Luc Schoonjans

Total inactivation of gamma-secretase activity in presenilin-deficient embryonic stem cells.

he question of how proteins such as the β-amyloid precursor protein (β-APP), Notch-1 and others can be cleaved in the plane of the membrane has challenged cell biologists and researchers into Alzheimer’s disease for some time. Processing of β-APP by γ-secretase is considered an important therapeutic target, as it constitutes the final step in the release of the amyloid βpeptide (Aβ), the principal constituent of the amyloid plaques in the brains of Alhzeimer’s patients. Recently, γ-secretase activity was linked to two proteins, presenilin-1 (PS1) and presenilin-2 (PS2; refs 1, 2). Presenilins are strongly hydrophobic proteins embedded in intracellular membranes, and missense mutations in their genes cause a rare, aggressive form of Alzheimer’s disease. It has been proposed that two aspartate residues in the transmembrane domains of presenilins constitute the catalytic site of γsecretase. One argument against this hypothesis is the important residual γ-secretase activity observed in cells derived from PS1or PS2-deficient mice. The question is therefore whether cells that are completely devoid of PS1 and PS2 maintain this γ-secretase activity or not. Unfortunately, PS-null mice die early in embryogenesis (after roughly 9.5 days of development; E9.5), making it impossible to generate sufficient cells to carry out the required biochemical experiments. We therefore generated pluripotent lines of embryonic stem cells from PS-null blastocysts obtained by mating of PS1PS2 mice. We genotyped the cell lines using the polymerase chain reaction (PCR) and Southern blotting (Fig. 1a). The 32% yield of double-deficient (PS-null) cell lines was slightly higher than the expected 25%; we confirmed the absence of PS1 (Fig. 1b) and PS2 (data not shown) by western blotting. We observed the accumulation of endogenous βand α-secretase-cleaved β-APP carboxy-terminal stubs, the direct substrates for γ-secretase (Fig. 1b and ref. 1). We then expressed β-APP695 harbouring the K595N/M596L (Swedish) mutation in the embryonic stem cells, using recombinant Semliki Forest virus (SFV). This mutation markedly increases production of Aβ , facilitating its detection in our assays. In contrast to results obtained with single-knockout cells, no Aβ was detected in the conditioned media of PS1PS2 cells (Fig. 1c). Using serial dilutions, we established that we would have detected Aβ even if its production was <0.5% of that in wildtype cells (Fig. 1c). We further confirmed the absence of γ-secretase activity using an enzyme-linked immunosorbent assay (ELISA) specific for the 42-amino-acid form of Aβ. mNotch∆E is another substrate for PS1-dependent γsecretase. Processing of this construct mimics that of full-length Notch-1, and results in release of the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates, together with transcription factors of the CBF-1 family, several genes, including HES-1 (ref. 9). We therefore co-transfected the embryonic stem cells with plasmids encoding mNotch∆E and luciferase under the control of the HES-1 promoter (kindly provided by R. Kopan and A. Israel), and measured induction of luciferase activity. As a control, we also transfected embryonic stem cells with plasmids encoding the NICD. Induction of HES-1 activity with mNotch∆E was observed in PS1PS2 cells, but not in PS1 PS2 cells (Fig. 1d). In contrast, NICD strongly induced luciferase activity, implying that the Notch signalling pathway downstream of the proteolytic processing of mNotch∆E was essentially intact in PS1PS2 cells. This was further confirmed by the observation that signalling in PS1PS2 cells was also restored after co-transfection with PS1 complementary DNA (data not shown). These observations extend to the molecular level our previous conclusion that the phenotype of PS1PS2 embryos is similar to that of Notch-deficient embryos. We further confirmed that proteolysis of mNotch∆E in PS1PS2 cells was completely inhibited using a biochemical assay, as before (Fig. 1e). Quantification of the obtained signals by phosphorimaging indicated that NICD production in PS1PS2 cells was <1% of that observed in PS1PS2 cells. In PS1PS2 cells, a weak NICD signal was already observed after 30 min of pulse labelling (Fig. 1f), whereas it was never observed in PS1PS2 cells. Studies involving overexpression of dominant-negative PS2 mutants have implicated PS2 in γ-secretase activity. However, here we have shown for the first time that γ-secretase activity is completely eliminated in embryonic stem cells that are devoid of presenilins. Although these results do not categorically prove that presenilins have catalytic activities, they constitute an important piece of indirect evidence in support of the ‘presenilin is γ-secretase’ hypothesis, by resolving one important counterargument. Our results also add to the emerging concept of ‘regulated intramembrane proteolysis’ (RIP). RIP cleaves integral membrane proteins in the plane of the membrane, releasing cytosolic fragments that enter the nucleus to control the transcription of genes involved in differentiation, lipid metabolism and the unfolded-protein response during endoplasmic reticulum stress. We anticipate that the cell lines generated in this study will greatly facilitate further investigation of the function of presenilins in RIP, and the identification of unknown presenilin substrates. h

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.

Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization.

Homozygous plasminogen activator inhibitor-1 (PAI-1)-deficient (PAI-1-/-) mice were generated by homologous recombination in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions of murine PAI-1 was demonstrated by Southern blot analysis. A 3.0-kb PAI-1-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-1+/+) but not from PAI-1-/- mice. Plasma PAI-1 levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63 +/- 2 ng/ml, 30 +/- 10 ng/ml, and undetectable (< 2 ng/ml) in PAI-1+/+, heterozygous (PAI-1+/-) and PAI-1-/- mice, respectively (mean +/- SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1+/+ but not of PAI-1-/- mice. SDS-gel electrophoresis of plasma incubated with 125I-labeled recombinant human tissue-type plasminogen activator revealed an approximately 115,000-M(r) component with plasma from endotoxin-stimulated (0.5 mg/kg) PAI-1+/+ but not from PAI-1-/- mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAI-1-/- mice were viable, produced similar sizes of litters as PAI-1+/+ mice, and showed no apparent macroscopic or microscopic histological abnormalities.

Caspase-14 protects against epidermal UVB photodamage and water loss.

Caspase-14 belongs to a conserved family of aspartate-specific proteinases. Its expression is restricted almost exclusively to the suprabasal layers of the epidermis and the hair follicles. Moreover, the proteolytic activation of caspase-14 is associated with stratum corneum formation, implicating caspase-14 in terminal keratinocyte differentiation and cornification. Here, we show that the skin of caspase-14-deficient mice was shiny and lichenified, indicating an altered stratum-corneum composition. Caspase-14-deficient epidermis contained significantly more alveolar keratohyalin F-granules, the profilaggrin stores. Accordingly, caspase-14-deficient epidermis is characterized by an altered profilaggrin processing pattern and we show that recombinant caspase-14 can directly cleave profilaggrin in vitro. Caspase-14-deficient epidermis is characterized by reduced skin-hydration levels and increased water loss. In view of the important role of filaggrin in the structure and moisturization of the skin, the knockout phenotype could be explained by an aberrant processing of filaggrin. Importantly, the skin of caspase-14-deficient mice was highly sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation, leading to increased levels of UVB-induced apoptosis. Removal of the stratum corneum indicate that caspase-14 controls the UVB scavenging capacity of the stratum corneum.

Cardiomyocyte-Specific Overexpression of Nitric Oxide Synthase 3 Improves Left Ventricular Performance and Reduces Compensatory Hypertrophy After Myocardial Infarction

Nitric oxide (NO) is an important modulator of cardiac performance and left ventricular (LV) remodeling after myocardial infarction (MI). We tested the effect of cardiomyocyte-restricted overexpression of one NO synthase isoform, NOS3, on LV remodeling after MI in mice. LV structure and function before and after permanent LAD coronary artery ligation were compared in transgenic mice with cardiomyocyte-restricted NOS3 overexpression (NOS3-TG) and their wild-type littermates (WT). Before MI, systemic hemodynamic measurements, echocardiographic assessment of LV fractional shortening (FS), heart weight, and myocyte width (as assessed histologically) did not differ in NOS3-TG and WT mice. The inotropic response to graded doses of isoproterenol was significantly reduced in NOS3-TG mice. One week after LAD ligation, the infarcted fraction of the LV did not differ in WT and NOS3-TG mice (34 ± 4% versus 36 ± 12%, respectively). Four weeks after MI, however, end-systolic LVID was greater, and fractional shortening and maximum and minimum rates of LV pressure development were less in WT than in NOS3-TG mice. LV weight/body weight ratio was greater in WT than in NOS3-TG mice (5.3 ± 0.2 versus 4.6 ± 0.5 mg/g; P < 0.01). Myocyte width in noninfarcted myocardium was greater in WT than in NOS3-TG mice (18.8 ± 2.0 versus 16.6 ± 1.6 μm; P < 0.05), whereas fibrosis in noninfarcted myocardium was similar in both genotypes. Cardiomyocyte-restricted overexpression of NOS3 limits LV dysfunction and remodeling after MI, in part by decreasing myocyte hypertrophy in noninfarcted myocardium.

Mice lacking factor VII develop normally but suffer fatal perinatal bleeding

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII−/−) embryos developed normally. FVII−/− mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal–fetal transfer of FVII was undetectable and survival of embryos did not depend on TF–FVII-initiated fibrin formation. Thus, the TF−/− embryonic lethal and the FVII−/− survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

Fatty acid carbon is essential for dNTP synthesis in endothelial cells

The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.

Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice

Background— Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG). Methods and Results— Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57±5 versus 39±4 and 65±6 versus 48±4 &mgr;L, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium. Conclusions— Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.

Improved Generation of Germline-Competent Embryonic Stem Cell Lines from Inbred Mouse Strains

Genetically altered mice may exhibit highly variable phenotypes due to the variation in genetic background, which can only be circumvented by generation of inbred, isogenic gene‐targeted and control mice. Here we report that an embryonic stem (ES) cell culture medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor allows efficient derivation and maintenance of ES cell lines from all of 10 inbred mouse strains tested, including some that were presumed to be nonpermissive for ES cell derivation (129/SvEv, 129/SvJ, C57BL/6N, C57BL/6JOla, CBA/CaOla, DBA/2N, DBA/1Ola, C3H/HeN, BALB/c, and FVB/N). Germline transmission was established by blastocyst injection of established ES cell lines after 10 or more passages from all of seven strains tested (129/SvJ, C57BL/6N, C57BL/6JOla, DBA/2N, DBA/1Ola, BALB/c, and FVB/N), by diploid aggregation of ES cell lines from all of four strains tested (129/SvEv, C57BL/6N, CBA/ CaOla, and FVB/N), or by tetraploid aggregation of ES cell lines from all of three strains tested (129/SvEv, C57BL/6N, and CBA/CaOla). Thus, these inbred ES cell lines may constitute useful tools to derive gene‐targeted mice and isogenic controls in selected genetic backgrounds.

Tumour hypoxia causes DNA hypermethylation by reducing TET activity

Hypermethylation of the promoters of tumour suppressor genes represses transcription of these genes, conferring growth advantages to cancer cells. How these changes arise is poorly understood. Here we show that the activity of oxygen-dependent ten-eleven translocation (TET) enzymes is reduced by tumour hypoxia in human and mouse cells. TET enzymes catalyse DNA demethylation through 5-methylcytosine oxidation. This reduction in activity occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, hypoxia-inducible factor activity or reactive oxygen species, and depends directly on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro. In patients, tumour suppressor gene promoters are markedly more methylated in hypoxic tumour tissue, independent of proliferation, stromal cell infiltration and tumour characteristics. Our data suggest that up to half of hypermethylation events are due to hypoxia, with these events conferring a selective advantage. Accordingly, increased hypoxia in mouse breast tumours increases hypermethylation, while restoration of tumour oxygenation abrogates this effect. Tumour hypoxia therefore acts as a novel regulator of DNA methylation.