EMBO/SourceData huggingface dataset:
Figure 3Steady state levels of endogenous AK2 in presence and absence of MIA40. 72 h before experiments, HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR. Cell lysates were analysed by immunoblot against the indicated proteins. MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins. ALR depletion did not affect AK2 levels. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.Stability of endogenous AK2 in the presence and absence of MIA40. 72 h before experiments HeLa, cells were transfected with control siRNA or siRNAs directed against MIA40. Cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by cell lysis, and AK2 was isolated by denaturing immunoprecipitation. Eluates were analyzed by SDS-PAGE and autoradiography. Stability of endogenous AK2 is significantly decreased upon depletion of MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed; * represents p-value <0.05 compared to wild-type.Stability of cytosolic AK2-HA cysteine variant. Experiment was performed as in (B) except that cells expressing AK2C40S,C42S,C92S-HA were analysed by IP against HA. AK2C40S,C42S,C92S-HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.Steady state levels of AK2-HA cysteine variants. AK2C40S,C42S,C92S-HA and AK2WT-HA were expressed in the presence or absence of proteasomal inhibitor MG132. Protein levels at steady state were analysed by immunoblot. All variants are stabilized by proteasomal inhibition. Reported values are the mean of 3 (AK2WT-HA) or 5 (AK2C40S,C42S,C92S-HA) independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. *** represents p<0.001.Steady state levels of AK2-HA cysteine variant. 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5. AK2C40S,C42S,C92S-HA was stabilized by decreased proteasome levels. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.
Figure 4Steady state levels of AK2C40S,C42S,C92S-HA in presence and absence of DPP8 and DPP9. 72 h before experiments, HEK293 cells stably expressing AK2C40S,C42S,C92S-HA were transfected with control siRNA or siRNAs directed against DPP8 or/and DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. DPP9 depletion led to an increase in AK2C40S,C42S,C92S-HA levels, while DPP8 depletion did not. Combined depletion of DPP8 and DPP9 increased AK2C40S,C42S,C92S-HA levels even further. This indicates that DPP9 is the main peptidase and DPP8 can serve as its backup. White and black arrow heads indicate the endogenous and HA-tagged AK2, respectively. The gray arrow head indicates the DPP9 band. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. * represents p<0.05, ,## represent p<0.01 and ***,### represent p<0.001.Steady state levels of AK2C40S,C42S,C92S-HA upon incubation with the DPP9 inhibitor 1G244 (Wu et al., 2009). As (B) except that cells were treated for 16 h with inhibitor or DMSO as control. AK2C40S,C42S,C92S-HA levels were strongly increased upon DPP8/9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. *** represents p<0.001.Steady state levels of endogenous AK2 in presence and absence of DPP9 and MIA40. 72 h before experiments HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 and/or DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. If import of endogenous AK2 was prevented by depletion of MIA40, then concomitant depletion of DPPs increased its levels. Strikingly, depletion of DPP9 alone also increased the levels of endogenous AK2. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01.Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244. As (D) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control. Endogenous AK2 levels were increased upon DPP8/9 inhibition. Reported values are the mean of 5 (3 d) and 6 (5 d) independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, and *** represents p<0.001.Steady state levels of AK2-HA variants with mutated N-termini upon incubation with either 1G244 or vehicle control. HEK293 cells stably expressing AK2-HA variants were lysed and subjected to immunoblot against the indicated proteins. Levels of DPP8/9-insensitive AK2 variants are strongly increased. Reported values are the mean of 4 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01 and ###, *** represents p<0.001. Asterisk denominate statistical comparison with respective control, and hash tag represents comparison to N-WT.
Figure 5Cell fractionation upon inhibition of DPP9 activity. HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO. Cells were subjected to fractionation into 'cytosol' (supernatant) and 'mitochondria' (pellet) by differential digitonin lysis and centrifugation. The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration (lanes 2,5,8,11) irrespective of 1G244 treatment. Likewise, the cytosolic marker protein LDH localized under all conditions to the same fractions. Conversely, endogenous AK2 appeared at this digitonin concentration in the 'cytosol' fraction but only upon 1G244 treatment (compare lanes 2 and 5). Thus, inhibition of DPP9 results in appearance of AK2 in the cytosol. Quantification shows loading-control adjusted AK2-signals of 1G244-treated 'cytosol' fraction (lane 5) over DMSO-treated 'cytosol' fraction (lane 2). White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed.Localization of DPP9-insensitive AK2-HA variants. Experiment was performed as described in Figure 2C. Compared to the wild type, DPP9-insensitive and import-competent AK2-HA variants co-localized with cytosolic markers indicating partial cytosolic localization. The colocalization of AK2-HA variants with Mitotracker was quantified using Fiji (Pearson's correlation coefficient). A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. ** represents p<0.01. Data shown in Figures 2C, 5B, S2 and S5 are derived from the same experiments. The black horizontal line represents the median. Data information: Scale bars: 10 μm.Localization of AK2C40S-HA upon proteasomal inhibition. Experiment was performed as described in Figure 2C. Upon MG132 treatment, colocalization of AK2C40S-HA with the cytosolic marker increased. A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. *** represents p<0.001.The black horizontal line represents the median. Data information: Scale bars: 10 μm.
Figure 6AK2 activity in dependence of its substrate AMP. Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0.012 mM AMP. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are superimposable. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   AK2 activity in dependence of its substrate ADP. Maximal activity was observed at around 1-2 mM ADP with an apparent KM of 0.12 mM and 0.1 mM ADP for AK2C40S,C232S and AK2C40S,C42S,C92S,C232S, respectively. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are thus highly similar. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   Activity assay for AK2-HA variants isolated from HEK293 cells. Indicated AK2-HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants. Then, the AK2 activity assay described in (B) was performed. While import-competent AK2 variants exhibited full activity, a cytosolic AK2 variant with the native N-terminus showed very low activity. This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9-insensitive cytosolic AK2 variants. The enzymatic inactive K28Q variants served as negative control indicating that only AK2-HA-dependent activity ways measured by our assay. Reported values are the mean of 2 independent experiments; error bars represent ±SD.
Figure 7Steady state levels of NDUFB10, NDUFS5, NDUFA8, COA6, CHCHD2, and TIMM9 upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. Proteins except TIMM9 are stabilized upon DPP9 inhibition. Reported values are the mean of 3-5 independent experiments; error bars represent ±SD. Student's t-test was performed. ** represents p<0.01.Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. NDUFB10C107S is stabilized upon DPP9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed for the respective control and treatment samples. ** represents p<0.01.Localization of NDUFB10 variants upon 1G244 treatment. Experiment was performed as in (C) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins. Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition. Reported values are the mean of 2 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.
Figure 2A Pup survival analysis between P0-P10, by sex. Each dot represents the absolute percent of remaining animals per group (Jag1CTRL n=211, male Jag1Ndr/Ndr n=26, female Jag1Ndr/Ndr n= 20, log-rank test, ****P<0.0001).B P10 plasma total bilirubin levels (n=6 per group, unpaired t test, **P=0.0091). C P10 plasma thrombin antithrombin levels (n=6 per group, unpaired t test, ns, P=0.2788).D P10 serum fibrinogen levels (n=6 per group, unpaired t test, ns, P=0.7585).E P10 whole blood prothrombin time (INR) (n=6 per group, unpaired t test, ns, P>0.9999).F P10 tail bleeding time (n=6 per group, unpaired t test, ns, P=0.8916).G-I µCT of P30 skulls. (G) Color map depicting wall thickness. Scale bar 3.5 mm. (H) Skull full thickness volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.7595; Sex P= 0.1164, Genotype **P= 0.0012. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0227; : Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.034), (I) Compact bone total volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.8631; Sex P= 0.0901, Genotype **P= 0.0014. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0305; Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0307).J-K (J) Provoked vascular accidents induced by portal vein resin injection, with (K) resin leakage quantification outside of blood vessels (red) in adult mice (n=6 per group, unpaired t test, ns, P=0.1088).L Adult brain Evans blue assay (Jag1CTRL n=7, Jag1Ndr/Ndr n=8, unpaired t test, ns, P=0.3807).M Hemorrhagic P3 Jag1Ndr/Ndr brain (n= 2/83 Jag1Ndr/Ndr mice, sex n.d.).N Hemorrhagic P15 Jag1Ndr/Ndr retina. Scale bar 20 µm. Sex n.d.O Red blood cells outside P10 Jag1Ndr/Ndr retinal arteriole. Scale bar 10 µm.
Figure 3(A) Radial arrangement of arterioles (magenta) and venules (green) from the optic nerve. Blue arrows label arteriovenous crossings. Magnification of crossing shows compression/narrowing of the underlying vein. Scale bar left and middle panels 50 µm, and right panel 20 µm.(B) Arteriovenous crossings number per retina (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.7074, Sex P =>0.9999, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr *P= 0.0201; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr **P= 0.0061).C-D (C) Number of major arterioles (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.4353, Sex P =0.1995, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr *P= 0.0146; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr **P= 0.0011) and (D) venules (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.044, Sex P =0.044, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1Ndr/Ndr vs. Female:Jag1Ndr/Ndr *P=0.0302; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr ***P= 0.0002).E-F (E) Arterial tortuosity at P30 and 1 year, irrespective of sex (n=6 per group, two-way ANOVA, not significant). (F) Arterial tortuosity in male and female mice, irrespective of age (n=5-7, two-way ANOVA, not significant). G-H (G) Venous tortuosity at P30 and 1 year, irrespective of sex (n=6-8 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.1166, Age P = 0.0787, Genotype **P=0.0013. Tukey's multiple comparisons test: P30:Jag1+/+ vs. P30:Jag1Ndr/Ndr **P=0.004; P30:Jag1Ndr/Ndr vs. 1 year:Jag1Ndr/Ndr P=0.0802; 1 year:Jag1+/+ vs. 1 year:Jag1Ndr/Ndr P= 0.5066). (H) Venous tortuosity in male and female mice, irrespective of age (n=5-7, two-way ANOVA with Tukey's multiple comparisons test. Interaction *P = 0.0223, Sex **P = 0.007, Genotype ***P= <0.0001. Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr P= 0.3247, Male:Jag1Ndr/Ndr vs. Female:Jag1Ndr/Ndr **P= 0.0031, Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr ****P= 0.0001).(J) Tortuosity of MCA, as assessed by number of diversions per length (n=6, t-test ns).(K) Visualization of MCA with alpha smooth muscle cell actin (αSMA) showed stereotype vasculature in 6 of 6 Jag1+/+ animals, but 2 of 6 Jag1Ndr/Ndr animals showed highly divergent MCA architecture (blue arrows).(L) Distances between arterial branches in MCA at P10 (unpaired t-test, not significant).
Figure 4A-E αSMA coverage of (A) P10, (B) P15, (C) P30, (D) 3-6 month old and € 1-year-old retinal arterioles. Brackets in A, B denote perpendicular VSMCs around arteriole. Yellow arrows in A, B, C. indicate VSMCs with an orientation parallel to the blood vessel. Green arrowheads in D denote stenosis. White arrowheads in E label αSMA-negative gaps and white arrows an aneurysm. Cropped images were placed on black background for esthetic purposes.(F) Incidence of α SMA negative gaps on arteries by age (Two-way ANOVA with Sidak's multiple comparisons test).(G) Number of α SMA negative gaps per retina and mouse by age (Two-way ANOVA with Sidak's multiple comparisons test. Dots represent biological replicates/individual retinas/mice. Two-way ANOVA not significant. Šídák's multiple comparisons test: 1 year Jag1+/+ vs 1 year Jag1Ndr/Ndr *P= 0.0189).H Transmission electron microscopy of retinal arterioles. VSMCs color-coded in magenta, EC in green. Scale bar 5 µm, boxed region 1 µm. Blue arrowheads denote the edges of VSMCs.I-J (I) One-year-old retinal arteriolar cCasp3+ apoptotic cells. Scale bar 10 µm. (J) cCasp3+ cells per field (n=6 per group, unpaired t test, *P=0.0109).(K) Left panel aSMA coverage of MCA of P10 mice. Scale bar 20 µm. Right panel magnification of area where number of nuclei was counted. Nuclei were color-coded blue.(L) MCA vascular smooth muscle cell nuclei quantification (n=6 per group, unpaired t test, *P=0.0421).(N) Blood pressure (BP) before treatment in male and female Jag1CTRL and Jag1Ndr/Ndr mice. Each dot represents one animal/one biological replicate. (Two-way ANOVA Interaction P=0.9407, Sex P=0.4957, Genotype *P= 0.0207. Šídák's multiple comparisons test not significant).(O) Blood pressure increase in Jag1CTRL and Jag1Ndr/Ndr mice treated with vehicle or AngII for two weeks (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparisons).(P) Detection of Evans blue leakage in brain in Jag1CTRL and Jag1Ndr/Ndr mice with or without AngII treatment. (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P=0.9687, Treatment **P=0.007, Genotype P=0.5058).(Q) One Jag1Ndr/Ndr mouse (male) displayed a macroscopic EB leakage, upon AngII treatment.® Retinal arteriolar αSMA coverage in vehicle or AngII-treated mice. Green arrowheads denote stenosis, blue arrowheads label αSMA-negative gap. (S) αSMA-negative gaps in arterioles per retina (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=3-5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P = 0.1054, treatment P=0.0815; Genotype P=0.0747. Ší'ák's multiple comparisons test: AngII treated Jag1+/+ vs AngII treated Jag1Ndr/Ndr *P= 0.0315).).
Figure 5A-D (A) P30 retina, three CD31+ capillary layers pseudo-colored for superficial, intermediate and deep layers. Scale bar 50 µm. (B) P30 SCP branching point number (n=6-8 per group, unpaired t test, ns, P=0.5346). (C) P30 ICP vascular length (n=6 per group, unpaired t test, *P=0.0124). (D) P30 ICP branching point number (n=6 per group, unpaired t test, **P=0.0057). E-H (E) 3-6 months old retina, three pseudo-colored CD31+ capillary layers. Scale bar 50 µm. (F) Adult SCP branching point number (n=6 per group, unpaired t test, ns, P=0.5365). (G) Adult ICP vascular length (n=6-7 per group, unpaired t test, **P=0.0078). (H) Adult ICP branching point number (n=6-7 per group, unpaired t test, **P=0.001I-J (I) ICP COLIV and CD31 (arrowhead labels COLIV+ CD31- capillary). Scale bar 50 µm. (J) Empty COLIV sleeve length in ICP per field (n=6 per group, unpaired t test, ns, P=0.1583).(K) Retinal capillary plexuses in 3-6 months old (top panels) and one-year-old mice (bottom panels). White arrowheads label discontinuous ICP. Blue arrowhead labels SCP, green ICP and magenta DCP.(L-M) Vertical sprout number between SCP (S) and ICP (I) and ICP (I) and DCP (D) in (L) adult (n=6 per group, two-way ANOVA, genotype ***P=0.0004, Sidak's multiple comparisons test S-I ns, P=0.0592, I-D ****P<0.0001) and (M) one-year-old mice (n=8 per group, two-way ANOVA, genotype ns, P=0.4507, Sidak's multiple comparisons test S-I ns, P=0.9483, I-D ns, P=0.6046).N-P (N) P10 and (O) P40 neurofilament (NF) and CD31 staining. Scale bar 50 µm. (P) % area positive for NF in P10 and P40 mice (n=6 per group, two-way ANOVA: Interaction ***P= 0.0001; Age *P=0.0428; Genotype ***P=0.0004. Sidak's multiple comparisons test: P10 Jag1+/+ vs P10 Jag1Ndr/Ndr P= 0.9246; P40 Jag1+/+ vs P40 Jag1Ndr/Ndr ****P= <0.0001).
Figure 6A Representative retinographs from a healthy adult, patient with CADASIL, healthy pediatric individual, patient with ALGS and patient with BA. Boxed regions and black arrowheads magnify arteriovenous crossing.B-F (B) Arteriovenous crossings per retina (n=14 per group, unpaired t test, ns, P=0.3854. (C) Major arterioles (n=14 per group, unpaired t test, ns, P=0.2902) and (D) venules (n=14 per group, unpaired t test, ns, P=0.1536). (E) Arterial (n=14 per group, unpaired t test, ns, P=0.0759) and (F) venous tortuosity (n=14 per group, unpaired t test, ns, P=0.1050).G-K (G) Arteriovenous crossings per retina (CTRL n=5, ALGS n=3, BA n=4, one-way ANOVA, ns, P=0.0608, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0492, ALGS vs. BA ns, P=0.0999). (H) Major arterioles (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.0026, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0039, ALGS vs. BA ns, P=0.9799) and (I) venules (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, ***P=0.0008, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0011, ALGS vs. BA ns, P>0.9999). (J) Arterial (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, *P=0.0486, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0392, ALGS vs. BA ns, P=0.1204) and (K) venous tortuosity (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.009, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0076, ALGS vs. BA *P=0.0327).
Figure 1(A) Upper panels- Immunofluorescence imaging of PRR7 with Shank (synaptic) and MAP2 (dendritic) markers in DIV21 hippocampal neurons. Lower panels- Single plane apotome images of nuclei (DAPI) show PRR7 can be present (arrow) or absent (arrowhead) from nuclei.(B) Synaptic and dendritic distribution of PRR7 and pre (Bassoon) and postsynaptic (PSD95) markers (arrowheads-triple colocalization).(C) PRR7-GFP expressed in 17DIV hippocampal neurons is present in dendritic spines (arrows). Scale bar is 15 µm.(D) PRR7 distribution across subcellular fractions (TOT = whole lysate, Syn = synaptosomes, Nuc = nuclei) isolated from rat hippocampal tissue. Fib (Fibrillarin), SNAP25 and PSD95 were used as nuclear, presynaptic and postsynaptic markers, respectively.