5 research outputs found
Cerebrovascular and blood-brain barrier impairments in Huntington's disease: Potential implications for its pathophysiology: Vascular impairments in HD
ObjectiveAlthough the underlying cause of Huntington's disease (HD) is well established, the actual pathophysiological processes involved remain to be fully elucidated. In other proteinopathies such as Alzheimer's and Parkinson's diseases, there is evidence for impairments of the cerebral vasculature as well as the bloodâbrain barrier (BBB), which have been suggested to contribute to their pathophysiology. We investigated whether similar changes are also present in HD.MethodsWe used 3â and 7âTesla magnetic resonance imaging as well as postmortem tissue analyses to assess blood vessel impairments in HD patients. Our findings were further investigated in the R6/2 mouse model using in situ cerebral perfusion, histological analysis, Western blotting, as well as transmission and scanning electron microscopy.ResultsWe found mutant huntingtin protein (mHtt) aggregates to be present in all major components of the neurovascular unit of both R6/2 mice and HD patients. This was accompanied by an increase in blood vessel density, a reduction in blood vessel diameter, as well as BBB leakage in the striatum of R6/2 mice, which correlated with a reduced expression of tight junctionâassociated proteins and increased numbers of transcytotic vesicles, which occasionally contained mHtt aggregates. We confirmed the existence of similar vascular and BBB changes in HD patients.InterpretationTaken together, our results provide evidence for alterations in the cerebral vasculature in HD leading to BBB leakage, both in the R6/2 mouse model and in HD patients, a phenomenon that may, in turn, have important pathophysiological implications. Ann Neurol 2015;78:160â17
Ătude de la barriĂšre hĂ©matoencĂ©phalique dans des modĂšles animaux de la maladie d'Alzheimer
Tableau dâhonneur de la FacultĂ© des Ă©tudes supĂ©rieures et postdoctorales, 2015-2016La barriĂšre hĂ©matoencĂ©phalique (BHE) est une barriĂšre biologique qui protĂšge le cerveau de molĂ©cules potentiellement toxiques pour le SNC. Plusieurs Ă©tudes laissent penser que des modifications de la BHE ont lieu au cours de la maladie dâAlzheimer (MA). Ainsi, la BHE demeure un grand dĂ©fi dans le dĂ©veloppement de mĂ©dicaments pour les maladies cĂ©rĂ©brales. Cependant, des systĂšmes de transport biologique au niveau de la BHE pourraient ĂȘtre ciblĂ©s par des anticorps afin dâamĂ©liorer la disponibilitĂ© cĂ©rĂ©brale de diffĂ©rents mĂ©dicaments. Notre objectif Ă©tait dâĂ©valuer la BHE dans la MA et de quantifier lâaccumulation cĂ©rĂ©brale dâun anticorps monoclonal ciblant le rĂ©cepteur de la transferrine (RTf). Dans la premiĂšre partie du projet, nous avons Ă©valuĂ© la BHE chez deux modĂšles de souris de la MA, des souris doubles (APP/PS1) et triples (3xTg-AD) transgĂ©niques. Une diminution du volume vasculaire a Ă©tĂ© observĂ©e dans lâhippocampe des souris 3xTg-AD ĂągĂ©es de 6 Ă 18 mois par rapport aux souris tĂ©moins. Ces souris ont dĂ©montrĂ© une diminution du transport cĂ©rĂ©bral du glucose et de l'expression de GLUT1 Ă lâĂąge de 18 mois. En revanche, aucune diffĂ©rence nâa Ă©tĂ© observĂ©e chez les souris APP/PS1. Dans la deuxiĂšme partie du projet, nous avons Ă©tudiĂ© lâeffet de lâallĂšle Δ4 de lâapolipoprotĂ©ine E, un facteur de risque gĂ©nĂ©tique majeur de la MA, sur la BHE. Les souris APOE4 ont dĂ©montrĂ© une rĂ©duction de la vascularisation cĂ©rĂ©brale et du transport cĂ©rĂ©bral du glucose de maniĂšre dĂ©pendante de lâĂąge. De plus, une rĂ©duction de lâĂ©paisseur de la lame basale, une augmentation de lâexpression de RAGE et une diminution du passage cĂ©rĂ©bral du DHA ont Ă©tĂ© observĂ©es chez les souris APOE4 par rapport aux souris APOE2 et APOE3. Finalement, nous avons dĂ©veloppĂ© la technique de la perfusion cĂ©rĂ©brale in situ pour quantifier lâaccumulation cĂ©rĂ©brale dâun anticorps fluorescent (Ri7) ciblant le RTf. Ainsi, nous avons confirmĂ© lâaccumulation cĂ©rĂ©brale de Ri7 au niveau des cellules endothĂ©liales en utilisant un mĂ©canisme saturable et sans affecter lâintĂ©gritĂ© physique de la BHE. Cet anticorps pourrait donc ĂȘtre utilisĂ© pour modifier lâexpression capillaire de diffĂ©rents rĂ©cepteurs/transporteurs, ce qui pourrait ĂȘtre efficace dans le traitement de la MA.The blood-brain barrier (BBB) is a biological barrier which protects the brain from potential toxic molecules. Several studies suggest that changes in the BBB occur in Alzheimer's disease (AD). On the other hand, the BBB is a major challenge in the development of drugs for cerebral diseases. However, biological transport systems at the BBB, such as the transferrin receptor (TfR), could be targeted with monoclonal antibodies to develop the technology of Trojan horse and improve the brain availability of different drugs. The aim of my PhD project was to assess the BBB in AD and quantify the brain uptake of a monoclonal antibody targeting the TfR. In the first part of the project, we evaluated the BBB in two mouse models of AD, doubles (APP / PS1) and triple (3xTg-AD) transgenic mice. A reduction of the vascular volume was observed in the hippocampus of 3xTg-AD mice aged 6-18 months compared to control mice. These mice showed a decrease in glucose cerebral transport and expression of GLUT1 at the age of 18 months. However, no difference was observed in APP/PS1 mice. In the second part of the project, we studied the effect of the Δ4 allele of apolipoprotein E, a major genetic risk factor of AD, on the BBB. The APOE4 mice showed a reduction of the cerebral vascularization and glucose cerebral transport in age dependent manner. Moreover, a reduction in the thickness of the basement membrane, an increase in the expression of RAGE and a decrease in cerebral passage of DHA were observed in APOE4 mice compared to APOE2 and APOE3 mice. Finally, we developed the in situ brain perfusion technique to quantify the brain uptake of a fluorescent antibody (RI7) targeting the TfR. In this section, we confirmed the cerebral accumulation of RI7 in the endothelial cells. This accumulation was made without affecting the physical integrity of the BBB and using a saturable mechanism. Hence, this antibody could be used to modify the expression of various receptors/transporters in the cerebral capillary, which may be effective in the treatment of neurodegenerative diseases such as AD
Human Apolipoprotein E Δ4 Expression Impairs Cerebral Vascularization and BloodâBrain Barrier Function in Mice
The proteome of the bloodâbrain barrier in rat and mouse: highly specific identification of proteins on the luminal surface of brain microvessels by in vivo glycocapture
Abstract Background The active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the bloodâbrain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins. Methods Here we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice. Results Using the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture. Conclusions The proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms