immunofluorescence co-localization analysis of LONP1, HMGCS2 and mitochondria in mPTC (up, scale bar: 20μm) and human kidney tissue (down, scale bar: 200μm).


Figure 6A Immunohistochemical analysis of HMGCS2 expression in CKD children . Scale bar : 50μm . B Immunohistochemical semi - quantitative IOD analysis of HMGCS2 ( n = 8 in Control group , n = 15 in CKD group ) . Western blot analysis of HMGCS2 in WT and cKI mice of UUO model ( n = 3 or 4 , biological replicates ) . western blotting analysis of Hmgcs2 expression in WT and Hmgcs2 + / - mice ( n = 3 , biological replicates ) .

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Figure 6A Immunohistochemical analysis of HMGCS2 expression in CKD children. Scale bar: 50μm. B Immunohistochemical semi-quantitative IOD analysis of HMGCS2 (n=8 in Control group, n=15 in CKD group).Western blot analysis of HMGCS2 in WT and cKI mice of UUO model (n=3 or 4, biological replicates).western blotting analysis of Hmgcs2 expression in WT and Hmgcs2+/- mice (n=3, biological replicates).


Figure 7G , H Representative immunoblot and densitometric analysis of HMGCS2 , FN1 , Collagen III , α - SMA and Vimentin in mPTCs treated as indicated . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) . J - N Representative Western blot and densitometric analysis of LONP1 , FN1 , α - SMA and Vimentin of each group was shown . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) . mPTCs were transfected with siNC or siLONP1 , and then pre - treated with 84 - B10 ( 10 μM ) 2h before TGF - β1 ( 10ng / mL ) treatment for another 24h .

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Figure 7G, H Representative immunoblot and densitometric analysis of HMGCS2, FN1, Collagen III, α-SMA and Vimentin in mPTCs treated as indicated. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group).J-N Representative Western blot and densitometric analysis of LONP1, FN1, α-SMA and Vimentin of each group was shown. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group). mPTCs were transfected with siNC or siLONP1, and then pre-treated with 84-B10 (10 μM) 2h before TGF-β1 (10ng/mL) treatment for another 24h.


Figure 8A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model . Scale bar : 50μm . B Quantification of fibrotic area in UUO model ( n = 10 in each group , biological replicates ) . G , H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model ( n = 10 in each group , biological replicates ) . Scale bar : 50μm .

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Figure 8A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model. Scale bar: 50μm. B Quantification of fibrotic area in UUO model (n=10 in each group, biological replicates).G, H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model (n=10 in each group, biological replicates). Scale bar: 50μm.


Figure 1B . Representative traces for C - H , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and shRNA - expressing neurons ( green traces ) . C , D . Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at − 60 mV ( AMPAR - mediated EPSCs ; C ) or at + 40 mV ( NMDAR - mediated EPSCs ; D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . E . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices at − 60 mV ( AMPAR - mediated EPSCs ; C ) or at + 40 mV ( NMDAR - mediated EPSCs ; D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from uninfected and infected slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . F , G . As C , D , for GSK3β . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . H . As E , for GSK3β . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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Figure 1B. Representative traces for C-H, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and shRNA-expressing neurons (green traces).C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. F, G. As C, D, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. H. As E, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


Figure 2B . Representative traces for C , where baseline responses ( dark trace ) are taken between 10 and 0 minutes before LTD induction , and responses after LTD induction ( light trace ) are taken between 40 and 45 minutes after the end of LTD induction . C . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of LTD by LFS ( 300 pulses at 1 Hz ) , in control ( uninfected ) and GSK3α ( blue ) or GSK3β ( orange ) shRNA - expressing neurons . Error bars represent SEM . D . Quantification of average synaptic depression from the last 5 minutes of the time course shown in C . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . E . Cumulative frequency distribution of the ESPC values plotted in D . Statistical significance was calculated according to the Kolmogorov - Smirnov test .

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Figure 2B. Representative traces for C, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (uninfected) and GSK3α (blue) or GSK3β (orange) shRNA-expressing neurons. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. E. Cumulative frequency distribution of the ESPC values plotted in D. Statistical significance was calculated according to the Kolmogorov-Smirnov test.


Figure 3A . Representative traces for B and C , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in neurons from inactive control - , GSK3α or GSK3β inhibitor - treated slices ( all at 10 µM ) . Slices were incubated with each compound for 1 hour before recording , and during whole - cell recording itself . Compounds were also included in the internal solution of the recording pipette ( all at 10 µM ) . B . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from inactive control , GSK3α or GSK3β inhibitor - treated slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . C . Cumulative frequency distribution of the AMPA / NMDA ratios plotted in B . Statistical significance was calculated using the Kolmogorov - Smirnov test . D . Representative traces for E and F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in neurons from hippocampal slices treated with DMSO ( vehicle control ) , AR - 18 ( 10 µM ) , CHIR ( 1 µM ) or SC79 ( 20 µM ) . E , F . As B , C , for DMSO , AR - 18 , CHIR and SC79 . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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Figure 3A. Representative traces for B and C, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from inactive control-, GSK3α or GSK3β inhibitor-treated slices (all at 10 µM). Slices were incubated with each compound for 1 hour before recording, and during whole-cell recording itself. Compounds were also included in the internal solution of the recording pipette (all at 10 µM).B. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from inactive control, GSK3α or GSK3β inhibitor-treated slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. C. Cumulative frequency distribution of the AMPA/NMDA ratios plotted in B. Statistical significance was calculated using the Kolmogorov-Smirnov test. D. Representative traces for E and F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from hippocampal slices treated with DMSO (vehicle control), AR-18 (10 µM), CHIR (1 µM) or SC79 (20 µM). E, F. As B, C, for DMSO, AR-18, CHIR and SC79. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


Figure 4B . Representative traces for C , D , where baseline responses ( dark trace ) are taken between 10 and 0 minutes before LTD induction , and responses after LTD induction ( light trace ) are taken between 40 and 45 minutes after the end of LTD induction . C . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of LTD by LFS ( 300 pulses at 1 Hz ) , in control ( inactive control ) , GSK3α ( blue ) and GSK3β ( orange ) inhibitor , and AR - 18 - treated neurons ( yellow ) . Drug treatment was for 1 hour before recording at 10 µM for all compounds . Error bars represent SEM . D . Quantification of average synaptic depression from the last 5 minutes of the time course shown in C . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . E . As B , for F and G . F . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of mGluR - dependent LTD by PP - LFS ( 900 paired pulses , separated by 50 ms , at 1 Hz ) , in control ( inactive control ) and GSK3α inhibitor - treated neurons ( blue ) . Drug treatment was for 1 hour before recording at 10 µM for all compounds . Representative traces for synaptic responses before ( dark trace ) and after ( light trace ) LTD induction are shown above the time course . Error bars represent SEM . G . Quantification of average synaptic depression from the last 5 minutes of the time course shown in F . Error bars represent SEM . No statistically significant difference was found , according to the Mann - Whitney U test .

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Figure 4B. Representative traces for C, D, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (inactive control), GSK3α (blue) and GSK3β (orange) inhibitor, and AR-18-treated neurons (yellow). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. E. As B, for F and G. F. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of mGluR-dependent LTD by PP-LFS (900 paired pulses, separated by 50 ms, at 1 Hz), in control (inactive control) and GSK3α inhibitor-treated neurons (blue). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Representative traces for synaptic responses before (dark trace) and after (light trace) LTD induction are shown above the time course. Error bars represent SEM. G. Quantification of average synaptic depression from the last 5 minutes of the time course shown in F. Error bars represent SEM. No statistically significant difference was found, according to the Mann-Whitney U test.


Figure 5B . Representative traces for C - F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and EGFP - GSK3α - or EGFP - GSK3β - expressing neurons ( green traces ) . C , D . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at − 60 mV ( AMPAR - mediated EPSCs ) in slices expressing EGFP - GSK3α ( C ) or EGFP - GSK3β ( D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . E , F . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at + 40 mV ( NMDAR - mediated EPSCs ) in slices expressing EGFP - GSK3α ( E ) or EGFP - GSK3β ( F ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test .

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Figure 5B. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α- or EGFP-GSK3β -expressing neurons (green traces).C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in slices expressing EGFP-GSK3α (C) or EGFP-GSK3β (D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E, F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at +40 mV (NMDAR-mediated EPSCs) in slices expressing EGFP-GSK3α (E) or EGFP-GSK3β (F). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test.


Figure 6A . Representative confocal images from EGFP - GSK3α FRAP experiments . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but for spines expressing EGFP - GSK3β . Scale bars = 1 µm . E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test . N = 12 , 11 and 11 spines for EGFP - GSK3α baseline , cLTD and washout groups and 20 , 11 and 9 spines for EGFP - GSK3β baseline , cLTD and washout groups , respectively . F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α or EGFP - GSK3β in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test . G . Representative western blots from whole extracts of organotypic slices incubated in ACSF ( untreated ; ' baseline ' ) , treated with 20 µM NMDA for 5 minutes ( ' cLTD ' ) or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ( ' washout ' ) . Blots were probed for pSer21 / 9 GSK3α / β , total GSK3α / β , pSer845 GluA1 , and total GluA1 . H . Quantification of experiments as those shown in G . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 4 independent experiments for all conditions . Asterisks ( * * ) denote P < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Representative confocal images from EGFP-GSK3α FRAP experiments. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but for spines expressing EGFP-GSK3β. Scale bars = 1 µm. E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test. N = 12, 11 and 11 spines for EGFP-GSK3α baseline, cLTD and washout groups and 20, 11 and 9 spines for EGFP-GSK3β baseline, cLTD and washout groups, respectively.F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α or EGFP-GSK3β in 'baseline', 'cLTD' or 'recovery' conditions. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test.G. Representative western blots from whole extracts of organotypic slices incubated in ACSF (untreated; 'baseline'), treated with 20 µM NMDA for 5 minutes ('cLTD') or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ('washout'). Blots were probed for pSer21/9 GSK3α/β, total GSK3α/β, pSer845 GluA1, and total GluA1. H. Quantification of experiments as those shown in G. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 4 independent experiments for all conditions. Asterisks (**) denote P < 0.01.


Figure 7A . Representative confocal images from EGFP - GSK3α FRAP experiments in organotypic slices cultured from C57 wild - type mice . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in wild - type mouse slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but in slices cultured from C57 tauKO mice . Scale bars = 1 µm . E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 13 , 11 and 12 spines for wt baseline , cLTD and recovery and 9 , 7 and 5 spines for tauKO baseline , cLTD and recovery groups , respectively . F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α in wild - type or tauKO slices in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Representative confocal images from EGFP-GSK3α FRAP experiments in organotypic slices cultured from C57 wild-type mice. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in wild-type mouse slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout ') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but in slices cultured from C57 tauKO mice. Scale bars = 1 µm. E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 13, 11 and 12 spines for wt baseline, cLTD and recovery and 9, 7 and 5 spines for tauKO baseline, cLTD and recovery groups, respectively.F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α in wild-type or tauKO slices in 'baseline', 'cLTD' or 'recovery' conditions. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


Figure 8B . Representative traces for C - F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and EGFP - GSK3α - expressing neurons ( green traces ) in C57 wt or tauKO slices . C - F . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at − 60 mV ( AMPAR - mediated EPSCs ) in C57 wt ( C ) or tauKO slices ( D ) , or at + 40 mV ( NMDAR - mediated EPSCs ) in wt ( E ) or tauKO slices ( F ) . Red circles represent the averages of all pairs in a given plot , and n denotes the number of cell pairs . In all cases , error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8B. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α-expressing neurons (green traces) in C57 wt or tauKO slices.C-F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in C57 wt (C) or tauKO slices (D), or at +40 mV (NMDAR-mediated EPSCs) in wt (E) or tauKO slices (F). Red circles represent the averages of all pairs in a given plot, and n denotes the number of cell pairs. In all cases, error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


Figure 1LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . CBA analysis of IL - 1β ( A ) in the culture supernatants . LPS - primed Abro1 + / + and Abro1 − / − BMDMs were treated with various doses of ATP . ELISA of IL - 18 ( B ) in the culture supernatants . CBA analysis of IL - 1β secretion from Abro1 + / + and Abro1 − / − BMDMs transduced with GFP - or ABRO1 - GFP - expressing lentiviruses prior to stimulation with LPS and ATP . Cell lysates were immunoblotted with anti - ABRO1 antibody . ELISA analysis of IL - 1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium ( Con ) , LPS or LPS plus ATP . Cell lysates were immunoblotted with anti - ABRO1 and anti - GAPDH antibodies . Abro1 + / + and Abro1 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with nigericin , silica , MSU , MDP , Alum , poly ( dA : dT ) , flagellin or LTx ( Anthrax Lethal Factor ) . CBA analysis of IL - 1β secretion in the culture supernatants . Representative immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Abro1 + / + and Abro1 − / − BMDMs treated with ATP , nigericin , MSU , flagellin or poly ( dA : dT ) . Flow cytometric analysis of caspase - 1 activity in LPS - primed Abro1 + / + and Abro1 − / − BMDMs stimulated with nigericin for indicated times . Immunostaining of endogenous ASC specks in LPS - primed Abro1 + / + and Abro1 − / − BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . Scale bars , 10 μm . Quantification of ASC specks from ( H ) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei . At least 100 cells from each treatment condition were quantified . UD , undetectable . LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti - ASC antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. CBA analysis of IL-1β (A) in the culture supernatants.LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. ELISA of IL-18 (B) in the culture supernatants.CBA analysis of IL-1β secretion from Abro1+/+ and Abro1−/− BMDMs transduced with GFP- or ABRO1-GFP-expressing lentiviruses prior to stimulation with LPS and ATP. Cell lysates were immunoblotted with anti-ABRO1 antibody.ELISA analysis of IL-1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium (Con), LPS or LPS plus ATP. Cell lysates were immunoblotted with anti-ABRO1 and anti-GAPDH antibodies.Abro1+/+ and Abro1−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with nigericin, silica, MSU, MDP, Alum, poly (dA: dT), flagellin or LTx (Anthrax Lethal Factor). CBA analysis of IL-1β secretion in the culture supernatants.Representative immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Abro1+/+ and Abro1−/− BMDMs treated with ATP, nigericin, MSU, flagellin or poly (dA: dT).Flow cytometric analysis of caspase-1 activity in LPS-primed Abro1+/+ and Abro1−/− BMDMs stimulated with nigericin for indicated times.Immunostaining of endogenous ASC specks in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin or poly (dA:dT). Scale bars, 10 μm. Quantification of ASC specks from (H) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei. At least 100 cells from each treatment condition were quantified. UD, undetectable.LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti-ASC antibody.


Figure 2WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( A ) . n = 6 for PBS groups and n = 12 for MSU groups . WT , Abro1 − / − and Nlrp3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . Flow cytometric analysis of peritoneal cell exudates ( B ) . n = 6 for PBS groups and n = 12 for MSU groups . Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) . Flow cytometric analysis of caspase - 1 activity in peritoneal exudate monocytes and neutrophils . n = 12 per group . Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β in the peritoneal lavage fluid ( D ) n = 6 for PBS groups and n = 12 for Alum groups . Abro1 + / + and Abro1 − / − mice were intraperitoneally injected with Alum ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( E ) . n = 6 for PBS groups and n = 12 for Alum groups . Survival of Abro1 + / + and Abro1 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group . ELISA of serum IL - 1β from Abro1 + / + and Abro1 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (A). n = 6 for PBS groups and n = 12 for MSU groups.WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups.Abro1+/+ and Abro1−/− mice were intraperitoneally injected with MSU (1 mg per mouse). Flow cytometric analysis of caspase-1 activity in peritoneal exudate monocytes and neutrophils. n=12 per group.Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β in the peritoneal lavage fluid (D) n = 6 for PBS groups and n = 12 for Alum groups.Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (E). n = 6 for PBS groups and n = 12 for Alum groups.Survival of Abro1+/+ and Abro1−/− mice subjected to LPS (15 mg/kg). n=20 per group.ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


Figure 3HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 , ASC or pro - casapase - 1 ( Pro - Casp - 1 ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose . BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody . BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody . BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody . BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody . HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 or NLRP3 deletion mutants as indicated in ( F ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose . HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 and Flag - tagged ABRO1 or ABRO1 deletion mutants as indicated in ( H ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3 and Flag-tagged ABRO1 or ABRO1 deletion mutants as indicated in (H). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


Figure 4HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - tagged NLRP3 ( WT ) , NLRP3 ( S194D ) , or NLRP3 ( S194A ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads . WT BMDMs were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody . WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody . HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194A ) were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads . HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194D ) were treated with or without 100 nM SP600125 for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads . WT and Nlrp3S194A / S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-tagged NLRP3 (WT), NLRP3 (S194D), or NLRP3 (S194A). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.WT BMDMs were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194A) were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194D) were treated with or without 100 nM SP600125 for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.WT and Nlrp3S194A/S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


Figure 5HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 , Flag - ABRO1 and HA - ubiquitin ( HA - Ub ) , HA - Ub K48 only or K63 only . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose . HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding HA - Ub , Flag - ABRO1 , and NLRP3 truncations as indicated . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of ubiquitination of NLRP3 truncations ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose . LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP ( left ) or nigericin ( right ) . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody . Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were left unstimulated or stimulated with LPS or LPS plus ATP . Immunoblot analysis of NLRP3 and NLRP3 ( A350V ) ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads . HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 ( A350V ) and HA - ubiquitin ( HA - Ub ) . Immunoblot analysis of NLRP3 ( A350V ) ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads . Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were stimulated with or without 100ng / ml LPS for 1 h . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody . HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 , and HA - ubiquitin ( HA - Ub ) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads . LPS - primed WT and Nlrp3S194A / S194A BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3 (A350V) and HA-ubiquitin (HA-Ub). Immunoblot analysis of NLRP3 (A350V) ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Figure 6WT BMDMs were stimulated with LPS for 1 h . Immunoblot analysis of NLRP3 , ABRO1 , and BRCC3 from the cell lysates immunoprecipitated with control IgG , anti - ABRO1 , anti - BRCC3 or anti - NLRP3 antibody . Brcc3 + / + and Brcc3 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Brcc3 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of ABRO1 . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody . Abro1 + / + and Abro1 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Abro1 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of BRCC3 . Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti - BRCC3 antibody . A constant amount of Myc - NLRP3 plasmid together with an increasing amount of Myc - ABRO1 plasmid were co - transfected in HEK - 293T cells . Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti - BRCC3 antibody . Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibodyAbro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibodyBrcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture supernatantsAbro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody.Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody.A constant amount of Myc-NLRP3 plasmid together with an increasing amount of Myc-ABRO1 plasmid were co-transfected in HEK-293T cells. Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti-BRCC3 antibody.Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibodyAbro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibodyBrcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatantsAbro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Figure 7Brcc3 + / + and Brcc3 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with ATP , nigericin , MSU , Alum , poly ( dA : dT ) or flagellin . CBA analysis of IL - 1β secretion in the culture supernatants . Immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs treated with ATP , nigericin , MSU , flagellin , and poly ( dA : dT ) . Immunostaining of endogenous ASC specks in LPS - primed Brcc3 + / + and Brcc3 - / - BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . ASC speck positive cells were counted and analyzed as described in Fig . 1I . UD , undetectable . LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs were left unstimulated or stimulated with nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody . Survival of Brcc3 + / + and Brcc3 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group . ELISA of serum IL - 1β from Brcc3 + / + and Brcc3 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups . Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( G ) Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( H ) . n = 6 for PBS groups and n = 12 for MSU groups .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT).Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable.LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group.ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G)Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.


Figure 1b ) Box plot showing the distribution of log2 ( FC ) of all or selected miR - 29a targets in the Data ref : Mazziotti et al , 2017 dataset of age - regulated genes in the visual cortex . Notches represent confidence intervals for the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance vs . all detected genes was assessed by Wilcoxon ' s test ( miR - 29a targets p = 4 . 9x10 - 14 , top 200 miR - 29a targets p = 3 . 7x10 - 38 ) . ( d , e ) Mature miR - 29a ( n = 3 / 4 mice for each age ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s test * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns not significant ) and Dnmt3a expression level ( normalized to P10 ) at different ages measured by qPCR ( n = 3 / 4 mice for each age ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s test : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns not significant ) . One way ANOVA p - values are reported . Error bars represent ± SEM . f ) Scatter plot showing correlation of miR - 29a and Dnmt3a levels in the visual cortex display a strong negative correlation ( Pearson correlation : r = - 0 . 817 , p < 0 . 001 ) . Each circle represents data from a single animal . g ) Representative examples of in situ hybridization on visual cortex ( left P25 , right P120 ) using a LNA probe against miR - 29a . Scale bar 180 µm . Cortical layers are delimited by roman numerals and bracketsh ) Example of co - staining for PV ( green ) , miR - 29a ( red ) and nuclear marker ( blue ) in a P120 mouse . White triangles represent PV + / miR - 29a + cells . Scale bar 20 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1b) Box plot showing the distribution of log2(FC) of all or selected miR-29a targets in the Data ref: Mazziotti et al, 2017 dataset of age-regulated genes in the visual cortex. Notches represent confidence intervals for the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes was assessed by Wilcoxon's test (miR-29a targets p=4.9x10-14, top 200 miR-29a targets p=3.7x10-38).(d,e) Mature miR-29a (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test ***p<0.001, ****p<0.0001, ns not significant) and Dnmt3a expression level (normalized to P10) at different ages measured by qPCR (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test: *p<0.05, **p<0.01, ****p<0.0001, ns not significant). One way ANOVA p-values are reported. Error bars represent ±SEM. f) Scatter plot showing correlation of miR-29a and Dnmt3a levels in the visual cortex display a strong negative correlation (Pearson correlation: r=-0.817, p<0.001). Each circle represents data from a single animal. g) Representative examples of in situ hybridization on visual cortex (left P25, right P120) using a LNA probe against miR-29a. Scale bar 180 µm. Cortical layers are delimited by roman numerals and bracketsh) Example of co-staining for PV (green), miR-29a (red) and nuclear marker (blue) in a P120 mouse. White triangles represent PV+/miR-29a+ cells. Scale bar 20 µm.


Figure 2Mature miR - 29a expression ( miR29 ) in visually deprived mice : a ) normal rearing ( NR , N = 5 ) vs dark rearing ( DRb , N = 5 ) ; t - test , p = 0 . 19 , b ) NR ( N = 4 ) vs dark rearing from P10 ( DRp10 , N = 5 ; t - test , p = 0 . 70 . In both experiments , analysis was performed at P25Mature miR - 29a expression ( miR29 ) in visually deprived mice : c ) one week period of dark rearing beginning at P30 ( DRw ) followed by a 4 hour exposure to light ( DRw + 4h light ) vs control animals ( ctr , N = 4 for each group ; one - way ANOVA , p = 0 . 68 ) Mature miR - 29a expression ( miR29 ) in visually deprived mice : d ) days of MD beginning at P25 , contralateral cortex vs ipsilateral cortex to deprived eye , N = 5 ; paired t test , p = 0 . 52 ) . Error bars represent ± SEM , symbols represent individual cases .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Mature miR-29a expression (miR29) in visually deprived mice: a) normal rearing (NR, N=5) vs dark rearing (DRb, N=5); t-test, p=0.19, b) NR (N=4) vs dark rearing from P10 (DRp10, N=5; t-test, p=0.70. In both experiments, analysis was performed at P25Mature miR-29a expression (miR29) in visually deprived mice: c) one week period of dark rearing beginning at P30 (DRw) followed by a 4 hour exposure to light (DRw+4h light) vs control animals (ctr, N=4 for each group; one-way ANOVA, p=0.68)Mature miR-29a expression (miR29) in visually deprived mice: d) days of MD beginning at P25, contralateral cortex vs ipsilateral cortex to deprived eye, N=5; paired t test, p=0.52). Error bars represent ±SEM, symbols represent individual cases.


Figure 3b ) MiR - 29a and Dnmt3a expression in the cortex treated with scrambled or miR - 29a mimic ( N = 3 mice ) . Data were normalized to the untreated cortex of the same animal . Mir - 29a : scr vs mim29a , t - test , p = 0 . 005 ; Dnmt3a scr vs mim29a t - test , p < 0 . 0001 . c ) IOS analysis . Ocular dominance index ( ODI ) of deprived animals treated with scrambled ( scr , N = 6 black lines ) or miR - 29a mimic ( mimic29 , N = 8 orange lines ) , or left untreated ( MD no treat , N = 5 grey lines ) before and after 3 days of MD . Dashed lines represent single animals , continuous lines the group average . two - way ANOVA RM , treatment X time , p = 0 . 028 ; post hoc Sidak ' s comparison , post - MD mim29a vs scr p < 0 . 05 , mim29a vs MD no treat p < 0 . 01 ; pre - MD scr vs post - MD scr p < 0 . 001 ; pre - MD MD no treat vs post - MD MD no treat p < 0 . 01 ; other comparisons not significant ) . d ) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled ( scr + MD ) and miR - 29a mimic ( mim29a + MD ) . Each symbol represents the result of a single mouse . ( MD + scr vs MD + mim29a ; N = 4 ; t - test , Npas4 : p = 0 . 038 ; Bdnf exIV : p = 0 . 049 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3b) MiR-29a and Dnmt3a expression in the cortex treated with scrambled or miR-29a mimic (N=3 mice). Data were normalized to the untreated cortex of the same animal. Mir-29a: scr vs mim29a, t-test, p=0.005; Dnmt3a scr vs mim29a t-test, p<0.0001.c) IOS analysis. Ocular dominance index (ODI) of deprived animals treated with scrambled (scr, N=6 black lines) or miR-29a mimic (mimic29, N=8 orange lines), or left untreated (MD no treat, N=5 grey lines) before and after 3 days of MD. Dashed lines represent single animals, continuous lines the group average. two-way ANOVA RM, treatment X time, p= 0.028; post hoc Sidak's comparison, post-MD mim29a vs scr p<0.05, mim29a vs MD no treat p<0.01; pre-MD scr vs post-MD scr p<0.001; pre-MD MD no treat vs post-MD MD no treat p<0.01; other comparisons not significant).d) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and miR-29a mimic (mim29a+MD). Each symbol represents the result of a single mouse. (MD+scr vs MD+mim29a; N=4; t-test, Npas4: p=0.038; Bdnf exIV: p=0.049).


Figure 4b ) Effects of amiR - 29a on the expression of miR - 29a and Dnmt3a . Fold change values with respect to the contralateral untreated cortex are reported ( ± SEM ) for mice treated with amiR - 29a or scr . scr vs amiR - 29a ; N = 3 t - test , miR - 29a * * p = 0 . 0010 ; Dnmt3a * * * * p = 0 . 0019 . c ) Distribution of log2 ( FC ) for the significant cases in transcriptomics ( N = 4 scr treated and N = 3 amiR - 29a treated biological replicates ) and proteomics ( N = 4 biological replicates per group ) datasets of animals treated with amiR - 29a versus scr animals . The black line represents the average . ( d , e ) Box plot showing distribution of log2 ( FC ) of miR29a targets as d ) transcripts ( RNA ) and e ) proteins ( PROT ) in transcriptomic and proteomic datasets of adult animals treated with amiR - 29a . Notches correspond to confidence intervals of the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance vs . all detected genes and proteins , respectively , assessed by Wilcoxon ' s test ( p - values shown in figure ) . f ) Box plots reporting distribution of log2 ( FC ) of age down - regulated miR29a targets after amiR - 29a treatment ( N = 4 scr treated and N = 3 amiR - 29a treated biological replicates ) . Notches correspond to confidence intervals of the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance was assessed with Wilcoxon ' s test ( p - values shown in figure ) . h ) Scatter plot correlating the expression changes of genes significantly regulated by age ( log2FC , x - axis ) and miR - 29a antagonization by amiR29a in adult mice ( log2FC , y - axis ) . ( Spearman r = - 0 . 22 p = 1 . 4x10 - 8 ; Fisher test showing significant enrichment in the IV quadrant p = 2 . 2x10 - 16 ) . The number of genes for each quadrant is reported in figure . i ) Scatter plot correlating the expression changes induced by miR - 29a antagonization in adult mice at the transcript - ( log2FC , x - axis ) and protein - level ( log2FC , y - axis ) . Spearman coefficient correlation , r = 0 . 11 ; p = 0 . 006 , Fisher test showing significant enrichment in the I quadrant p = 0 . 03 . The number of genes for each quadrant is reported in figure .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4b) Effects of amiR-29a on the expression of miR-29a and Dnmt3a. Fold change values with respect to the contralateral untreated cortex are reported (±SEM) for mice treated with amiR-29a or scr. scr vs amiR-29a; N=3 t-test, miR-29a **p=0.0010; Dnmt3a ****p=0.0019.c) Distribution of log2(FC) for the significant cases in transcriptomics (N=4 scr treated and N=3 amiR-29a treated biological replicates) and proteomics (N=4 biological replicates per group) datasets of animals treated with amiR-29a versus scr animals. The black line represents the average. (d,e) Box plot showing distribution of log2(FC) of miR29a targets as d) transcripts (RNA) and e) proteins (PROT) in transcriptomic and proteomic datasets of adult animals treated with amiR-29a. Notches correspond to confidence intervals of the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes and proteins, respectively, assessed by Wilcoxon's test (p-values shown in figure). f) Box plots reporting distribution of log2(FC) of age down-regulated miR29a targets after amiR-29a treatment (N=4 scr treated and N=3 amiR-29a treated biological replicates). Notches correspond to confidence intervals of the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance was assessed with Wilcoxon's test (p-values shown in figure).h) Scatter plot correlating the expression changes of genes significantly regulated by age (log2FC, x-axis) and miR-29a antagonization by amiR29a in adult mice (log2FC, y-axis). (Spearman r=-0.22 p=1.4x10-8; Fisher test showing significant enrichment in the IV quadrant p=2.2x10-16). The number of genes for each quadrant is reported in figure.i) Scatter plot correlating the expression changes induced by miR-29a antagonization in adult mice at the transcript- (log2FC, x-axis) and protein-level (log2FC, y-axis). Spearman coefficient correlation, r=0.11; p=0.006, Fisher test showing significant enrichment in the I quadrant p=0.03. The number of genes for each quadrant is reported in figure.


Figure 5a ) VEP analysis . Ratio between contralateral and ipsilateral eye responses ( C / I ratio ± SEM ) in animals treated with saline and not deprived ( sal no MD ) , with scrambled LNA and MD ( scr MD ) , with amiR - 29a LNA and MD ( amiR - 29a MD ) . N = 6 / 7 ; one - way ANOVA , p = 0 . 0003 ; post hoc Tukey ' s test : sal noMD vs scr + MD , ns ; sal noMD vs amiR - 29a + MD , p = 0 . 0002 ; scr + MD vs amiR - 29a + MD , p = 0 . 0169 . b ) IOS analysis . ODI ± SEM of deprived animals treated with scr ( N = 5 , black lines ) and amiR - 29a LNA ( N = 6 , blue lines ) . Dashed lines connect pre - and post - MD ODI values of each animal . Solid lines represent the group average . Two - way ANOVA RM , treatment X time , p = 0 . 0074 ; post hoc Sidak ' s test , pre - MD scr vs post - MD scr , ns ; pre - MD vs post - MD amiR - 29a , p = 0 . 0005 . c ) IOS amplitude ( ± SEM ) in response to contralateral eye ( contra ) and ipsilateral eye ( ipsi ) stimulation . Other conventions as in b ) . amiR - 29a ( N = 6 ) vs scr ( N = 5 ) . Contra eye : two - way ANOVA RM , time X treatment interaction p = 0 . 034 ; post hoc Sidak ' s test ; pre - MD vs post - MD scr , ns ; pre - MD vs post - MD amiR - 29a , p = 0 . 0008 . Ipsi eye : two - way ANOVA RM , time X treatment interaction , ns . d ) Average time course of the IOS response to visual stimulation of the contralateral eye ( contra ) pre - and post - MD ( thick black line ) . Grey area delimited by thin black lines envelops traces from scr animals ( 95 % confidence interval ) ; blue area envelop traces from amiR - 29a animals ( 95 % confidence interval ) . e ) Same as in d ) for ipsilateral eye ( ipsi ) pre - and post - MD responses . f ) The effects of MD on Npas IV and Bdnf exIV expression is reported as the ratio between expression levels of deprived and nondeprived cortex for animals treated with scrambled ( scr + MD ) and amiR - 29a ( amiR - 29a + MD ) . Each symbol represent the result of one animal . Error bars represent ± SEM . scr vs amiR - 29a ; t - test ; N = 7 - 9 , Npas4 : p = 0 . 035 ; N = 4 - 5 ; Bdnf exIV : p = 0 . 026 . g ) Scatter plot correlating the changes in protein abundance induced by MD in young mice ( MD CP , x - axis ) with the effects observed in the adult deprived mice treated with amiR - 29a ( MD + LNA _ AD , y - axis ) . Proteins present in both data sets and showing an absolute expression fold change ( FC > 0 . 1 ) are represented . Green symbols correspond to proteins with concordant direction of regulation ; red symbols correspond to discordant regulation . Pearson correlation : r = 0 . 19 , p = 0 . 019 . The number of genes in each quadrant is reported in figure .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5a) VEP analysis. Ratio between contralateral and ipsilateral eye responses (C/I ratio ± SEM) in animals treated with saline and not deprived (sal no MD), with scrambled LNA and MD (scr MD), with amiR-29a LNA and MD (amiR-29a MD). N=6/7; one-way ANOVA, p=0.0003; post hoc Tukey's test: sal noMD vs scr+MD, ns; sal noMD vs amiR-29a+MD, p=0.0002; scr+MD vs amiR-29a+MD, p=0.0169.b) IOS analysis. ODI ± SEM of deprived animals treated with scr (N=5, black lines) and amiR-29a LNA (N=6, blue lines). Dashed lines connect pre- and post-MD ODI values of each animal. Solid lines represent the group average. Two-way ANOVA RM, treatment X time, p= 0.0074; post hoc Sidak's test, pre-MD scr vs post-MD scr, ns; pre-MD vs post-MD amiR-29a, p=0.0005.c) IOS amplitude (± SEM) in response to contralateral eye (contra) and ipsilateral eye (ipsi) stimulation. Other conventions as in b). amiR-29a (N=6) vs scr (N=5). Contra eye: two-way ANOVA RM, time X treatment interaction p=0.034; post hoc Sidak's test; pre-MD vs post-MD scr, ns; pre-MD vs post-MD amiR-29a, p=0.0008. Ipsi eye: two-way ANOVA RM, time X treatment interaction, ns.d) Average time course of the IOS response to visual stimulation of the contralateral eye (contra) pre- and post-MD (thick black line). Grey area delimited by thin black lines envelops traces from scr animals (95% confidence interval); blue area envelop traces from amiR-29a animals (95% confidence interval). e) Same as in d) for ipsilateral eye (ipsi) pre- and post-MD responses. f) The effects of MD on Npas IV and Bdnf exIV expression is reported as the ratio between expression levels of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and amiR-29a (amiR-29a+MD). Each symbol represent the result of one animal. Error bars represent ±SEM. scr vs amiR-29a; t-test; N=7-9, Npas4: p=0.035; N=4-5; Bdnf exIV: p=0.026.g) Scatter plot correlating the changes in protein abundance induced by MD in young mice (MD CP, x-axis) with the effects observed in the adult deprived mice treated with amiR-29a (MD+LNA_AD, y-axis). Proteins present in both data sets and showing an absolute expression fold change (FC>0.1) are represented. Green symbols correspond to proteins with concordant direction of regulation; red symbols correspond to discordant regulation. Pearson correlation: r=0.19, p=0.019. The number of genes in each quadrant is reported in figure.


Figure 6a ) Images of WFA - labeled PNNs of scrambled treated visual cortex ( left ) and miR - 29a mimic ( right ) in young mice . Scale bar 60 µm . ( b , c ) Quantification of PNN density ( scr vs mim29a , N = 3 mice per group ; t - test , p = 0 . 20 ) and intensity for each animal ( scr vs mim29a , N = 3 mice per group ; t - test , p = 0 . 014 ) . d ) Cumulative frequency of PNNs staining intensity in scrambled and mimic treated young mice scr ( N = 1438 cells vs mim29a N = 1749 cells , Kolmogorov - Smirnov test , p < 0 . 0001 ) . e ) PNN distribution in classes of intensity in mice treated with scrambled and mimic 29a . f ) Images of WFA - labeled PNNs of scrambled treated visual cortex ( left ) and amiR - 29a LNA ( right ) in adult mice . Scale bar 60 µm . ( g , h ) Quantification of PNN density ( scr vs amiR - 29a , N = 3 mice per group ; t - test , p = 0 . 03 ) and intensity for each animal scr vs amiR - 29a , N = 3 mice per group ; t - test , p = 0 . 047 . i ) Cumulative frequency of PNNs intensity in scrambled and amiR - 29a treated adult mice . scr N = 1883 cells vs amiR - 29a N = 1937 cells , Kolmogorov - Smirnov test , p < 0 . 0001 . j ) PNN distribution in classes of intensity in mice treated with scrambled and amiR - 29a . k ) Biochemical quantification of C4S in adult amiR - 29a treated mice ( N = 5 ) as compared to scrambled animals ( N = 4 ) . t test , p = 0 . 0008 . l ) Example of Arylsulfatase B ( ArsB ) western blot . Left : The lanes corresponding to the treated ( treat scr or treat amiR - 29a ) and untreated ( untreat scr or untreat amiR - 29a ) cortex ( ctx ) of two amiR - 29a treated and two scr treated mice are reported . Right : quantification of the data shows ArsB upregulation by amiR - 29a . The results are expressed as fold change normalized to untreated cortex . Each symbol represents the result of one animal . N = 4 , scr vs amiR - 29a treated cortex , unpaired t test , p = 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6a) Images of WFA-labeled PNNs of scrambled treated visual cortex (left) and miR-29a mimic (right) in young mice. Scale bar 60 µm. (b,c) Quantification of PNN density (scr vs mim29a, N=3 mice per group; t-test, p=0.20) and intensity for each animal (scr vs mim29a, N=3 mice per group; t-test, p=0.014). d) Cumulative frequency of PNNs staining intensity in scrambled and mimic treated young mice scr (N=1438 cells vs mim29a N=1749 cells, Kolmogorov-Smirnov test, p<0.0001). e) PNN distribution in classes of intensity in mice treated with scrambled and mimic 29a. f) Images of WFA-labeled PNNs of scrambled treated visual cortex (left) and amiR-29a LNA (right) in adult mice. Scale bar 60 µm. (g,h) Quantification of PNN density (scr vs amiR-29a, N=3 mice per group; t-test, p=0.03) and intensity for each animal scr vs amiR-29a, N=3 mice per group; t-test, p=0.047. i) Cumulative frequency of PNNs intensity in scrambled and amiR-29a treated adult mice. scr N=1883 cells vs amiR-29a N=1937 cells, Kolmogorov-Smirnov test, p< 0.0001. j) PNN distribution in classes of intensity in mice treated with scrambled and amiR-29a. k) Biochemical quantification of C4S in adult amiR-29a treated mice (N=5) as compared to scrambled animals (N=4). t test, p=0.0008.l) Example of Arylsulfatase B (ArsB) western blot. Left: The lanes corresponding to the treated (treat scr or treat amiR-29a) and untreated (untreat scr or untreat amiR-29a) cortex (ctx) of two amiR-29a treated and two scr treated mice are reported. Right: quantification of the data shows ArsB upregulation by amiR-29a. The results are expressed as fold change normalized to untreated cortex. Each symbol represents the result of one animal. N=4, scr vs amiR-29a treated cortex, unpaired t test, p=0.043