EMBO/soda-nel huggingface dataset:
Paraspeckles are constructed as block copolymer micelles (2021)
Figure 3

Figure 3. Deletion of both the 5′ and 3′ terminal domains of NEAT1_2 causes random distribution of both the 5′ and 3′ ends of NEAT1_2 within the paraspeckle.

A The schematics of the WT and the mutant (∆5′/∆3′) with deletion of the 5′ and 3′ terminal regions are shown as Figure 1A. The positions of the NEAT1 probes used in SRM (blue) and EM (orange) are shown.

B SRM images of the paraspeckles in MG132-treated (5 μM for 6 h) HAP1 NEAT1 ∆5′/∆3′ cells detected by NEAT1_2k (green) and NEAT1_15k FISH probes (left, magenta) or NONO immunofluorescence (IF) (right, magenta). Scale bar, 500 nm.

C Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ (upper; WT: n = 41, ∆5′/∆3′ kb: n = 44) and 3′ (lower: WT, n = 167, ∆5′/∆3′ kb: n = 34) ends to the core and shell or the shell in WT and ∆5′/∆3′ kb cells treated with MG132 (5 μM for 6 h).

D (upper) EM observation of the paraspeckles in MG132-treated (5 μM for 17 h) ∆5′/∆3′ cells using NEAT1_5′ (left) and NEAT1_D2 probes (right). Scale bar, 100 nm. (middle) Graph showing the proportion of the localization of NEAT1_5′ (left, 310 gold particles) and NEAT1_D2 (right, 294 gold particles) probes within the paraspeckles in HAP1 NEAT1 ∆5′/∆3′ cells. (lower) Graph showing the proportion of the localization of NEAT1_5′ (left, 19 cells) and D2 (right, 22 cells) probes in each paraspeckle in ∆5′/∆3′ cells. Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).

E Schematics of the NEAT1_2 configuration in the WT and ∆5′/∆3′ mutant.
Paraspeckles are constructed as block copolymer micelles (2021)
Figure 6

Figure 6. The transcription level of NEAT1_2 is a key determinant for the size and organization of paraspeckles.

A Theoretical calculation of the rescaled radius of paraspeckles versus the rescaled transcription rate for the case in which the A and C blocks were deleted by CRISPR/Cas9 in the steady state. The graph is derived by assuming that NEAT1_2 is produced at a constant rate. The produced NEAT1_2 RNPs diffuse in the solution and the free diffusion is hindered by the attractive interactions between NEAT1_2 RNPs with the interaction parameter χ (we used χ = 1.0). NEAT1_2 was degraded at the constant rate k0. The radius was rescaled by the length scale $\sqrt{D/k_{0}}$ and the transcription rate was rescaled by the inverse time scale k0(D/k0)3/2/v0.

B Quantitation of NEAT1_2 by RT-qPCR in WT and ∆5′/∆3′/∆PAS cells with or without MG132 treatment (6 h). Data are represented as mean ± SD (n = 3).

C The paraspeckles in WT and ∆5′/∆3′/∆PAS cells detected by SRM using NEAT1_5′ and 2k FISH probes (green), and NONO IF (magenta) under the conditions shown in (B). Scale bar, 500 nm.

D Quantitation of the diameter (Sx) observed by SRM in WT and ∆5′/∆3′/∆PAS cells under the conditions shown in (B). WT [mean size: MG132 (-), 354.2 nm (n = 32); MG132 0.25 μM, 380.5 nm (n = 68); MG132 1 μM, 394.1 nm (n = 108); MG132 5 μM, 409.6 nm (n = 103)], ∆5′/∆3′/∆PAS [mean size: MG132 (-), 344.4 nm (n = 18); MG132 0.25 μM, 472.1 nm (n = 27); MG132 1 μM, 601.4 nm (n = 27); MG132 5 μM, 721.2 nm (n = 26)]. (*: P = 0.029, ****: P < 0.0001, compared with WT: Kruskal-Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).

E Theoretical calculation of the fraction of A (or C) blocks in the shell vs the logarithm of the transcription rate (rescaled by the rate at which the transcripts are spontaneously incorporated in the paraspeckle). The numbers of segments of A blocks were 5.0 (cyan), 7.5 (orange), 10.0 (green), and 12.5228 (black). The parameters used for the calculations are shown in the Materials and Methods.

F Quantitation of NEAT1_2 by RT-qPCR in ∆0-0.8 kb cells with or without MG132 treatment (5 μM for 6 h). Data are represented as mean ± SD (n = 3).

G The paraspeckles detected by SRM with NEAT1_5′, 1k, and 2k (green), and NEAT1_3′ (magenta) FISH probes in the ∆0-0.8 kb cells under the conditions shown in (H). Scale bar, 500 nm.

H Graph showing proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in ∆0-0.8 kb cells under the conditions shown in (F) [MG132 (-): n = 78, MG132 (+): n = 127].
4'-Phosphopantetheine corrects CoA, iron and dopamine metabolic defects in mammalian models of PKAN (2019)
Figure 1

Figure 1 Isolating disease-vulnerable brain tissue from disease-protected reveals CoA pathway defects in Pank2 KO animals. (A) The CoA synthesis pathway, including intermediates and enzymes (boxed). (B) Brain dissection yielding three study regions (red dotted lines) including GP, SN, and cerebellum. Captured and modified from Brain Atlas 2 (Allen Institute for Brain Science) (C) Relative quantification of mRNA for Ppcs, Ppcdc, and Coasy by genotype from each of the three study regions. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) (D) Western blot and quantification for Coasy from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as a loading control. n=8, 4 for WT and n=8, 6 for KO (vehicle and pPanSH groups, respectively). Data shown here were evaluated by either one or two-way ANOVA depending on the number of variables. **;P<0.01, ***;P<0.001. All graphs represent mean ±s.e.m.
4'-Phosphopantetheine corrects CoA, iron and dopamine metabolic defects in mammalian models of PKAN (2019)
Figure 2

Figure 2 Regional brain differences in iron homeostasis suggest a mechanism for iron overload in PKAN. (A) Relative mRNA expression of Tfrc, Ireb2, and Hamp by genotype and brain region. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) (B) Total iron quantity in cytosol and mitochondria from GP using ICP-MS. The data distribution is presented using a box plot, including minimum, first quartile, median, third quartile, and maximum. The magenta-colored line represent mean. n=6 for both genotypes (C) Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase (D) and complex I (E) in GP by genotype and treatment status (veh = vehicle; pPanSH = 4'-phosphopantetheine). n=5 for both genotypes and treatment groups. (F) Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control. n=6, 4 for WT and n=6, 6 for KO (vehicle and pPanSH groups, respectively). Data were evaluated by one or two-way ANOVA depending on the number of variables. *P<0.05, **P<0.01, ***P<0.001. All graphs except B represent mean ±s.e.m.
4'-Phosphopantetheine corrects CoA, iron and dopamine metabolic defects in mammalian models of PKAN (2019)
Figure 3

Figure 3 Dopamine metabolism is impaired in Pank2 KO GP. (A) Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype, brain region and treatment status (pPanSH = 4'-phosphopantetheine). n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) QRT-PCR. n=5 for Western blot for all groups (B-C) Relative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region. n= same as 3A. (D) Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype. n=5 for WT and n=3 for KO. Statistical significance was determined using ANOVA. *P<0.05, **P<0.01, ***P<0.001. All graphs represent mean ±s.e.m.
4'-Phosphopantetheine corrects CoA, iron and dopamine metabolic defects in mammalian models of PKAN (2019)
Figure 4

Figure 4 Human cells with PANK2 mutations show perturbations in the CoA synthesis pathway and diminished mitochondrial respiration. (A) Relative quantification of COASY mRNA in human PKAN primary cell lines. n= 5 for both genotypes and cell types (B) Relative quantification of COASY/Coasy in fresh, blood-derived lymphocytes from human and mouse compared by genotype. n=51,35 for human (control, PKAN, respectively) and n=5 for both mouse genotypes. (C) Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; Oligomycin, FCCP; Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy-glucose. Quantification of baseline OCR and ECAR is shown, as well. n=3 per genotype with four technical replicates. (D) Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n=4 for both genotypes and treatment groups. (E) Relative mRNA expression of mitochondrially-encoded genes important for oxidative phosphorylation from complex I (MT-ND1), complex III (MT-CYB), cytochrome C (MT-CO1), and ATP synthase (MT-ATP6) in human mutant fibroblasts versus control cells. n=3 for both genotypes. (F) Calculation of active mitochondria using the JC-1 assay by genotype in control and PKAN lymphoblasts. n=2. Data were analyzed using one-way or two-way ANOVA for statistical significance. *P<0.05; **P<0.01, ***P<0.001. All graphs represent mean ±s.e.m.
4'-Phosphopantetheine corrects CoA, iron and dopamine metabolic defects in mammalian models of PKAN (2019)
Figure 5

Figure 5 Pank1 expression differences and recovery attempt using PPARα agonists. (A-B) Relative expression of Pank1α, Pank1β, total Pank1 and Pank3 in mouse brain regions. n=8,8,4 for both genotypes (GP, SN, Cerebellum, respectively). (C) Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate (0.8 mg/g body weight) or gemfibrozil (1.2 mg/g body weight). n=8 for both genotypes treated with vehicle, n=5 for both KO groups treated with either bezafibrate or gemfibrozil. Data were evaluated by one-way or two-way ANOVA depending on the number of variables. *P<0.05, **P<0.01, ***P<0.001. All graphs represent mean ±s.e.m.
4'-Phosphopantetheine corrects CoA, iron and dopamine metabolic defects in mammalian models of PKAN (2019)
Figure 6

Figure 6 4'-phosphopantetheine corrects markers of CoA, iron, and dopamine dyshomeostasis and mitochondrial dysfunction in Pank2 KO mice. (A) Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds: 4'-phosphopantetheine (pPanSH), pantetheine, vitamin B5, and CoA. n=8 (WT-veh), 5 (KO-veh), 5 (KO-pPanSH), 4 (KO-all other intermediates). (B) Correction of Coasy, Tfrc, Ireb2, and Drd1 expression in GP by 4'-phosphopantetheine over a range of doses. n=8 (WT-veh), 5 (KO-veh), 5 (KO-0.82), 4 (WT-5), 5 (KO-5), 5 (KO-8.2), 5 (KO-10). 5 (WT-20), 5 (KO-20) (C) Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH. n=3 for both genotypes. (D) ECAR measured in cultured human fibroblasts by genotype and treatment status. Cells were treated for three days with 4'-phosphopantetheine (pPanSH) or vehicle (veh). n=3 for both genotypes. Data were evaluated by one-way or two-way ANOVA depending on the number of variables. Asterisks shown in B-D represent statistical significance compared to the vehicle-treated control groups. *P<0.05, **P<0.01, ***P<0.001. All graphs represent mean ±s.e.m.
4'-Phosphopantetheine corrects CoA, iron and dopamine metabolic defects in mammalian models of PKAN (2019)
Figure 7

Figure 7 Withdrawal of 4'-phosphopantetheine causes brain biomarkers to return to pre-treatment levels. (A) Relative quantification of Coasy, Tfrc and Drd1 mRNA from GP by number of days post-cessation of treatment with 5μg of 4'-phosphopantetheine per g of body weight. n= 11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-pPanSH-D7), 4 (KO-pPanSH-D7), Data shown were evaluated by one-way ANOVA. Asterisks represent statistical analyses compared to the WT vehicle group. *P<0.05, **P<0.01, ***P<0.001. All graphs represent mean ±s.e.m.
NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients (2020)
Figure 1

Figure 1. Cortical malformations in BBSOAS patients with NR2F1 haploinsufficiency.

(A-F) Cortical malformations observed in six novel BBSOAS patients (See Expanded View Figure EV1A-F for additional MRI panel and Table 1 for clinical features). Patient (P)1 shows abnormally elongated occipital convolutions (arrowheads in A), whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6; B-F). B', C' and C'' highlight the abnormally convoluted regions boxed in B and C.

(G) Schematic representation of the human NR2F1 protein sequence, showing novel variants of a new BBSOAS patient cohort. Key aminoacids for the functioning of the DNA-binding domain (DBD) or the ligand-binding domain (LBD) are listed in boxes.

(H,H') TBR2 (green in H), SOX2 (red in H) and NR2F1 (yellow in H') immunofluorescence (IF) of a GW11 section of human neocortex (see also Figures EV1G-G''').

(I) NR2F1 expression (quantified by pixel intensity) at different L-M levels (see also Figures EV1G-G''') and in different regions along the A-P extent of the cortex, as indicated. n ≥ 3 sections from n = 1 fetal brain.

(J-L') NR2F1 (red) IF of human GW14 neocortex, showing expression levels in the CP (K,L) and VZ (K',L') around primary convolutions in the posterior-most cortex (see also Figure EV1H). High NR2F1 is detected in the neurogenic area of a gyrus (L').

(M,M') NR2F1 level as measured on single cells in the VZ (M) and CP (M') of sulci and gyri, as in (J-L'). n ≥ 4 sections from n = 1 fetal brain.

(N-O) NR2F1 (red) and TBR2 (green) immunostaining of the same regions shown in (J-L'). NR2F1 level as measured in single TBR2+ IPs by pixel intensity is shown in graph (O). n ≥ 4 convolutions from n = 1 fetal brain (n = 2 sections).

(P-R) NR2F1 (red) and HOPX (green) immunostaining of the same regions shown in (J-L') (see also Figure EV1H). Virtually all HOPX+ bRGs are also NR2F1+ (magnification in P'') and their number is greatly increased in gyri (P'') compared to sulci (P'), as quantified in (R). At a single cell level, NR2F1 pixel intensity is higher in oSVZ HOPX+ cells in gyri compared to sulci (Q). n ≥ 4 sections from n = 1 fetal brain.

Data information: Nuclei (blue) were stained with DAPI. In (I, R) the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. 2-way ANOVA (I) and Student t-test (M,M',O,Q,R) (*P<0.05, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.
NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients (2020)
Figure 2

Figure 2. Nr2f1 regulates neural progenitor self-renewal in a neurosphere assay.

(A) Representative images of wild-type (WT) and mutant (KO) neurospheres obtained from E15.5 neocortices and cultured in vitro. The culture passage ("step") corresponds to 3 days of culture after dissociation and re-plating. KO aggregates are more numerous and larger in size.

(B) Graph showing the number of neurospheres per P100 cell plate at different steps, as indicated. Nr2f1KO neurospheres proliferate for a longer time (up to 30 passages tested), whereas WT cells exhaust around step 9. n ≥ 3 culture wells from n = 2 batches.

(C) Panel of WT and KO isolated neurospheres grown from day 1 to day 10. Blue and red lines represent the diameter of WT and KO neurospheres, respectively.

(D,E) PH3 (green; dividing cells) IF on cross-sections of day 4 neurospheres. White arrowheads point at mitotic figures, which are quantified in (E) as percentage of dividing cells over DAPI-stained nuclei (blue). n ≥ 3 neurospheres from n = 2 batches.

Data information: In (B,E) data are represented as means ± SEM. 2-way ANOVA (B) and Student t-test (E) (**P<0.01, ***P<0.001). Scale bars: 50µm.
NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients (2020)
Figure 3

Figure 3. Nr2f1-mediated control of progenitor amplification in vivo.

(A-D) Ki67 (green; mitotically active progenitors) and Tuj1 (red; post-mitotic neurons) IF at E14.5 (A,A'), E16.5 (B,B') and E18.5 (C,C') of lateral pallia of wild-type (WT) versus mutant (KO) brains. Ki67+ proliferating cells (quantified in D) accumulate in mutant cortices. n ≥ 3 brains.

(E-F') Tbr2 (green; IPs) and Pax6 (red; radial glia cells) IF of E13.5 (E,E') and E17.5 (F,F') lateral pallia of WT and KO brains. See Expanded View Figure EV3 for Tbr2 Pax6 double staining in E17.5 HET embryos.

(G-I) Quantification of different classes of cortical progenitors: apical radial glia cells (aRGs) as Pax6+ NPs in VZ (G), basal intermediate progenitors (IPs) as Tbr2+ cells in SVZ (H), basal RGs as Pax6+ cells in outer SVZ (I). See Expanded View Figure EV3 for quantification of distinct progenitor classes in E17.5 HET embryos. n ≥ 3 brains.

(J-L) E12.5 dorsal brain views (J,J') and representative sections of posterior hemispheres (K,K'), showing a vesicle enlargement in mutant brains (K'). The extension of the ventricular surface was quantified along the A-P axis (L); red color code represents Nr2f1 gradient in WT brains. 12µm-thick sections were collected on series of 10 slides; consecutive measurement levels are 120µm apart from each other. n ≥ 3 brains.

(M) Schematic representation of different aRG cell divisions, as visualized in vivo after co-in utero electroporation (IUE) of Tis21-RFP and Sox2-GFP plasmids into E12.5-old embryos. Green: symmetric proliferative divisions; Yellow: asymmetric differentiative divisions; Red: symmetric differentiative divisions.

(N-P) GFP (green; expressed under Sox2 promoter) and RFP (red; expressed under Tis21 promoter) IF of E13.5 lateral pallia, 18-hours after IUE. The proportion of single or double positive NPs is shown in pie charts in (P). n = 3 electroporated brains.

(Q-R') Ki67 (green; progenitors) and EdU (red) IF of E13.5 WT (Q,Q') and KO (R,R') embryos, injected with EdU at E12.5. Differentiating cells (EdU+Ki67-) are located in the IZ/CP; percentages shown in (Q',R') were obtained from n ≥ 4 sections of n ≥ 2 brains. See Expanded View Figure EV3 for neural differentiation index of E13.5 HET embryos.

Data information: Nuclei (blue) were stained with DAPI. In (D,G,H,I) the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. In graphs, data are represented as means ± SEM. Student t-test (Q',R'; E13.5, EdU injected at E12.5; WT/KO: *=0.03967) and 2-way ANOVA (D,G,H,I,L,P) (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.
NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients (2020)
Figure 4

Figure 4. Nr2f1 orchestrates cell cycle dynamics in posterior hemispheres.

(A-D) Graphs showing the average number of PH3+ mitotic figures in the posterior lateral pallium of WT and mutant cortices. The total number of mitoses (A) as well as the number of dividing cells in VZ (Pax6+Sox2+ aRGs; B), SVZ (Tbr2+ IPs; C) and outer (basal-most) region of SVZ (oSVZ/IZ; Pax6+ oRGs; D) are shown. For graphs (B-D), statistical analysis by 2-way ANOVA is shown in Appendix Table S5. n ≥ 3 brains per age/genotype.

(E) Cell cycle duration at different embryonic ages (from E10.5 to E16.5) in WT (blue bars) or KO embryos (orange line), as quantified upon a double EdU/BrdU injection protocol in posterior-most LP (See Figure EV4D-E'' and Methods). n ≥ 3 brains per age/genotype.

(F-G') Ki67 (green; NPs) and EdU (red) IF in WT (F,F') and KO (G,G') after 6 hours consecutive EdU incorporation. Note the higher number of labelled NPs in KO cortices (G') suggesting faster cell cycle progression and/or longer S-phase.

(H) Best linear fit of EdU+Ki67+ NP percentage after 8 hours EdU cumulative labelling in WT (blue line) and KO (orange line) brains. The x- and y-intercept are proportional to the S-phase length and to the number of cycling cells, respectively, while the steepness of the line is proportional to cell cycle duration. (see Methods; WT: y = 11.66x +24.62; KO: y = 13.54x +32.96). n ≥ 2 brains per time point/genotype.

(I-K) PH3 (green; dividing NPs) and Pax6 (red; aRGs) IF of E13.5 WT (I) and KO (J) lateral pallia. Strong and weak/punctate PH3 patterns show M-phase and G2-phase cells, respectively. G2-phase NP percentage is quantified in (K). As a result of shorter G1-phase and globally shorter cell cycle time, the percentage of G2-phase cells is increased in the KO NP pool. n ≥ 2 brains.

(L) Cell cycle time of E13.5 NPs in the lateral pallia of WT (upper column) and KO cells (lower column), as calculated by EdU cumulative labelling and PH3 staining (see Methods). KO NPs have a 16.1% faster cell cycle time, due to a 30.9% shorter G1-phase compensating a 14.3% longer S-phase. n ≥ 3 brains.

Data information: Nuclei (blue) were stained with DAPI. In (A-E,H,K) the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. Student t-test (K; *P<0.05) and 2-way ANOVA (A-E,H,L) (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.