Mice co-expressing human APPSwe/Ind TGF-B1 as a tool for the identification of Alzheimer's disease therapies

Elevated brain levels of amyloid-β (Aβ) and transforming growth factor- β1 (TGF-β1) are thought to contribute to the cognitive and cerebrovascular alterations of Alzheimer's disease (AD). Here, we sought to investigate the role of simultaneous increases of Aβ and TGF-β1 on cerebrovascular, neuronal, glial and mnemonic function using a bitransgenic mouse model concurrently overexpressing a mutated form of the human amyloid precursor protein (APPSwe,Ind) and a constitutively active form of TGF-β1 (A/T mice). In addition, we attempted to counter these deficits using three different pharmacological agents: the peroxisome proliferator-activated receptor (PPARγ) agonist pioglitazone (20mg/kg/day), the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor simvastatin (40mg/kg/day) and the angiotensin II receptor type 1 antagonist losartan (10 and 25mg/kg/day), all of which with previously demonstrated efficacy in singly APP and TGF mouse models. We found that A/T mice exhibited spatial learning and memory impairments, together with an early progressive decline in cerebrovascular dilatory capacity, preserved contractility, and reduction in constitutive nitric oxide (NO) synthesis. Altered levels of vasodilator-synthesizing enzymes and fibrotic proteins were also apparent with increasing age. A/T mice featured glial and neuronal dysfunctions, as well as neurovascular and neurometabolic coupling deficits to sensory stimulation that were normalized only by pioglitazone. The latter also improved reversal learning in adult, but not in aged A/T mice, despite worsening effects on vasodilatory function. Similar negative effects were observed with simvastatin on dilatory function, and simvastatin additionally failed to improve neurovascular coupling and cognitive deficits. In contrast, losartan at the low, but not at the high dose displayed minor benefits on the learning capacity of A/T mice and significantly improved spatial memory. Although neurovascular coupling to sensory stimulation was not normalized by either dose, the low and high doses of losartan improved vasodilatory function without normalizing the baseline NO production essential to maintain blood vessel tone. Together, these findinds suggest that A/T mice, which integrate the comorbid factor of AD cerebrovascular dysfunction to the amyloid pathology, may well reflect the complexity that one faces when attempting to rescue function in AD patients. It may also represent an improved model to test new therapeutic strategies.Des taux élevés d'amyloide-β (Aβ) et du 'transforming growth factor-β1' (TGF-β1) dans le cerveau contribuent aux altérations cognitives et cérébrovasculaires de la maladie d'Alzheimer. Dans cette thèse, nous avons tenté de cerner le rôle d'une augmentation simultanée d'Aβ et de TGF-β1 sur les fonctions cérébrovasculaires, neuronales, gliales et mnémoniques chez une souris bitransgénique qui surexprime ces deux peptides (souris A/T). Nous avons aussi tenté de remédier aux anomalies identifiées chez la souris A/T en utilisant trois agents pharmacologiques différents. Ces traitements sont : la pioglitazone (20mg/kg/jour), un agoniste des récepteurs (PPARγ), la simvastatin (40mg/kg/jour), un inhibiteur de la '3-hydroxy-3-methyl-glutaryl-CoA réductase', le losartan (10 et 25mg/kg/jour), un antagoniste du récepteur de type 1 de l'angiotensine II, dont l'efficacité a été démontrée dans des modèles de souris transgéniques qui surproduisent indépendamment l'Aβ ou le TGF-β1. Nous avons trouvé que les souris A/T avaient des déficits d'apprentissage et de mémoire, et qu'elles présentaient un déclin précoce et progressif de la capacité dilatatrice des vaisseaux cérébraux, et une réduction de la synthèse constitutive du monoxyde d'azote (NO), mais que la contractilité était préservée. Des niveaux altérés d'enzymes impliquées dans la synthèse de molécules vasodilatatrices et des protéines médiatrices de la fibrose vasculaire étaient également apparents. Les souris A/T présentaient aussi des dysfonctions gliales et neuronales, ainsi que des déficits de couplages neurovasculaire et neurométabolique, qui étaient contrés uniquement par la pioglitazone. La pioglitazone améliorait aussi la capacité d'apprentissage de type 'reversal learning' chez les souris adultes et non chez les souris âgées, tout en exerçant des effets négatifs sur les fonctions vasodilatatrices. Des effets néfastes sur la capacité des vaisseaux à se dilater ont aussi été observés avec la simvastatine qui, par contre, n'a pu reproduire les bienfaits cognitifs et sur le couplage neurovasculaire observés avec la pioglitazone. Seul le traitement avec la faible dose de losartan a amélioré de façon modeste les capacités d'apprentissage des souris A/T et a significativement rétabli leur mémoire spatiale. Cependant, l'augmentation de perfusion cérébrale induite par une stimulation sensorielle n'était pas normalisée par le losartan à aucune dose, et le losartan a complètement (dose faible) ou partiellement (dose élevée) rétabli les fonctions dilatatrices sans toutefois améliorer la production constitutive du NO essentiel pour le maintain du tonus vasculaire. L'ensemble de ces résultats suggère que les souris A/T, qui combinent la pathologie de l'Aβ aux dysfonctions cerébrovasculaires induites par le TGF-β1, reflètent la complexité de la maladie humaine et mettent en évidence les défis à relever pour traiter efficacement les patients atteints d'Alzheimer. Les souris A/T pourraient donc représenter un modèle amélioré afin de tester de nouvelles approches thérapeutiques

Augmented aortic atherosclerosis in ApoE deficient mice with targeted overexpression of urotensin-II receptor

Urotensin-II (U-II) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study, we hypothesized that selective overexpression of UT in an SMC-specific fashion would increase atherosclerotic lesion formation in a hypercholesterolemic mouse model. The objectives were to demonstrate the role of UT in this mouse model of atherosclerosis, and to elucidate some of the mechanism involved in the process. We used four strains of mice; wildtype (WT), UT+ (a transgenic strain expressing human UT driven by the alpha-SM22 promoter), ApoE knockout (ko), and UT+/ApoE ko. All animals were fed a high-fat diet for 12 weeks. Western blot analysis revealed a significant increase in UT expression in UT+ and ApoE ko mice (P<0.05). Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT+/ApoE ko but not in UT + mice when compared to wild type mice (P<0.0001). Analysis of aortas showed a significant increase in atherosclerotic lesion in the UT +, ApoE ko and UT+/ApoE ko compared to WT mice (P<0.05). Oral administration of the UT receptor antagonist SB-657510A for 10 weeks in a group of ApoE ko mice fed a high fat diet resulted in a significant reduction of lesion (P<0.001). Immunohistochemistry revealed the presence of strong expression of UT and U-II proteins in the atheroma of UT+, ApoE ko and UT+/ApoE ko mice, particularly in foam cells. SB-657510A also significantly reduced ACAT-1 protein expression in the atherosclerotic lesion of ApoE ko mice (P<0.05). The present findings suggest that the use of UT receptor antagonists may reduce lesion formation through reduced foam cell formation and lipid uptake, demonstrating an important role for UT in the pathogenesis of atherosclerosis

Effects of pioglitazone (pio) on cerebrovascular reactivity and protein alterations in adult A/T mice.

<p><b><i>A</i></b>, The impaired cerebrovascular dilatations to acetylcholine (ACh) and calcitonin gene-related peptide (CGRP) in A/T mice (▼) were aggravated or reversed to weak constrictions by pioglitazone (▽). This was accompanied by a full recovery of baseline NO synthesis in adult A/T mice measured during NOS inhibition (L-NNA, 10^-5M). Dilatations induced by the NO donor SNP were unimpaired in A/T mice, but were reversed to small constrictions or shifted to the right at high SNP concentrations in pioglitazone-treated A/T mice. Contractile responses to 5-HT and ET-1 were unaltered in A/T mice and pioglitazone had no detrimental effects. Error bars represent SEM (n=3-6 mice/group). <b><i>B</i></b>, Vasodilator synthesizing enzyme COX-2 and proteins associated with vascular fibrosis VEGF, CTGF and MMP-9 were not significantly altered by genotype or treatment, as measured by Western blot in pial vessels of adult A/T mice relative to their treated counterparts and WT mice. Actin was used as a reference for loading (n=4 mice/group). Similar results were obtained in the aged cohort, but data were not illustrated for clarity purposes. Error bars represent SEM. ★p<0.05, ★★, ** p<0.01, ★★★, *** p<0.001 when compared to untreated WT controls (★) or A/T mice (*) using two-way ANOVA (part A) and repeated measures ANOVA (part B) followed by Newman-Keuls post-hoc test.</p

Pioglitazone (pio) restored stimulus-evoked CBF and CGU in A/T mice.

<p><b><i>A</i></b>, The impaired hyperemic response to whisker stimulation in adult and aged A/T mice was rescued by pioglitazone compared to age-matched WT controls, as measured by LDF (n=4 mice/group) (traces of adult mice shown, green: WT; turquoise: WTpio; violet: A/T and pink: A/Tpio). Values represent the percent increase in CBF induced by whisker stimulation relative to baseline. <b><i>B</i></b>, Pioglitazone also improved the decreased CGU response to whisker stimulation in the somatosensory cortex of aged A/T compared to WT mice (n=3-6 mice/group). The % activation ratio denotes the percentage of corrected standard uptake value (SUV) in the activated contralateral (left side (L)) vs. ipsilateral (right side (R)) somatosensory cortex. Error bars represent SEM. ★p<0.05, ★★p<0.01, ★★★p<0.001 using two-way ANOVA followed by Newman-Keuls post-hoc test.</p

Pioglitazone (pio) had no effect on amyloidosis in A/T mice.

<p><b><i>A</i></b>, Levels of soluble and insoluble Aβ_1-40 and Aβ_1-42 in adult A/T mice, as assayed in cortex and hippocampus by ELISA, were comparable between pioglitazone-treated- and untreated A/T mice. <b><i>B</i></b>, Western blot analysis with 6E10 antibody confirmed no effect of pioglitazone on soluble Aβ species in cortex of adult A/T mice. On the gel, (★) represents the APP band and (*), the 4kDa band of monomeric Aβ including Aβ_1-40 and Aβ_1-42, as quantified in the bar graph. <b><i>C</i></b>, Thioflavin S-stained dense-core Aβ plaque load in cortex and hippocampus were unaltered by pioglitazone in adult A/T mice. <b><i>D</i></b>, Similarly, 6E10-immunostaining of diffuse and dense-core Aβ plaques in 5µm-thick paraffin sections showed no effect of pioglitazone on cortical and hippocampal Aβ plaque load in adult A/T mice. Error bars represent SEM. ★p<0.05, ★★p<0.01, ★★★p<0.001 for comparison to A/T mice using two-way ANOVA followed by Newman-Keuls post-hoc test (n=4 mice/group).</p

Effects of pioglitazone (pio) on spatial learning and memory in adult A/T mice.

<p><b><i>A</i></b>, A/T mice (▼) displayed impaired learning during hidden-platform testing compared to aged-matched wild-type (WT) littermates (●) in the Morris watermaze. These deficits were not due to visual or motor disabilities as all groups had comparable abilities to find the visible platform (days 1-3). Pioglitazone did not improve the deficit in A/T mice (▽) and did not affect the performance of WT controls in the hidden platform training (days 4-8). (○).B, A/T mice, treated (▽) and non-treated (▼) with pioglitazone displayed significant deficit in memory retention as assessed during the probe trial in the target quadrant. <b>C</b>, When the probe trial was subdivided in 15s segments for better understanding of the pattern of behavior throughout the 60s-long probe trial in target quadrant, pioglitazone did not exert any benefit in A/T mice. <b><i>D</i></b>, A/T mice showed an inclination for the original quadrant during the probe trial, as shown by the increased time spent in this quadrant and smaller average distance to the platform location. <b><i>E</i></b>, Patterns of behaviour in original quadrant segmented in 4 slices of 15s each during the probe trial showed that pioglitazone had a positive effect in A/T mice (▽) in increasing percent time spent and average distance to platform during in the last quarter of the probe trial. <b><i>F</i></b>, Pie-representation of typical swimming patterns of % time spent and % distance traveled in target quadrant (red, #4) compared to the original quadrant (yellow, #2) during the probe trial. The drawings (on the left) are representations of single mouse swimming pattern best matching the average of of each group, as displayed by the pie patterns (on the right). Error bars represent SEM (n=6-9 mice/group). ★p<0.05 ★★, ** p<0.01, ★★★p<0.001 when compared to untreated WT controls (★) or A/T mice (*) using two-way ANOVA and repeated measures ANOVA followed by Newman-Keuls post-hoc test.</p

Brain endothelial TAK1 and NEMO safeguard the neurovascular unit

Inactivating mutations of the NF-kappa B essential modulator (NEMO), a key component of NF-kappa B signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood-brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-kappa B and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-kappa B-independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1-NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP