EMBO/SourceData huggingface dataset:
f4(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments.(b) These samples were subjected to immunoprecipitation with the anti-VDAC antibody to evaluate ubiquitination. Representative immunoblots are shown from three independent experiments.(c,d) Representative images of YFP-Parkin overexpressing p53−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm.(e-g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300-400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P0.05, compared with the corresponding control (two-tailed unpaired Student's t-test).
f5(a) Representative electron micrographs of DOX-treated hearts. Arrows indicate abnormal mitochondria defined by marked swelling, vacuolation (loss of electron density) or disrupted cristae; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents abnormal mitochondria (blue line) and an autophagic vacuole containing mitochondria (red line); original magnification × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).(d) The 8-hydroxy-2′-deoxyguanosine (8-OHdG), popular marker for oxidative DNA damage, in mitochondria DNA from DOX-treated hearts. Results are shown from 6-8 hearts analysed in duplicate.(e) Maximum dP/dt was examined using cardiac catheterization under graded dobutamine infusion in PBS-treated (control) and DOX-treated mice (n=6-8 per group). DKO indicates double knockout. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).
f6We used 20-month-old heterozygous littermates surviving grossly tumor free. (a,b) The sequential state III oxygen consumption rate of heart isolated mitochondria (300 μg) was monitored with a fibre-optic system. Substrates and inhibitors were added sequentially at the final concentrations indicated as follow. FCCP, 1 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone; M/P, 5 mM malate+5 mM pyruvate; Rot, 100 nM rotenone; Suc, 5 mM succinate; AA, 50 nM antimycin A; TMPD/Asc, 0.4 mM N,N,N′,N′-tetramethyl-p-phenylenediamine +1 mM ascorbate; KCN, 5 mM potassium cyanide. Representative results are shown (a) and oxygen consumption by each complex was calculated (n=5-8 per group, b).(c,d) Mitochondrial particle size (c) and membrane potential (d) were determined using flow cytometry for TMRE in cardiac mitochondria preparations. Results are shown from 4-8 hearts analysed in duplicate.(e) H2O2 production with 0.5 mM NADH, complex I-linked substrate, was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red (n=5-8 per group).(f) Myocardial high-energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC (n=5 per group).(g) Maximum dP/dt was examined using cardiac catheterization with graded a dobutamine infusion (n=5 per group).(h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section. HT indicates heterozygous; DHT double heterozygous. Data are shown as the means±s.d. *P0.05; **P0.01; ***P0.001 (two-tailed unpaired Student's t-test)
f7(a) Representative electron micrographs of the hearts of 20-month-old aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph shows an autophagic vacuole containing mitochondria; original magnification, × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).(d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e).(d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e).(f) NADH-driven H2O2 production was determined using Amplex Red. Results are shown from five hearts analysed in duplicate.(g) Maximum dP/dt was examined using cardiac catheterization with graded dobutamine infusion in WT and Parkin Tg aged littermates (n=8 per group).(h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section.(i) Real-time PCR assessing the expression of cell cycle inhibitors and proinflammatory cytokines (n=8 per group).(j) Representative immunoblots assessing the expression of cell cycle inhibitors are shown; Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).
Figure 1A-F Brain MRI of the proband at 23 years. (A) Sagittal fast spin echo (FSE) T1-weighted image shows thinning of the corpus callosum, dilation of the IV ventricle and of the cisterna magna and reduced volume of vermis and brainstem; two lacunar lesions are evident in the dorsal pons. (B) T2-weighted axial fluid-attenuated inversion recovery (FLAIR) images show hyperintense periventricular white matter with several lacunar lesions also in the basal ganglia and thalami. (C) MR angiography 3D TOF (time of flight) reconstruction shows two saccular aneurisms in the right M1 (ø 4 mm) and left M2 (ø 2.5 mm) segments of middle cerebral arteries (arrows). (D) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging (DTI) in the right side of the dorsal pons. Severe cavitations and lateral ventricles dilation (left > right) on FLAIR images (B, E) and diffuse microbleeds as small hypointense foci on SWI are visible (F) in the same slices of (E). R = right, L = left.G, H Brain MRI scans of the asymptomatic parents of the proband show multiple focal hyperintensities on T2-weighted images in the periventricular and subcortical cerebral white matter, expression of gliosis secondary to chronic small vessel ischemic changes, more evident in the father (G), 56 years, than in the mother (H), 54 years.
Figure 2Gene expression of NOTCH3, HES1, HEY1, HEYL and the 17 recently identified targets were established by a real-time PCR panel assay in skeletal muscle of controls (n = 6), proband (II:1), parents (mother I:1, father I:2) and CADASIL patients (n = 3). Graph shows gene expression fold changes relative to controls and normalized on GAPDH (reference gene), expressed as mean of two experiments ± SEM. Among the 17 novel assayed target genes, RCAN2, ANGPT4, HP and SORBS were not detected in any sample and were therefore not reported. Green and red lines indicate 2.0 and 0.5 fold changes, respectively. Statistical significances and P-values are reported in Supplementary Table S1.
Figure 3A-C Histopathological examination of skin (A) and skeletal muscle biopsies (B, C) of control (left), proband (middle) and CADASIL patient (right). (A) Collagen IV staining (in green) in skin vessels: Collagen wall appears compact in the control, while it is disorganized in the skin vessels of the proband and of the CADASIL patient. Derangement of the collagen wall in single collagen fibers is more evident in the proband than in the CADASIL patient, where collagen wall is quite compact. Vessel's endothelium is delineated by ULEX staining (in red). Collagen IV staining in skeletal muscle vessels (B) recapitulates the skin picture. Smooth muscle actin immunostaining of skeletal muscle biopsies (C) shows attenuated SMCs in the tunica media of both the proband and the CADASIL patient, with foci characterized by a complete SMCs loss (particularly in the CADASIL patient) and thinning of the vessel wall (arrows).D, E Ultrastructural analysis of skin biopsies. (D) Skin biopsy of the proband: (left panel) At low magnification, the tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal membrane with a multilayered aspect (arrows); SMCs are identified by the presence of "focal adhesions" (square boxes); (middle panel) collagen fibrils appear organized in a parallel pathway along SMC (arrow), note the "focal adhesions" (square box); (right panel) collagen fibrils ("CF") are more represented in dense bundles than elastic ones ("E"). No GOM is detected, in contrast to CADASIL patient (E), showing deposits of granular osmiophilic material (arrows) located between SMC plasmalemma and basal lamina.F KCNA5 immunostaining of skeletal muscle biopsies of control (left), proband (middle) and CADASIL patient (right). A global decrease in reactivity is evident in the vessels of both the proband and the CADASIL patient. Note multiple areas in which KCNA5 is barely detectable (arrows).
Figure 1 (b) 2D-[1H;15N]-TROSY spectrum of 400 µM 2H,13C,15N-labeled TREM2-TMH (residues R161-H206) in DPC micelles at 37 °C. Assigned resonances are labeled. (c) Secondary chemical shifts plotted against the residue number indicates an α-helical region between residues 173 and 198. The used TREM2-TMH construct along with the predicted transmembrane helix content (aa 175-195) is also indicated. (d) Circular dichroism (CD) spectrum of TREM2-TMH in DPC micelles indicates an α-helical secondary structure content of ~50%.  (e) 3D-15N-edited NOESY spectra show that the transmembrane helix is interrupted at residues 190-192. Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α-helical secondary structure.
Figure 2(a, b) Overlay of 2D-[1H,15N]-TROSY spectra of 2H,15N-labeled TREM2-TMH in DPC micelles (black) and in complex with (a) unlabeled DAP12 (red) or (b) TREM2-TMH lacking its positively charged lysine residue (K186A, red).(c) Chemical shift perturbations (CSPs) between the two spectra in panels (a) or (b). The α-helical secondary structure as determined for wild-type TREM2-TMH is indicated above and the position of the K186A mutation is labeled in green. (f) For TREM2-K186A-TMH a ~7% increase in α-helical secondary structure can be detected by CD spectroscopy.  (g) A continuous α-helical secondary structure in the kink region (aa 189-192) was probed by sequential amide NOE contacts in TREM2-K186A-TMH. Red dots indicate the chemical shift of intra-residual (i) amide resonance and red broken lines the inter-residual connectivity (i±1).
Figure 3 (a,b) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild-type TREM2-TMH (a) and the K186A variant (b), indicative of motions in the nanosecond to picosecond time scale. S2 can adopt values between 1 (rigid) and 0 (unrestricted motion). Error bars have been obtained by a Monte-Carlo-simulation-based error estimation and motional model selection procedure implemented in the software Relax
Figure 4 (a) Paramagnetic relaxation enhancement (PRE) with a free-radical-modified stearic fatty acid in DPC micelles. Low values indicate location inside whereas values close to one suggest a location outside the detergent micelle. Orange bars: TREM2-TMH wt, green bars: K186A variant. The N-terminal residues of TREM2-TMH-wt are located deeper inside the micelles as in the K186A case, whereas the C-terminal end is positioned at the outside for wild-type and inside for the K186A variant. Negative bars indicate missing assignments. (b) NOE-cross peaks between backbone amides of TREM2-TMH wt (top) or the K186A variant (bottom) and selected regions of DPC (see color coding of the structure of the DPC molecule on the right). The K186A variant shows strong contacts to the hydrophobic aliphatic regions of the detergent throughout the transmembrane helix whereas no such contacts can be seen at the kink site of the wild-type helix, indicating a location outside of the micelle for this part.
Figure 5 (b) Immunoblot detection of ICD accumulation of TREM2 wildtype, K186A, K186P, and K186L variants mediated by γ-secretase cleavage upon overnight incubation of membrane preparations at 37 °C. ICD formation is blocked by 1 µM of the transition state analogue L-685,458. Note that inhibition of ICD formation from wt TREM2 was less efficient. Due to the charge within the TMH wt TREM2 is less efficiently transported to the cell surface (Lanier et al, 1998) and therefore retained within the endoplasmic reticulum, where other proteases not related to γ-secretase and therefore not inhabitable by L-685,458 may cleave. In vitro generation of the ICD of APP (AICD) was used as a positive control. Immunoblot detection of mature nicastrin and the presenilin 1 NTF, which results from presenilin endoproteolysis necessary to activate γ-secretase (Thinakaran et al, 1996), proves comparable amounts of γ-secretase in the individual assays.  (c) MALDI MS analysis of the ε-cleavage sites of wt and mutant TREM2 C-terminal fragments. Mass spectrometry reveals ε-cleavage C-terminal of A192 for the wt CTF. MS determination of the ε-cleavage site of TREM2 CTF K186A reveals major and minor ε-cleavage sites C-terminal of A195 and L193, respectively. The exact same cleavage pattern is observed for the K186L mutant. MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε-cleavage sites C-terminal of L193 and S191. Minor peaks that could not be assigned are indicated by an asterisk.
Figure 6(d, e) Chemically induced unfolding of TREM2-TMH wild-type (d) and the K186A variant (e) demonstrates that the less dynamic variant is more stable than the wild-type protein.(f) Water exchange peak as observed in a 3D-15N-edited-[1H,1H]-NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond.
Figure 1Representative images of the size of Mex3a mutant mice and control littermates at postnatal day (P)15. Scale bar, 1 cm. Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue (right panels). Scale bars, 50 μm. The offspring number (n) and observed genotype frequencies (%) resulting from heterozygous crosses are indicated below.Absolute weight of Mex3a KO mice and control littermates at different ages. Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one animal (WT: P1, n = 15; P8, n = 36; P15, n = 43; P22, n = 14; Adult, n = 14; Mex3a+/-: P1, n = 24; P8, n = 90; P15, n = 90; P22, n = 37; Adult, n = 21; Mex3a-/-: P1, n = 9; P8, n = 24; P15, n = 26; P22, n = 14; Adult, n = 8) ***P = 0.0003, ****P < 0.0001, two-way ANOVA test.Kaplan-Meier survival curves for the different Mex3a genotypes (n = 80 for each genotype) ****P < 0.0001, Log-rank (Mantel-Cox) test.
Figure 2Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice. These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days. Boxed areas depict the distal small intestinal section (ileum) used for subsequent immunohistochemical analyses.H&E staining of a Mex3a KO and WT littermate at P19. Inserts depict high magnification of the boxed areas.Average crypt depth (C) of WT animals (n = 3, > 40 crypts counted per animal) and Mex3a mutants (n = 6, > 20 crypts counted per animal). Also shown is the average villi height (D) of WT animals (n = 3, > 25 villi counted per animal) and Mex3a mutants (n = 6, > 15 villi counted per animal). Data is represented as mean ± standard error between P16 and P18. *P = 0.03, ***P = 0.0006, Student`s t test.Immunohistochemistry staining for (E) the tuft cell marker DCLK1 (black arrowheads indicate positive cells), (F) the Paneth cell marker LYZ1Immunohistochemistry staining for the proliferation marker KI67.mRNA ISH staining for the stem cell markers (H) Lgr5, (I) Olfm4, (J) Lrig1, and (K) Hopx in ileal sections of Mex3a mutants and littermate controls at P19. Inserts depict high magnification of the boxed areas.qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals (n = 3 of each genotype). Data is represented as mean fold-change plus standard error in Mex3a KO mice relatively to WT animals (dashed line). *P = 0.0208, **P < 0.01, ***P = 0.0007, Student`s t test.
Figure 3Quantification of the size (diameter length) of Mex3a KO and WT organoid-initiating structures 2 days after crypt isolation (n = 3 for each genotype, > 150 structures counted in total). Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one organoid-initiating structure. ****P < 0.0001, Student`s t test. Phase contrast microscopy images of intestinal organoids generated by Mex3a-/- and WT crypts 6 days after crypt isolation. Scale bars, 100 μm.Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at days 2 and 4 of culture after 10 weekly passages. Scale bars, 100 μm.qPCR analysis of crypt lineage marker genes in organoid cultures at day 2. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line). *P < 0.05, Student`s t test.Immunohistochemistry staining for the Paneth cell marker LYZ1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.qPCR analysis of crypt lineage marker genes in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).mRNA ISH staining for the stem cell markers Mex3a, Lgr5, Hopx and Lrig1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals. Mice were injected at P17 and sacrificed at P20 (n = 3 for each genotype). Scale bars, 50 μm.
Figure 4Hierarchical clustering of samples (n = 3 for each genotype) and heatmap of differentially expressed genes between the Mex3a-/- and WT mice. The red colour represents over-expression, while the blue colour under-expression.GSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against (E) the Mex3ahighGSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against the Lgr5high and (G) the Wnt signalling pathway gene signatures.qPCR analysis of selected genes in a cohort of independent mice. Data is presented as the mean fold-change of target gene expression in 2 biological replicates (Mex3a-/- Vs WT #1 and Mex3a-/- Vs WT #2) of Mex3a KO intestinal crypts relative to the respective WT controls (dashed line).
Figure 5Immunohistochemistry staining for PPARγ in Mex3a KO and WT mice ileum. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.Pparg mRNA ISH in ileal sections of Mex3a mutants and littermate controls at P18. Inserts depict high magnification of the boxed areas. Scale bars, 50 μm.qPCR analysis of PPARγ transcriptional targets in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 3 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle (DMSO). Scale bars, 100 μm.qPCR analysis of Lgr5, Pcna and Angptl4 expression in organoid cultures after 5 days of treatment with 50 μM Pioglitazone. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Pioglitazone-treated organoids relative to the DMSO vehicle (dashed line). * P = 0.02, Student`s t test.Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at day 1 after passaging Pioglitazone- or vehicle (DMSO)-treated organoids. Scale bars, 100 μm.
Figure 1C. Coomassie staining of SDS-PAGE-separated pulldown samples with/without cell extracts (C.E) using GST-tagged FET-NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E -). Several high-molecular weight proteins are retained by the FET-NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometryD. Immunoblot analysis (IB) of pulldown samples with FET-NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1 and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control.E. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3 and C-terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS-DDIT3/BRG1 and FUS-DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645-94 and 402-91. C-terminal parts of FUS are not present in the FUS-DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm.