39 research outputs found
Characterization of early ultrastructural changes in the cerebral white matter of CADASIL small vessel disease using high pressure freezing/freeze-substitution
AIMS: The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease. METHODS: We used high pressure freezing and freeze substitution (HPF/FS) in combination with high resolution electron microscopy (EM), immunohistochemistry and confocal microscopy of brain specimens from control and CADASIL (TgNotch3R169C ) mice aged 4 to 15 months to study white matter lesions in the corpus callosum. RESULTS: We first optimized the HPF/FS protocol in which samples were chemically prefixed, frozen in a sample carrier filled with 20% polyvinylpyrrolidone and freeze-substituted in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. EM analysis showed that CADASIL mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6 months, then vesiculation of the inner tongue and myelin sheath thinning at 15 months of age. Immunohistochemistry revealed an increased number of oligodendrocyte precursor cells, although only in older mice, but no reduction in the number of mature oligodendrocytes at any age. The number of Iba1 positive microglial cells was increased in older but not in younger CADASIL mice, but the number of activated microglial cells (Iba1 and CD68 positive) was unchanged at any age. CONCLUSION: We conclude that early WM lesions in CADASIL affect first and foremost the myelin sheath and the inner tongue, suggestive of a primary myelin injury. We propose that those defects are consistent with a hypoxic/ischaemic mechanism
Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease
Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke in humans. Dominant mutations in NOTCH3 cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a genetic archetype of cerebral ischemic SVD. Progress toward understanding the pathogenesis of this disease and developing effective therapies has been hampered by the lack of a good animal model. Here, we report the development of a mouse model for CADASIL via the introduction of a CADASIL-causing Notch3 point mutation into a large P1-derived artificial chromosome (PAC). In vivo expression of the mutated PAC transgene in the mouse reproduced the endogenous Notch3 expression pattern and main pathological features of CADASIL, including Notch3 extracellular domain aggregates and granular osmiophilic material (GOM) deposits in brain vessels, progressive white matter damage, and reduced cerebral blood flow. Mutant mice displayed attenuated myogenic responses and reduced caliber of brain arteries as well as impaired cerebrovascular autoregulation and functional hyperemia. Further, we identified a substantial reduction of white matter capillary density. These neuropathological changes occurred in the absence of either histologically detectable alterations in cerebral artery structure or blood-brain barrier breakdown. These studies provide in vivo evidence for cerebrovascular dysfunction and microcirculatory failure as key contributors to hypoperfusion and white matter damage in this genetic model of ischemic SVD
Notch3 Is a Major Regulator of Vascular Tone in Cerebral and Tail Resistance Arteries
Objectiveâ Notch3, a member of the evolutionary conserved Notch receptor family, is primarily expressed in vascular smooth muscle cells. Genetic studies in human and mice revealed a critical role for Notch3 in the structural integrity of distal resistance arteries by regulating arterial differentiation and postnatal maturation.Methods and Resultsâ We investigated the role of Notch3 in vascular tone in small resistance vessels (tail and cerebral arteries) and large (carotid) arteries isolated from Notch3-deficient mice using arteriography. Passive diameter and compliance were unaltered in mutant arteries. Similarly, contractions to phenylephrine, KCl, angiotensin II, and thromboxane A2 as well as dilation to acetylcholine or sodium nitroprusside were unaffected. However, Notch3 deficiency induced a dramatic reduction in pressure-induced myogenic tone associated with a higher flow (shear stress)-mediated dilation in tail and cerebral resistance arteries only. Furthermore, RhoA activity and myosin light chain phosphorylation, measured in pressurized tail arteries, were significantly reduced in Notch3KO mice. Additionally, myogenic tone inhibition by the Rho kinase inhibitor Y27632 was attenuated in mutant tail arteries. Conclusionsâ Notch3 plays an important role in the control of vascular mechano-transduction, by modulating the RhoA/Rho kinase pathway, with opposite effects on myogenic tone and flow-mediated dilation in the resistance circulation
Notch2 and Notch3 Function Together to Regulate Vascular Smooth Muscle Development
Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping
Prox1 Regulates the Notch1-Mediated Inhibition of Neurogenesis
During development of the spinal cord, Prox1 controls the balance between proliferation and differentiation of neural progenitor cells via suppression of Notch1 gene expression
J022 Effecteurs du récepteur Notch3 dans la cellule musculaire lisse des petites artÚres
Notch3 code pour un rĂ©cepteur transmembranaire dont lâexpression est fortement restreinte aux cellules musculaires lisses (CML) des petites artĂšres. Des Ă©tudes gĂ©nĂ©tiques chez lâhomme et la souris ont dĂ©montrĂ© que Notch3 Ă©tait un acteur clĂ© dans la physiologie normale et la pathologie des petites artĂšres. Chez lâhomme, des mutations dominantes de Notch3 sont responsables de la maladie CADASIL, une forme hĂ©rĂ©ditaire de maladie des petites artĂšres cĂ©rĂ©brales. Chez la souris, Notch3 est requis pour la maturation postnatale des petites artĂšres, en contrĂŽlant lâidentitĂ© artĂ©rielle et le remodelage du cytosquelette des CML. Notch3 joue Ă©galement un rĂŽle clĂ© dans la fonction normale des petites artĂšres, en contrĂŽlant les rĂ©ponses myogĂ©niques Ă la pression artĂ©rielle. Lâactivation de Notch3, dans la voie canonique, induit, par clivage protĂ©olytique, la libĂ©ration de son domaine intracellulaire qui se lie dans le noyau Ă RBP-Jk, favorisant la formation dâun complexe activateur de la transcription.Notre objectif est dâidentifier et de caractĂ©riser les effecteurs de Notch3 dans les petites artĂšres.Par une approche combinant transcriptome et Q-PCR sur des artĂšres de souris Notch3KO et WT, nous avons identifiĂ© 11 gĂšnes candidats. Leur niveau dâexpression est significativement diminuĂ© dans les artĂšres de souris Notch3KO, Ă un stade oĂč il nâexiste pas encore de lĂ©sions cellulaires visibles, et, cette diminution est corrigĂ©e par la rĂ©introduction spĂ©cifiquement dans les CML dâune protĂ©ine Notch3WT mais pas par celle dâune protĂ©ine mutĂ©e dĂ©fective pour la signalisation RBP-Jk. Les 6 gĂšnes dont nous avons pu Ă©tudier le patron dâexpression sont tous exprimĂ©s dans les CML artĂ©rielles. De façon remarquable, lâexpression vasculaire est artĂ©rielle prĂ©dominante pour les 6 gĂšnes et « petite artĂšre » prĂ©fĂ©rentielle pour 4 dâentre eux. De plus, chacun de ces gĂšnes a un profil dâexpression unique au niveau de lâarborisation artĂ©rielle et capillaire, mais, la superposition des diffĂ©rents profils recouvre celui de Notch3.Les travaux en cours ont pour but de dĂ©terminer si les gĂšnes candidats identifiĂ©s sont ou non des cibles primaires, RBPJk-dĂ©pendantes, et de caractĂ©riser histologiquement et fonctionnellement les mutants souris perte-de-fonction de ces gĂšnes
Blood brain barrier leakage is not a consistent feature of white matter lesions in CADASIL
The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo.
Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy
Notch3 is required for arterial identity and maturation of vascular smooth muscle cells.
Formation of a fully functional artery proceeds through a multistep process. Here we show that Notch3 is required to generate functional arteries in mice by regulating arterial differentiation and maturation of vascular smooth muscle cells (vSMC). In adult Notch3-/- mice distal arteries exhibit structural defects and arterial myogenic responses are defective. The postnatal maturation stage of vSMC is deficient in Notch3-/- mice. We further show that Notch3 is required for arterial specification of vSMC but not of endothelial cells. Our data reveal Notch3 to be the first cell-autonomous regulator of arterial differentiation and maturation of vSMC