EMBO/soda-nel huggingface dataset:
MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex (2020)
Figure 4

Figure 4: Transcriptomic and proteomic effects of miR-29a downregulation.

a) Experimental design for amiR-29a treatment in adult mice.

b) 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).

g) DAVID functional annotation clustering analysis showing the most significantly overrepresented GO terms of miR29a targets downregulated with age and upregulated by amiR-29a treatment.

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.

j) DAVID functional annotation clustering analysis showing the most overrepresented GO terms among genes showing consistent regulation at the transcript and protein level.
MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex (2020)
Figure 5

Figure 5: miR-29a downregulation promotes experience-dependent plasticity in adult mice with similar physiological and proteomics features of juvenile mice.

a) 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.

h,i) Hierarchical dendrograms of synaptic proteins (SynGO database) showing significant enrichment by colour-code: h) proteins with concordant regulation. i) proteins with discordant regulation.

Data information: Asterisks report statistical significance, *p<0.05, ***p<0.001.
MiR-29 coordinates age-dependent plasticity brakes in the adult visual cortex (2020)
Figure 6

Figure 6: Manipulations of miR-29a levels promote changes in PNNs

a) 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

Data information: Error bars represent ±SEM. Asterisk statistical significance, *p<.05 **p<0.001.

EXPANDED VIEW FIGURE LEGENDS
SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs (2020)
Figure 3

Figure 3: SARS-CoV-2 nucleocapsid protein undergoes LLPS at physiological conditions. A) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at B) pH 7.4 or C) pH 5.5, DIC micrographs of 50 μM MBP-N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.
SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs (2020)
Figure 5

Figure 5: The N-terminal and linker intrinsically disordered domains and the C-terminal dimerization domain are essential for robust N LLPS in vitro. A) DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7.4 without and with TEV protease (to cleave MBP from N) and without or with 0.5 mg/mL desalted total torula yeast RNA. Scale bars represent 80 μm. Phase separation over time as monitored by turbidity of 50 μM full-length N or deletion variants after addition of TEV protease in the absence (B) or presence (C) of 0.5 mg/ml desalted total torula yeast RNA. Error bars represent standard deviation of three replicates.
SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs (2020)
Figure 7

Figure 7: SARS-CoV-2 N partitions into liquid condensates of hnRNPs. A) N partitions into hnRNPA2 LC droplets even with the MBP tag attached. B) N partitions into TDP-43 CTD droplets even with the MBP tag attached. C) N partitions into FUS LC droplets only after cleavage of the MBP tag. D-F) N partitions into hnRNPA2 FL (D), TDP-43 FL (E), FUS FL (F) droplets. Source data for panels D, E, F with N and TEV are available online for this figure.
AP-2 reduces amyloidogenesis by promoting BACE1 trafficking and degradation in neurons (2020)
Figure 2

Figure 2 | AP-2 is required for lysosomal targeting of BACE1.

(A, B) BACE1 protein levels in cortical lysates of WT and AP-2µ KO mice. Protein levels in the KO were normalized to the WT set to 100% (KO: 447.64±48.78%, p<0.000, N=7 biological replicates).

(C, D) Immunofluorescence levels of BACE1 are significantly increased in the cortex of AP-2µ KO mice when normalized to the WT set to 100% (KO: 162.714±11.082%, p=0.005, N=4, biological replicates). Scale bar: 100µm.

(E) Localization of BACE1 in cultured WT and AP-2µ KO neurons. Scale bars: 20 µm, 5 µm (inserts).

(F) Line plot of BACE1 immunofluorescence along the neurites indicated in (E).

(G, H) Percentage of BACE1 puncta colocalizing with the LC3b (WT: 21.75±2.88, KO: 30.28±2.97, p=0.043, WT=41, KO=45 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within LC3b-positive organelles.

(I, J) Percentage of BACE1 puncta colocalizing with the eGFP-RAB7 (WT:19.54±1.91, KO: 37.09±3.13%, p<0.000, WT=44 and KO=43 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within RAB7-positive organelles.

(K, L) Colocalization of BACE1 and RAB7A in cortical sections of WT and AP-2μ ΚΟ brains, quantified using Pearson's colocalization coefficient (WT: 0.18±0.04, KO: 0.35±0.05, p=0.038, N=4 biological replicates). Scale bar: 10 µm.

(M, N) Representative fluorescence images of WT and AP-2µ KO neurons transfected with HA-BACE1-mCherry and immunostained for Cathepsin D. Channels were false color-coded to better illustrate the colocalization. Scale bars: 10µm. Arrowheads indicate BACE1 accumulation in lysosomes in the WT, and its absence in the KO. Dotted line outlines the neuron.

(O) Lysosomal localization of BACE1 is decreased in KO neurons (WT: 73.05±3.80, KO: 57.68±4.28, p=0.008, 45 WT and 44 KO neurons, N=4 biological replicates).

(P) Schematic illustration of BACE1 delivery to lysosomes monitored via BACE1-mKeima-Red.

(Q, R) Representative fluorescence images of WT and AP-2μ KO neurons transfected with BACE1-mKeima-Red and co-transfected with eBFP to reveal the neuronal morphology. Scale bars: 10µm, 2µm in zoomed images.

(S) The 550nm/438nm signal intensity ratio of BACE1-mKeima-Red quantified over the entire cell is significantly reduced in the KO condition compared to the WT (WT: 0.59±0.04, KO: 0.48±0.04, p=0.041, 28 WT and 25 KO neurons, N=3 biological replicates).

Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test in (H,J,L,O,S) and one-sample Student's t‐test in (B,D). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.
AP-2 reduces amyloidogenesis by promoting BACE1 trafficking and degradation in neurons (2020)
Figure 4

Figure 4 | AP-2 prevents amyloidogenic processing of APP in neurons.

(A, B) The eGFP/mCherry signal intensity ratio is significantly increased in mCherry-APP-P1-eGFP-expressing KO neurons comparing to the WT (WT: 0.94±0.06%, KO: 1.65±0.18, p=0.000, WT=39 and KO=41 neurons, N=5 biological replicates). Scale bars: 2μm.

(C, D) Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 100 µm (C), 20µm (D).

(E-G) Levels of APP-CTFs are significantly increased in AP-2µ KO cortex compared to the WT set to 100% (KOC99: 156.65±8.37%, p=0.004, N=4 biological replicates; KOC83: 154.79±15.6%, p=0.012; N=5 biological replicates).

(H) Levels of full-length (FL) APP are unaltered in AP-2µ KO cortex (KOAPP FL: 106.64±12.96%, p=0.315, N=6 biological replicates).

(I) Levels of Nicastrin (NCT) are unaltered in the AP-2µ KO cortex (see also Figure EV4K).

(J, K) Intracellular levels of Aβ1-40 are significantly increased in AP-2µ KO axons compared to the WT (WTAβ1-40: 1.82±0.12, KOAβ1-40: 2.45±0.12, p=0.000, WT=24 and KO=24 neurons, N=3 biological replicates). Scale bars, 5µm.

(L, M) Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm.

(N) Significantly increased ratio of Αβ1-42/ Αβ1-40 measured by ELISA in the media of cultured AP-2μ KO neurons compared to the WT set to 100% (KO:124.57±10.97%, p=0.037, N=6 biological replicates).

(O-Q) Accumulation of intracellular Aβ1−42 in neurons overexpressing the AP2-α-Mut comparing to neurons expressing the AP2-α−WT (WT: 1.14±0.18, Mut: 2.06±0.29, p=0.011, WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts).

(R-T) Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down in AP-2µ KO neurons (WTScr: 2.76±0.20, KOScr: 3.94±0.21; WTshBACE1: 2.52±0.12; KOshBACE1: 2.74±0.21; pWTScr vs pKOScr=0.000, pKOscr vs pKOshBACE1=000; pWTscr vs pWTshBACE1=0.826; pWTshBACE1 vs pKOshBACE1=0.863; 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm.

(U, V) Levels of intracellular Aβ1−42 are significantly upregulated in neurons overexpressing the HA-BACE1-LL/AA-GFP comparing to neurons expressing the HA-BACE1-GFP (BACE1: 0.78±0.90, BACE1-LL-AA: 1.99±0.33, p=000, 28-29 neurons per condition, N=3 biological replicates). Scale bar, 50µm.

Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test in (B,K,M,N,Q,V), two-way ANOVA in (T) and by one-sample Student's t‐test in (F-H). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.
AP-2 reduces amyloidogenesis by promoting BACE1 trafficking and degradation in neurons (2020)
Figure 5

Figure 5 | Synaptic dysfunctions in AP-2µ KO mice are mitigated by BACE1 haploinsufficiency.

(A, B) AP-2α levels normalized to GAPDH are significantly decreased in lysates of iPSC-derived neurons from late-onset AD patients carrying the TREM2 p.R47H (AD-TREM2-2 and AD-TREM2-4) variant compared to healthy controls (CON8 and CON9) set to 100% (ADAP-2α: 52.42±8.04%, p=0.020, N=3 biological replicates).

(C) Confocal images of either AP-2 WT (AAV-GFPCamkIIα-labelled) or AP-2 KO (AAV-Cre-GFPCamkIIα-labelled) granule neurons in the dentate gyrus of 13-week-old mice. Scale bars: 20µm, 2µm (inserts).

(D) Loss of AP-2µ significantly decreases the number of spines on granule neurons (WT: 12.63±0.88, KO: 8.07±0.45, p=0.004, N=4 biological replicates).

(E, F) Overlap coefficient between the presynaptic marker Bassoon and the postsynaptic marker PSD95 is significantly decreased in the entorhinal cortex of AP-2µ KO mice compared to the WT (WT: 0.45±0.02, KO: 0.33±0.06, p=0.034, N=3 biological replicates). Scale bar, 750nm.

(G, H) Lost preference for the novel object (nov) comparing to the familiar object (fam) in AP-2µ KO mice tested 24h after training in NOR (WTfam: 27.51±6.77, WTnov: 72.49±6.77, pWTfam vs WTnov=0.000; KOfam: 43.9±13.12, KOnov: 59.91±13.12, pKOfam vs KOnov=0.469, N=7 WT and 8 KO mice). Representative heatmaps are shown in (G).

(I, J) AP-2µ KO mice are significantly more active in the open field compared to the WT (WT: 1663±139.08%, KO: 3976±802.30%, p=0.020, N=7 WT and 8 KO mice). Representative heatmaps are shown in (I).

(K, L) Overlap coefficient between Bassoon and PSD95 is rescued upon BACE1 KD in AP-2µ KO neurons (WTscr: 0.39±0.02; KOscr: 0.24±0.01; WTshBACE1: 0.37±0.02; KOshBACE1: 0.33±0.02; pWTscr vs pKOscr<0.000; pKOscr vs pKOshBACE1=0.010; pWTscr vs pWTshBACE1=0.813; pWTshBACE1 vs pKOshBACE1=0.623, 22-24 neurons for each condition, N=3). Scale bars: 10µm.

(M-O) Neuronal loss is rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 (AP-2WT/BACEWT: 2.25±0.08, AP-2KO/BACEWT: 1.51±0.08, AP-2WT/BACEHET: 1.80±0.07, AP-2KO/BACEHET: 2.00±0.07; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT<0.000; pAP-2WT/BACE1WT vs pAP-2WT/BACE1HET=0.001; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.000; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.268, N=9 sections from N=3 for each genotype). Scale bar: 100 μm.

(P, Q) APP-CTF levels are rescued in the entorhinal cortex of AP-2µ KO mice haploinsufficient for BACE1 (AP-2WT/BACEWT:427574±24021, AP-2KO/BACEWT: 538895±34662, AP-2WT/BACEHET: 389870±19842, AP-2KO/BACEHET: 395389±21853; pAP-2WT/BACE1WT vs pAP-2KO/BACE1WT=0.017; pAP-2KO/BACE1WT vs pAP-2KO/BACE1HET=0.001; p AP-2WT/BACE1HET vs AP-2KO/BACE1HET=0.979; 13-15 sections, N=3 mice for each genotype). Scale bars: 40µm.

Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test in (B), unpaired two-tailed Student's t‐test in (D,F,H,J) and two-way ANOVA in (L,O,Q). n.s.-non-significant.* indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.
Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity (2021)
Figure 2

Figure 2. The CCT complex interacts with APOBEC3A in multiple cell types

(A) A3A co-precipitates with CCT complex subunits. U2OS and HepaRG cells with dox-inducible A3A-HA transgenes were treated with dox prior to immunoprecipitation of HA. HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex.

(B) Immunoprecipitation of CCT. IP of the CCT complex using antibodies to endogenous CCT subunits was performed in lysates from U2OS-A3A (top) and HepaRG-A3A (bottom) cells after dox treatment. Immunoblot analysis with probes for A3A-HA and indicated subunits of CCT with IgG as a control is shown.

(C) A3A interacts with CCT in leukemia cells. HA immunoprecipitation in hematopoietic cell lines with inducible A3A-HA was analyzed by immunoblot. HA, CCT1, and CCT5 antibodies were used to evaluate co-precipitation of CCT subunits with A3A.

(D) The CCT-A3A interaction occurs in breast cancer cells. HA (top) and CCT (bottom) immunoprecipitation in the MDA-MB-231 cell line with inducible A3A-HA expression was analyzed by immunoblot. Anti-HA and anti-CCT1 antibodies were used to evaluate reciprocal co-IPs.
Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity (2021)
Figure 3

Figure 3. The APOBEC3-CCT interaction is specific to A3A

(A) CCT co-precipitates with A3A but not A3B or A3G. 293T cells were transfected with empty vector (V), A3A-HA, A3B-HA, or A3G-HA. CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA.

(B) A3A, but not other APOBEC3 enzymes, interacts with CCT complex subunits. After transfection of 293T cells with HA-tagged APOBEC3 expression plasmids, IP was performed with anti-HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits. Arrows mark location of each APOBEC3 protein on HA immunoblot.

(C) HA immunoprecipitation was performed from U2OS cells with dox-inducible A3A, A3A-C106S (catalytically inactive mutant), and A3B transgenes. IP lysates were evaluated by immunoblot using antibodies to HA and CCT1.

(D) Immunoprecipitation of HA-tagged A3B constructs transfected into 293T cells. IP of A3B-HA and A3B-E24R-HA (cytoplasmic localization) was evaluated by immunoblot analysis using antibodies to A3B and CCT1. Arrow indicates CCT5 band.

(E) CCT interacts with A3A both with and without interferon. Immunoprecipitation of A3A-HA in K562 cells treated with and without type I interferon (IFN). Anti-ISG15 antibody serves as a control for IFN induction.

(F) CCT co-precipitates with endogenous A3A. Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1. Chemiluminescent exposure times described as short and long are indicated beside the blot. IgG antibody was used as a control throughout.