The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Anne Joutel - One of the best experts on this subject based on the ideXlab platform.
-
Notch3ECD immunotherapy improves cerebrovascular responses in CADASIL mice.
Annals of neurology, 2018Co-Authors: Lamia Ghezali, Céline Baron-menguy, Valérie Domenga-denier, Carmen Capone, Julien Ratelade, Søren Christensen, Lars Østergaard Pedersen, Jan Torleif Pedersen, Anne JoutelAbstract:CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), caused by dominant mutations in the Notch3 Receptor, is the most aggressive small vessel disease of the brain. A key feature of its pathogenesis is accumulation of the extracellular domain of Notch3 Receptor (Notch3ECD ) in small vessels, with formation of characteristic extracellular deposits termed granular osmiophilic material (GOM). Here, we investigated the therapeutic potential of a mouse monoclonal antibody (5E1) that specifically recognizes Notch3ECD . The binding affinity of 5E1 toward purified Notch3 was assessed using Octet analysis. The ability of 5E1 to bind Notch3ECD deposits in brain vessels and its effects on disease-related phenotypes were evaluated in the CADASIL mouse model, which overexpresses a mutant rat Notch3. Notch3ECD and GOM deposition, white matter lesions, and cerebral blood flow deficits were assessed at treatment initiation (10 weeks) and study completion (30 weeks) using quantitative immunohistochemistry, electron microscopy, and laser-Doppler flowmetry. 5E1 antibody bound recombinant rat Notch3 with an average affinity of 317nM. A single peripheral injection of 5E1 robustly decorated Notch3ECD deposits in the brain vasculature. Chronic administration of 5E1 did not attenuate Notch3ECD or GOM deposition and was not associated with perivascular microglial activation. It also failed to halt the development of white matter lesions. Despite this, 5E1 treatment markedly protected against impaired cerebral blood flow responses to neural activity and topical application of vasodilators and normalized myogenic responses of cerebral arteries. This study establishes immunotherapy targeting Notch3ECD as a new avenue for disease-modifying treatment in CADASIL that warrants further development. Ann Neurol 2018;84:246-259. © 2018 American Neurological Association.
-
Increased Notch3 Activity Mediates Pathological Changes in Structure of Cerebral Arteries.
Hypertension (Dallas Tex. : 1979), 2016Co-Authors: Céline Baron-menguy, Valérie Domenga-denier, Lamia Ghezali, Frank M. Faraci, Anne JoutelAbstract:CADASIL (Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy), the most frequent genetic cause of stroke and vascular dementia, is caused by highly stereotyped mutations in the Notch3 Receptor, which is predominantly expressed in vascular smooth muscle. The well-established TgNotch3R169C mouse model develops characteristic features of the human disease, with deposition of Notch3 and other proteins, including TIMP3 (tissue inhibitor of metalloproteinase 3), on brain vessels, as well as reduced maximal dilation, and attenuated myogenic tone of cerebral arteries, but without elevated blood pressure. Increased TIMP3 levels were recently shown to be a major determinant of altered myogenic tone. In this study, we investigated the contribution of TIMP3 and Notch3 signaling to the impairment of maximal vasodilator capacity caused by the archetypal R169C mutation. Maximally dilated cerebral arteries in TgNotch3R169C mice exhibited a decrease in lumen diameter over a range of physiological pressures that occurred before myogenic tone deficits. This defect was not prevented by genetic reduction of TIMP3 in TgNotch3R169C mice and was not observed in mice overexpressing TIMP3. Knock-in mice with the R169C mutation (Notch3R170C/R170C) exhibited similar reductions in arterial lumen, and both TgNotch3R169C and Notch3R170C/R170C mice showed increased cerebral artery expression of Notch3 target genes. Reduced maximal vasodilation was prevented by conditional reduction of Notch activity in smooth muscle of TgNotch3R169C mice and mimicked by conditional activation of Notch3 in smooth muscle, an effect that was blood pressure-independent. We conclude that increased Notch3 activity mediates reduction in maximal dilator capacity of cerebral arteries in CADASIL and may contribute to reductions in cerebral blood flow.
-
potassium channelopathy like defect underlies early stage cerebrovascular dysfunction in a genetic model of small vessel disease
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Fabrice Dabertrand, Christel Kroigaard, Emmanuel Cognat, Thomas Dalsgaard, Valerie Domengadenier, David C Hilleubanks, Adrian D Bonev, Joseph E Brayden, Anne Joutel, Mark NelsonAbstract:Abstract Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the Notch3 Receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3R169C mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3R169C mice. This effect was associated with an ∼60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor Receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3R169C mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3R169C mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.
-
CADASIL and CARASIL
Brain pathology (Zurich Switzerland), 2014Co-Authors: Saara Tikka, Emmanuel Cognat, Minna Pöyhönen, Matti Viitanen, Marc Baumann, Maija Siitonen, Petra Pasanen, Liisa Myllykangas, Toshio Fukutake, Anne JoutelAbstract:CADASIL and CARASIL are hereditary small vessel diseases leading to vascular dementia. CADASIL commonly begins with migraine followed by minor strokes in mid-adulthood. Dominantly inherited CADASIL is caused by mutations (n > 230) in Notch3 gene, which encodes Notch3 Receptor expressed in vascular smooth muscle cells (VSMC). Notch3 extracellular domain (N3ECD) accumulates in arterial walls followed by VSMC degeneration and subsequent fibrosis and stenosis of arterioles, predominantly in cerebral white matter, where characteristic ischemic MRI changes and lacunar infarcts emerge. The likely pathogenesis of CADASIL is toxic gain of function related to mutation-induced unpaired cysteine in N3ECD. Definite diagnosis is made by molecular genetics but is also possible by electron microscopic demonstration of pathognomonic granular osmiophilic material at VSMCs or by positive immunohistochemistry for N3ECD in dermal arteries. In rare, recessively inherited CARASIL the clinical picture and white matter changes are similar as in CADASIL, but cognitive decline begins earlier. In addition, gait disturbance, low back pain and alopecia are characteristic features. CARASIL is caused by mutations (presently n = 10) in high-temperature requirement. A serine peptidase 1 (HTRA1) gene, which result in reduced function of HTRA1 as repressor of transforming growth factor-β (TGF β) -signaling. Cerebral arteries show loss of VSMCs and marked hyalinosis, but not stenosis.
-
Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model
Acta neuropathologica communications, 2014Co-Authors: Emmanuel Cognat, Valérie Domenga-denier, Sabine Cleophax, Anne JoutelAbstract:Introduction Small vessel disease (SVD) of the brain is a leading cause of age- and hypertension-related cognitive decline and disability. Cerebral white matter changes are a consistent manifestation of SVD on neuroimaging, progressing silently for many years before becoming clinically evident. The pathogenesis of these changes remains poorly understood, despite their importance. In particular, their pathological correlate at early stages remains largely undefined. Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), caused by dominant mutations of the Notch3 Receptor, is regarded as a paradigm for the most common form of sporadic SVD. In this study, we used immunohistochemistry, confocal microscopy and electron microscopy, together with qualitative and quantitative analyses to assess oligodendroglial, axon and myelin damage in TgPAC-Notch3R169C mice, a model of preclinical CADASIL.
Elisabeth Tournier-lasserve - One of the best experts on this subject based on the ideXlab platform.
-
Distinct phenotypic and functional features of CADASIL mutations in the Notch3 ligand binding domain
Brain - A Journal of Neurology, 2009Co-Authors: Marie Monet-leprêtre, Valérie Domenga, Elisabeth Tournier-lasserve, Martin Dichgans, Boris Bardot, Barbara Lemaire, Ophélia Godin, Michel Cohen-tannoudji, Hugues Chabriat, Anne JoutelAbstract:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant small-vessel disease of the brain caused by mutations in the Notch3 Receptor. The highly stereotyped nature of the mutations, which alter the number of cysteine residues within the epidermal growth factor-like repeats (EGFR), predicts that all mutations share common mechanisms. Prior in vitro assays and genetic studies in the mouse support the hypothesis that common mutations do not compromise canonical Notch3 function but instead convey a non-physiological and deleterious activity to the Receptor through the unpaired cysteine residue. Intriguingly, in vitro studies predict that mutations located in the Delta/Serrate/LAG-2 ligand binding domain-(EGFR10-11) may result in a loss of Notch3 Receptor function. However, the in vivo relevance and functional significance of this with respect to the pathogenic mechanisms and clinical expression of the disease remain largely unexplored. To ascertain, in vivo, the functional significance of EGFR10-11 mutations, we generated transgenic mice with one representative mutation (C428S) in EGFR10 of Notch3. These mice, like those with a common R90C mutation, developed characteristic arterial accumulation of Notch3 protein and granular osmiophilic material upon aging. By introducing the mutant C428S transgene into a Notch3 null background, we found that, unlike the R90C mutant protein, the C428S mutant protein has lost wild-type Notch3 activity and exhibited mild dominant-negative activity in three different biological settings. From a large prospectively recruited cohort of 176 CADASIL patients, we identified 10 patients, from five distinct pedigrees carrying a mutation in EGFR10 or 11. These mutations were associated with significantly higher Mini-Mental State Examination and Mattis Dementia Rating Scale scores (P < 0.05), when compared with common mutations. Additionally, we found a strong effect of this genotype on the burden of white matter hyperintensities (P < 0.01). Collectively, these results highlight distinctive functional and phenotypic features of EGFR10-11 mutations relative to the common CADASIL mutations. Our findings are compatible with the hypothesis that EGFR10-11 mutations cause the disease through the same gain of novel function as the common mutations, and lead us to propose that reduced Notch3 signalling acts as a modifier of the CADASIL phenotype.
-
Activating Notch3 mutation in a patient with small-vessel-disease of the brain
Human Mutation, 2008Co-Authors: Charles Fouillade, Elisabeth Tournier-lasserve, Hugues Chabriat, Florence Riant, Manuele Mine, Minh Arnoud, Laurent Magy, Marie-germaine Bousser, Anne JoutelAbstract:The most common causative diagnosis of hereditary small-vessel-disease of the brain, CADASIL, is due to highly stereotyped mutations in the Notch3 Receptor. Notch3 has 33 exons but all CADASIL mutations occur within the Epidermal Growth Factor-like Repeats encoded by exons 2-24, lead to an odd number of cysteine residues and are associated with GOM deposits and abnormal Notch3 protein accumulation. The majority of CADASIL mutations appear to retain normal level of signaling activity, while very few mutations show reduced activity. Herein we identified a novel heterozygous missense mutation (c.4544T>C) in exon 25 of Notch3 in a patient with cerebral small-vessel-disease but lacking GOM deposits and Notch3 accumulation. The mutation should result in a p.L1515P substitution in the evolutionarily highly conserved juxtamembranous region of Notch3, which constitutes the heterodimerization domain. The p.L1515P mutant exhibits increased canonical Notch3 signaling, although in a ligand-independent fashion. Biochemical analysis suggests that the mutation renders Notch3 hyperactive through destabilization of the heterodimer. Therefore, our study suggests that the p.L1515P mutation falls in a novel mechanistic class of Notch3 mutations and that Notch3 activating mutations should be further considered for molecular analysis of patients with cerebral small-vessel-disease.
-
Activating Notch3 mutation in a patient with small‐vessel‐disease of the Brain
Human mutation, 2008Co-Authors: Charles Fouillade, Elisabeth Tournier-lasserve, Hugues Chabriat, Florence Riant, Manuele Mine, Minh Arnoud, Laurent Magy, Marie-germaine Bousser, Anne JoutelAbstract:The most common causative diagnosis of hereditary small-vessel-disease of the brain, CADASIL, is due to highly stereotyped mutations in the Notch3 Receptor. Notch3 has 33 exons but all CADASIL mutations occur within the Epidermal Growth Factor-like Repeats encoded by exons 2-24, lead to an odd number of cysteine residues and are associated with GOM deposits and abnormal Notch3 protein accumulation. The majority of CADASIL mutations appear to retain normal level of signaling activity, while very few mutations show reduced activity. Herein we identified a novel heterozygous missense mutation (c.4544T>C) in exon 25 of Notch3 in a patient with cerebral small-vessel-disease but lacking GOM deposits and Notch3 accumulation. The mutation should result in a p.L1515P substitution in the evolutionarily highly conserved juxtamembranous region of Notch3, which constitutes the heterodimerization domain. The p.L1515P mutant exhibits increased canonical Notch3 signaling, although in a ligand-independent fashion. Biochemical analysis suggests that the mutation renders Notch3 hyperactive through destabilization of the heterodimer. Therefore, our study suggests that the p.L1515P mutation falls in a novel mechanistic class of Notch3 mutations and that Notch3 activating mutations should be further considered for molecular analysis of patients with cerebral small-vessel-disease.
-
Pathogenic Mutations Associated with Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Differently Affect Jagged1 Binding and Notch3 Activity via the RBP/JK Signaling Pathway
American journal of human genetics, 2004Co-Authors: Anne Joutel, Valérie Domenga, Florence Riant, Marie Monet, Elisabeth Tournier-lasserveAbstract:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterized by the degeneration of smooth-muscle cells in small cerebral arteries. CADASIL is caused by mutations in Notch3, one of the four mammalian homologs to the Drosophila melanogaster NOTCH gene. Disease-associated mutations are distributed throughout the 34 epidermal growth factor-like repeats (EGFRs) that compose the extracellular domain of the Notch3 Receptor and result in a loss or a gain of a cysteine residue in one of these EGFRs. In human adults, Notch3 expression is highly restricted to vascular smooth-muscle cells. In patients with CADASIL, there is an abnormal accumulation of Notch3 in the vessel. Molecular pathways linking Notch3 mutations to degeneration of vascular smooth-muscle cells are as yet poorly understood. In this study, we investigated the effect of CADASIL mutations on Notch3 activity. We studied five naturally occurring mutations: R90C and C212S, located in the previously identified mutational hotspot EGFR2-5; C428S, shown in this study to be located in the ligand-binding domain EGFR10-11; and C542Y and R1006C, located in EGFR13 and EGFR26, respectively. All five mutant proteins were correctly processed. The C428S and C542Y mutant Receptors exhibited a significant reduction in Jagged1-induced transcriptional activity of a RBP/JK responsive luciferase reporter, relative to wild-type Notch3. Impaired signaling activity of these two mutants arose through different mechanisms; the C428S mutant lost its Jagged1-binding ability, whereas C542Y retained it but exhibited an impaired presentation to the cell surface. In contrast, the R90C, C212S, and R1006C mutants retained the ability to bind Jagged1 and were associated with apparently normal levels of signaling activity. We conclude that mutations in Notch3 differently affect Jagged1 binding and Notch3 signaling via the RBP/JK pathway.
-
Transgenic Mice Expressing Mutant Notch3 Develop Vascular Alterations Characteristic of Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy
The American journal of pathology, 2003Co-Authors: Marie Magdeleine Ruchoux, Valérie Domenga, Elisabeth Tournier-lasserve, Peggy Brulin, Jacqueline Maciazek, Sylvie Limol, Anne JoutelAbstract:CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; MIM 125310) is an increasingly recognized autosomal dominant small-artery disease of the brain. 1 The onset of symptoms occurs generally in mid-life although it can range from 25 to >60 years. The symptoms typically include recurrent ischemic strokes and/or cognitive impairment. Progression of the disease leads to dementia and premature death ∼15 to 20 years after clinical onset. Magnetic resonance imaging of the brain displays T2-weighted hyperintensities within the white matter in all patients and small deep infarcts in up to 60% of the patients. Such magnetic resonance imaging abnormalities can also be detected in asymptomatic patients, indicating that brain parenchyma damages are already present at the preclinical stage of the disease. 2-5 CADASIL is caused by highly stereotyped mutations in the Notch3 Receptor. 6 Notch3 belongs to the highly conserved Notch Receptor family involved in cell fate specification. 7 It contains all typical Notch motifs, including a characteristic extracellular domain exhibiting 34 tandem epidermal growth factor-like repeats. All CADASIL mutations result in the addition or the loss of a cysteine residue within 1 of the 34 epidermal growth factor-like repeats and therefore to an odd number of cysteine residues in the affected epidermal growth factor domain. 8-10 Prevalence of CADASIL is unknown but more than 400 affected families all over the world as well as sporadic cases carrying a de novo mutation have been identified. 11 On neuropathological examination, CADASIL brains show a diffuse myelin loss and multiple, small deep infarcts located within the white matter and basal ganglia. The pathological hallmark of CADASIL is a nonamyloid and nonarteriosclerotic angiopathy, which affects predominantly the small penetrating arteries. Vascular lesions are characterized by degeneration and loss of smooth muscle cells and the presence of a granular osmiophilic material (GOM) accumulating within the smooth muscle cell basement membrane and the surrounding extracellular matrix. 12-14 Examination of heart, muscle, skin, and many other peripheral organs revealed vessel changes, including the presence of GOM deposits, that were identical, although less severe, to those of cerebral arteries, providing evidence that CADASIL is indeed a systemic arteriopathy. 15-17 GOM deposits are highly specific of CADASIL and their detection in cerebral and peripheral vessels from skin or muscle biopsy material has been widely used as a diagnostic marker of this disease. 15,18,19 Importantly, VSMC alterations and GOM deposits have been detected in skin vessels of asymptomatic mutation carriers having a normal brain magnetic resonance imaging, indicating that arterial lesions are present at a very early stage of the disease before brain parenchyma damages and clinical symptoms occurrence (MMR, ETL, and AJ; unpublished data). 18 First clues to the pathogenic mechanisms underlying CADASIL came recently from the expression analysis of Notch3 in tissues from healthy individuals and from CADASIL patients. Data indicate that vascular smooth muscle cells (VSMCs) are the primary target of the pathogenic process in CADASIL. In healthy human adults, expression of Notch3 is highly restricted to the vessel wall and to VSMCs. Notch3, like the other Notch Receptors, undergoes a constitutive proteolytical cleavage generating an extracellular (Notch3ECD) and a transmembrane/cytosolic (Notch3TMIC) fragment, that remain associated at the cell surface to form a heterodimeric Receptor. In CADASIL patients, there is an abnormal accumulation of Notch3ECD in both brain vessels and peripheral tissue arteries. Accumulation takes place at the cell membrane of smooth muscle cells in very close vicinity but not within the GOM and results from an impaired clearance of the Receptor from the cell membrane. 20 Notch3 accumulation is a specific hallmark of CADASIL and its detection by simple immunostaining of skin biopsy is now commonly used as an easy and reliable diagnostic marker of CADASIL. 21 Notch3 accumulation, like GOM deposits, can be detected in mutation carriers during the long preclinical stage of the disease (ETL and AJ, unpublished observation). So far the mechanisms underlying pathological alterations in CADASIL remain unclear. Vascular smooth muscle cell degeneration may be caused by a defect in Notch3 signaling or a toxic effect of Notch3 or GOM accumulation. A defect in Notch3 signaling may result from a defect in ligand binding or a defect in transmitting the appropriate signal. It has also been proposed that the Notch3ECD, because of its failure to be cleared from the cell membrane, may dominantly inhibit the normal Notch3 pathway through competitive inhibition of ligand binding. 22,23 However, no evidence of such defects has been detected so far using in vitro assays (AJ, unpublished results). 24 In late-onset neurodegenerative disorders, such as Alzheimer’s or Huntington’s diseases, a toxic role of abnormal protein deposition is suspected to play a major role in pathogenesis. By analogy, one might hypothesize that smooth muscle cell degeneration in CADASIL arises from a toxic effect of Notch3ECD and/or GOM accumulation. To investigate the mechanisms of smooth muscle cell degeneration caused by Notch3 mutations, we generated transgenic mice in which the SM22α promoter drove the expression of the full-length human Notch3 with the Arg90Cys mutation, one CADASIL archetypal mutation, in VSMCs. We report that transgenic mice demonstrated vascular changes, including GOM deposits, Notch3 accumulation, and evidence of smooth muscle cell degeneration, strikingly similar to those observed in CADASIL patients. Vessel changes were detected in cerebral and peripheral vessels, including the tail arteries where alterations were the most prominent. Time-course analysis of vessel changes revealed that early signs of VSMC damages were present before Notch3 and GOM accumulation occurred, and that the first ultrastructural defects included the disruption of normal VSMC anchorage to adjacent extracellular matrix and cells as well as VSMC cytoskeleton changes.
Emmanuel Cognat - One of the best experts on this subject based on the ideXlab platform.
-
potassium channelopathy like defect underlies early stage cerebrovascular dysfunction in a genetic model of small vessel disease
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Fabrice Dabertrand, Christel Kroigaard, Emmanuel Cognat, Thomas Dalsgaard, Valerie Domengadenier, David C Hilleubanks, Adrian D Bonev, Joseph E Brayden, Anne Joutel, Mark NelsonAbstract:Abstract Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the Notch3 Receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3R169C mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3R169C mice. This effect was associated with an ∼60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor Receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3R169C mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3R169C mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.
-
CADASIL and CARASIL
Brain pathology (Zurich Switzerland), 2014Co-Authors: Saara Tikka, Emmanuel Cognat, Minna Pöyhönen, Matti Viitanen, Marc Baumann, Maija Siitonen, Petra Pasanen, Liisa Myllykangas, Toshio Fukutake, Anne JoutelAbstract:CADASIL and CARASIL are hereditary small vessel diseases leading to vascular dementia. CADASIL commonly begins with migraine followed by minor strokes in mid-adulthood. Dominantly inherited CADASIL is caused by mutations (n > 230) in Notch3 gene, which encodes Notch3 Receptor expressed in vascular smooth muscle cells (VSMC). Notch3 extracellular domain (N3ECD) accumulates in arterial walls followed by VSMC degeneration and subsequent fibrosis and stenosis of arterioles, predominantly in cerebral white matter, where characteristic ischemic MRI changes and lacunar infarcts emerge. The likely pathogenesis of CADASIL is toxic gain of function related to mutation-induced unpaired cysteine in N3ECD. Definite diagnosis is made by molecular genetics but is also possible by electron microscopic demonstration of pathognomonic granular osmiophilic material at VSMCs or by positive immunohistochemistry for N3ECD in dermal arteries. In rare, recessively inherited CARASIL the clinical picture and white matter changes are similar as in CADASIL, but cognitive decline begins earlier. In addition, gait disturbance, low back pain and alopecia are characteristic features. CARASIL is caused by mutations (presently n = 10) in high-temperature requirement. A serine peptidase 1 (HTRA1) gene, which result in reduced function of HTRA1 as repressor of transforming growth factor-β (TGF β) -signaling. Cerebral arteries show loss of VSMCs and marked hyalinosis, but not stenosis.
-
Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model
Acta neuropathologica communications, 2014Co-Authors: Emmanuel Cognat, Valérie Domenga-denier, Sabine Cleophax, Anne JoutelAbstract:Introduction Small vessel disease (SVD) of the brain is a leading cause of age- and hypertension-related cognitive decline and disability. Cerebral white matter changes are a consistent manifestation of SVD on neuroimaging, progressing silently for many years before becoming clinically evident. The pathogenesis of these changes remains poorly understood, despite their importance. In particular, their pathological correlate at early stages remains largely undefined. Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), caused by dominant mutations of the Notch3 Receptor, is regarded as a paradigm for the most common form of sporadic SVD. In this study, we used immunohistochemistry, confocal microscopy and electron microscopy, together with qualitative and quantitative analyses to assess oligodendroglial, axon and myelin damage in TgPAC-Notch3R169C mice, a model of preclinical CADASIL.
-
Archetypal Arg169Cys Mutation in Notch3 Does Not Drive the Pathogenesis in Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leucoencephalopathy via a Loss-of-Function Mechanism
Stroke, 2014Co-Authors: Emmanuel Cognat, Céline Baron-menguy, Valérie Domenga-denier, Sabine Cleophax, Charles Fouillade, Marie Monet-leprêtre, Mieke Dewerchin, Anne JoutelAbstract:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy, the most common heritable small vessel disease of the brain, is caused by dominant mutations in the Notch3 Receptor that stereotypically lead to age-dependent Notch3ECD deposition in the vessels. Notch3 loss of function has been demonstrated for few mutations. However, whether this finding applies to all mutations and whether a loss-of-function mechanism drives the manifestations of the disease remain yet unknown. This study investigated the in vivo functionality of the Arg169Cys archetypal mutation. We used mice with constitutive or conditional reduction of Notch3 activity, mice harboring the Arg169Cys mutation at the endogenous Notch3 locus (Notch3Arg170Cys), and mice overexpressing the Arg169Cys Notch3 mutant (TgPAC-Notch3R169C) on either a Notch3 wild-type or a null background. Notch3 activity was monitored in the brain arteries by measuring the expression of Notch3 target genes using real-time polymerase chain reaction. Notch3ECD deposits were assessed by immunohistochemistry. Brain parenchyma was analyzed for vacuolation and myelin debris in the white matter and infarcts. We identified a subset of genes appropriate to detect Notch3 haploinsufficiency in the adult. Expression of these genes was unaltered in Notch3Arg170Cys mice, despite marked Notch3ECD deposits. Elimination of wild-type Notch3 did not influence the onset and burden of white matter lesions in 20-month-old TgPAC-Notch3R169C mice, and 20-month-old Notch3-null mice exhibited neither infarct nor white matter changes. These data provide strong evidence that cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy can develop without impairment of Notch3 signaling and argue against a loss of Notch3 function as a general driving mechanism for white matter lesions in cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy.
-
Early white matter changes in CADASIL: evidence of segmental intramyelinic oedema in a pre-clinical mouse model
Acta Neuropathologica Communications, 2014Co-Authors: Emmanuel Cognat, Valérie Domenga-denier, Sabine Cleophax, Anne JoutelAbstract:Introduction Small vessel disease (SVD) of the brain is a leading cause of age- and hypertension-related cognitive decline and disability. Cerebral white matter changes are a consistent manifestation of SVD on neuroimaging, progressing silently for many years before becoming clinically evident. The pathogenesis of these changes remains poorly understood, despite their importance. In particular, their pathological correlate at early stages remains largely undefined. Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), caused by dominant mutations of the Notch3 Receptor, is regarded as a paradigm for the most common form of sporadic SVD. In this study, we used immunohistochemistry, confocal microscopy and electron microscopy, together with qualitative and quantitative analyses to assess oligodendroglial, axon and myelin damage in TgPAC-Notch3R169C mice, a model of preclinical CADASIL. Results The principal cerebral white matter changes in TgPAC-Notch3R169C mice are microvacuoles (≤1 μm diameter) in the myelin sheaths associated with focal myelin degradation and occurring in the absence of oligodendrocyte loss. Half the damaged myelin sheaths still contain an apparently intact axon. Clearance of myelin debris appears inefficient, as demonstrated by the significant but mild microglial reaction, with occasional myelin debris either contacted or internalized by microglial cells. Conclusion Our findings suggest that segmental intramyelinic oedema is an early, conspicuous white matter change in CADASIL. Brain white matter intramyelinic oedema is consistently found in patients and mouse models with compromised ion and water homeostasis. These data provide a starting point for novel mechanistic studies to investigate the pathogenesis of SVD-related white matter changes.
Martin Dichgans - One of the best experts on this subject based on the ideXlab platform.
-
Distinct phenotypic and functional features of CADASIL mutations in the Notch3 ligand binding domain
Brain - A Journal of Neurology, 2009Co-Authors: Marie Monet-leprêtre, Valérie Domenga, Elisabeth Tournier-lasserve, Martin Dichgans, Boris Bardot, Barbara Lemaire, Ophélia Godin, Michel Cohen-tannoudji, Hugues Chabriat, Anne JoutelAbstract:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant small-vessel disease of the brain caused by mutations in the Notch3 Receptor. The highly stereotyped nature of the mutations, which alter the number of cysteine residues within the epidermal growth factor-like repeats (EGFR), predicts that all mutations share common mechanisms. Prior in vitro assays and genetic studies in the mouse support the hypothesis that common mutations do not compromise canonical Notch3 function but instead convey a non-physiological and deleterious activity to the Receptor through the unpaired cysteine residue. Intriguingly, in vitro studies predict that mutations located in the Delta/Serrate/LAG-2 ligand binding domain-(EGFR10-11) may result in a loss of Notch3 Receptor function. However, the in vivo relevance and functional significance of this with respect to the pathogenic mechanisms and clinical expression of the disease remain largely unexplored. To ascertain, in vivo, the functional significance of EGFR10-11 mutations, we generated transgenic mice with one representative mutation (C428S) in EGFR10 of Notch3. These mice, like those with a common R90C mutation, developed characteristic arterial accumulation of Notch3 protein and granular osmiophilic material upon aging. By introducing the mutant C428S transgene into a Notch3 null background, we found that, unlike the R90C mutant protein, the C428S mutant protein has lost wild-type Notch3 activity and exhibited mild dominant-negative activity in three different biological settings. From a large prospectively recruited cohort of 176 CADASIL patients, we identified 10 patients, from five distinct pedigrees carrying a mutation in EGFR10 or 11. These mutations were associated with significantly higher Mini-Mental State Examination and Mattis Dementia Rating Scale scores (P < 0.05), when compared with common mutations. Additionally, we found a strong effect of this genotype on the burden of white matter hyperintensities (P < 0.01). Collectively, these results highlight distinctive functional and phenotypic features of EGFR10-11 mutations relative to the common CADASIL mutations. Our findings are compatible with the hypothesis that EGFR10-11 mutations cause the disease through the same gain of novel function as the common mutations, and lead us to propose that reduced Notch3 signalling acts as a modifier of the CADASIL phenotype.
-
CADASIL mutations enhance spontaneous multimerization of Notch3
Human molecular genetics, 2009Co-Authors: Christian Opherk, Nils Peters, Marco Duering, Anna Karpinska, Stefanie Rosner, Elisabeth Schneider, Benedikt Bader, Armin Giese, Martin DichgansAbstract:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic cause of stroke and vascular dementia. Disease-causing mutations invariably affect cysteine residues within epidermal growth factor-like repeat domains in the extracellular domain of the Notch3 Receptor (N3(ECD)). The biochemical and histopathological hallmark of CADASIL is the accumulation of N3(ECD) at the cell surface of vascular smooth muscle cells which degenerate over the course of the disease. The molecular mechanisms leading to N3(ECD) accumulation remain unknown. Here we show that both wild-type and CADASIL-mutated N3(ECD) spontaneously form oligomers and higher order multimers in vitro and that multimerization is mediated by disulfide bonds. Using single-molecule analysis techniques ('scanning for intensely fluorescent targets'), we demonstrate that CADASIL-associated mutations significantly enhance multimerization compared with wild-type. Taken together, our results for the first time provide experimental evidence for N3 self-association and strongly argue for a neomorphic effect of CADASIL mutations in disease pathogenesis.
-
Spectrum of mutations in biopsy-proven CADASIL: implications for diagnostic strategies.
Archives of neurology, 2005Co-Authors: Nils Peters, Christian Opherk, Tanja Bergmann, Mirna Castro, Jürgen Herzog, Martin DichgansAbstract:Background Mutations in the Notch3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is an important cause of stroke in young adults. Mutations are typically located within epidermal growth factor–like repeat domains in the extracellular part of the Notch3 Receptor. Identification of the mutation is critical for genetic counseling and testing of relatives at risk. Objectives To identify the spectrum of Notch3 mutations in CADASIL and to discuss the implications for diagnostic strategies. Design Screening for Notch3 mutations was performed in 125 unrelated German CADASIL patients with biopsy-proven disease by direct sequencing of exons coding for epidermal growth factor–like repeats. Results were compared with those of previously published studies. Results We detected 54 distinct mutations (117 missense mutations and 3 in-frame deletions) in 120 (96.0%) of the 125 patients. Of the mutations, 58.3% were located in exon 4 and 85.8% in exons 2 through 6. In 5 patients (4.0%), no mutation was identified. Conclusions Almost 90% of mutations could be detected within a few exons (exons 2-6). Thus, genetic testing should initially be focused on these exons, with some variation depending on the population in whom it is being performed. Yet, genetic testing for CADASIL is associated with a nameable proportion of false-negative results. Cases with a high index of clinical suspicion should be investigated by skin biopsy if genetic testing is negative.
-
CADASIL-associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 Receptor signaling through RBP-Jk.
Experimental cell research, 2004Co-Authors: Nils Peters, Christian Opherk, Simone Zacherle, Anja Capell, Petra Gempel, Martin DichgansAbstract:Mutations in the Notch3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary angiopathy leading to strokes and dementia. Pathogenic mutations remove or insert cysteine residues within epidermal growth factor (EGF) repeats in the extracellular domain of the Notch3 Receptor (N3ECD). Vascular smooth muscle cells (VSMC) are the predominant site of Notch3 expression in adults. In CADASIL patients, VSMC degenerate and N3ECD is deposited within the vasculature. However, the mechanisms underlying VSMC degeneration and N3ECD accumulation are still unknown. In this study, we investigated the consequences of three pathogenic Notch3 mutations on the biological activity of the Receptor by analyzing ligand (Delta-/Jagged-)-induced signaling via RBP-Jk. Two mutations (R133C and C183R) that are located outside the putative ligand binding domain (LBD) of the Receptor were found to result in normal Jagged1-induced signaling in A7r5 VSMC, whereas the third mutation (C455R located within the putative LBD) showed strongly reduced signaling activity. Ligand binding assays with soluble Delta1 and Jagged1 revealed that C455R interferes with ligand binding through disruption of the LBD which, as we show here, is located in EGF repeats 10/11 of Notch3. All mutant Receptors including Notch3C455R were targeted to the cell surface but showed an elevated ratio between the unprocessed full-length 280-kDa Receptor and S1-cleaved Receptor fragments. Taken together, these data indicate that CADASIL-associated Notch3 mutations differ with respect to their consequences both on ligand binding and ligand-induced signaling through RBP-Jk, whereas they have similar effects on Receptor maturation. Moreover, the data suggest that ligand-induced Receptor shedding may not be required for N3ECD deposition in CADASIL.
Thao P. Dang - One of the best experts on this subject based on the ideXlab platform.
-
Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo.
Cell death and differentiation, 2011Co-Authors: Lavinia Raimondi, Roberta Ciarapica, M De Salvo, Federica Verginelli, M. Gueguen, C Martini, L De Sio, G Cortese, Mattia Locatelli, Thao P. DangAbstract:Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch Receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 Receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.
-
Targeting Specific Regions of the Notch3 Ligand-Binding Domain Induces Apoptosis and Inhibits Tumor Growth in Lung Cancer
Cancer research, 2010Co-Authors: Luping Lin, David P. Carbone, Raymond L. Mernaugh, David L. Blum, Thao P. DangAbstract:Like many signaling pathways in development, the Notch Receptor pathway plays an important role in cancer pathobiology when it is dysregulated. Potential ligand-binding sites within the epidermal growth factor (EGF)–like repeats of Notch1 have been identified, but the ligand-binding domains in Notch3, which is implicated in lung cancer, are not known. In screening a library of 155 peptides representing all 34 EGF-like repeats in Notch3, we discovered two distinct ligand-binding regions involving the 7–10 and 21–22 repeats that are distinct from the putative ligand-binding domain of Notch1. In cell-based assays, peptides from these regions induced apoptosis and reduced expression of the Notch3-dependent gene Hey1. They also bound directly to the Notch ligand Jagged1, suggesting that their mechanism of action involves disrupting interactions between Notch3 and Jagged1. Recombinant Fc fusion peptides engineered for in vivo testing showed that the Notch3 peptides defined could trigger apoptosis and suppress tumor growth in tumor xenograft assays. These findings rationalize a mechanistic approach to lung cancer treatment based on Notch3 Receptor–targeted therapeutic development. Cancer Res; 70(2); 632–8
-
Dominant-negative Notch3 Receptor inhibits mitogen-activated protein kinase pathway and the growth of human lung cancers.
Cancer research, 2005Co-Authors: Nobuhiro Haruki, Keiko S. Kawaguchi, Shannon Eichenberger, Pierre P. Massion, Sandra J. Olson, Adriana Gonzalez, David P. Carbone, Thao P. DangAbstract:Notch3 is a member of an evolutionarily conserved family of cell surface Receptors important in cell-fate determination in both vertebrates and invertebrates. Significant data support the role of Notch pathway in cancer development, although the conflicting role of Notch signaling pathways in tumorigenesis suggests that its action is highly context-dependent. Furthermore, although Notch Receptors signal primarily through the regulation of hairy enhancer of split (HES) and HES-related (HRT) genes, they are known to crosstalk with other signaling pathways, including the epidermal growth factor (EGF) and the mitogen-activated protein kinase pathways. Whereas much is known about the role of Notch1 in human cancer, the role of Notch3 in epithelial tumors, such as lung carcinomas, has not been well established. In this study, we show that Notch3 is expressed in 80 of 207 (39%) resected human lung tumors and that its expression is positively correlated with EGF Receptor expression. Inhibition of the Notch3 pathway using a dominant-negative Receptor dramatically reduces growth in soft agar and increases growth factor dependence. We also find that Notch inhibition increases sensitivity to EGF Receptor tyrosine kinase inhibition and decrease in phosphorylation of the mitogen-activated protein kinase. These observations support a role for Notch3 signaling in lung cancer, and one potential mechanism of maintaining the neoplastic phenotype is through the modulation of the EGF pathway.
