Potential Cross Talk between Autism Risk Genes and Neurovascular Molecules: A Pilot Study on Impact of Blood Brain Barrier Integrity.
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ABSTRACT:
SUBMITTER: Jagadapillai R
PROVIDER: S-EPMC9315816 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe monocarboxylate transporter 8 (MCT8) plays a vital role in maintaining brain thyroid hormone homeostasis. This transmembrane transporter is expressed at the brain barriers, as the blood-brain barrier (BBB), and in neural cells, being the sole known thyroid hormone-specific transporter to date. Inactivating mutations in the MCT8 gene (SLC16A2) cause the Allan-Herndon-Dudley Syndrome (AHDS) or MCT8 deficiency, a rare X-linked disease characterized by delayed neurodevelopment and severe psychomotor disorders. The underlying pathophysiological mechanisms of AHDS remain unclear, and no effective treatments are available for the neurological symptoms of the disease.MethodsNeurovascular unit ultrastructure was studied by means of transmission electron microscopy. BBB permeability and integrity were evaluated by immunohistochemistry, non-permeable dye infiltration assays and histological staining techniques. Brain blood-vessel density was evaluated by immunofluorescence and magnetic resonance angiography. Finally, angiogenic-related factors expression was evaluated by qRT-PCR. The studies were carried out both in an MCT8 deficient subject and Mct8/Dio2KO mice, an AHDS murine model, and their respective controls.ResultsUltrastructural analysis of the BBB of Mct8/Dio2KO mice revealed significant alterations in neurovascular unit integrity and increased transcytotic flux. We also found functional alterations in the BBB permeability, as shown by an increased presence of peripheral IgG, Sodium Fluorescein and Evans Blue, along with increased brain microhemorrhages. We also observed alterations in the angiogenic process, with reduced blood vessel density in adult mice brain and altered expression of angiogenesis-related factors during brain development. Similarly, AHDS human brain samples showed increased BBB permeability to IgG and decreased blood vessel density.ConclusionsThese findings identify for the first time neurovascular alterations in the MCT8-deficient brain, including a disruption of the integrity of the BBB and alterations in the neurovascular unit ultrastructure as a new pathophysiological mechanism for AHDS. These results open a new field for potential therapeutic targets for the neurological symptoms of these patients and unveils magnetic resonance angiography as a new non-invasive in vivo technique for evaluating the progression of the disease.
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Project description:Obstructive sleep apnea syndrome (OSAS) is characterized by repeated episodes of hypoxia during the night. The severity of the disorder can be evaluated using an apnea-hypopnea index (AHI). The physiological consequences are mainly cardiovascular and neuronal dysfunctions. One hypothesis to explain such associated neurological disorders is disruption of the blood-brain barrier (BBB), which protects the brain from endovascular cytotoxic compounds. We selected two subgroups of volunteers from the PROOF cohort study (France), a group of patients suffering newly diagnosed severe OSAS (AHI > 30/h) and a group showing no sleep apnea (AHI < 5/h). We exposed a human in vitro BBB model of endothelial cells (HBEC-5i) with sera of patients with and without OSAS. After exposure, we measured the apparent BBB permeability as well as tight junction and ABC transporter expression using whole cell ELISA. We showed that after incubation with sera from OSAS patients, there was a loss of integrity in the human in vitro BBB model; this was reflected by an increase in permeability (43%; p < 0.001) and correlated with a 50% and 40% decrease in tight junction protein expression of ZO-1 and claudin-5, respectively. At the same time, we observed an upregulation in Pgp protein expression (52%) and functionality, and a downregulation in BCRP expression (52%). Our results demonstrated that severe BBB disorder after exposure to sera from OSAS patients was reflected by an opening of the BBB.
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Project description:Impairment of brain endothelial barrier integrity is critical for cerebral cavernous malformation (CCM) lesion development. The current study investigates changes in tight junction (TJ) complex organization when PDCD10 (CCM3) is mutated/depleted in human brain endothelial cells. Analysis of lesions with CCM3 mutation and brain endothelial cells transfected with CCM3 siRNA (CCM3-knockdown) showed little or no increase in TJ transmembrane and scaffolding proteins mRNA expression, but proteins levels were generally decreased. CCM3-knockdown cells had a redistribution of claudin-5 and occludin from the membrane to the cytosol with no alterations in protein turnover but with diminished protein-protein interactions with ZO-1 and ZO-1 interaction with the actin cytoskeleton. The most profound effect of CCM3 mutation/depletion was on an actin-binding protein, cortactin. CCM3 depletion caused cortactin Ser-phosphorylation, dissociation from ZO-1 and actin, redistribution to the cytosol and degradation. This affected cortical actin ring organization, TJ complex stability and consequently barrier integrity, with constant hyperpermeability to inulin. A potential link between CCM3 depletion and altered cortactin was tonic activation of MAP kinase ERK1/2. ERK1/2 inhibition increased cortactin expression and incorporation into the TJ complex and improved barrier integrity. This study highlights the potential role of CCM3 in regulating TJ complex organization and brain endothelial barrier permeability.
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Project description:Autism spectrum disorders (ASD) are complex conditions whose pathogenesis may be attributed to gene-environment interactions. There are no definitive mechanisms explaining how environmental triggers can lead to ASD although the involvement of inflammation and immunity has been suggested. Inappropriate antigen trafficking through an impaired intestinal barrier, followed by passage of these antigens or immune-activated complexes through a permissive blood-brain barrier (BBB), can be part of the chain of events leading to these disorders. Our goal was to investigate whether an altered BBB and gut permeability is part of the pathophysiology of ASD.Postmortem cerebral cortex and cerebellum tissues from ASD, schizophrenia (SCZ), and healthy subjects (HC) and duodenal biopsies from ASD and HC were analyzed for gene and protein expression profiles. Tight junctions and other key molecules associated with the neurovascular unit integrity and function and neuroinflammation were investigated.Claudin (CLDN)-5 and -12 were increased in the ASD cortex and cerebellum. CLDN-3, tricellulin, and MMP-9 were higher in the ASD cortex. IL-8, tPA, and IBA-1 were downregulated in SCZ cortex; IL-1b was increased in the SCZ cerebellum. Differences between SCZ and ASD were observed for most of the genes analyzed in both brain areas. CLDN-5 protein was increased in ASD cortex and cerebellum, while CLDN-12 appeared reduced in both ASD and SCZ cortexes. In the intestine, 75% of the ASD samples analyzed had reduced expression of barrier-forming TJ components (CLDN-1, OCLN, TRIC), whereas 66% had increased pore-forming CLDNs (CLDN-2, -10, -15) compared to controls.In the ASD brain, there is an altered expression of genes associated with BBB integrity coupled with increased neuroinflammation and possibly impaired gut barrier integrity. While these findings seem to be specific for ASD, the possibility of more distinct SCZ subgroups should be explored with additional studies.
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Project description:Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.
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Project description:The metalloprotease meprin β (Mep1b) is capable of cleaving cell-adhesion molecules in different tissues (e.g. skin, kidney and intestine) and is dysregulated in several diseases associated with barrier breakdown (Alzheimer´s disease, kidney disruption, inflammatory bowel disease). In this study, we demonstrate that Mep1b is a novel regulator of tight junction (TJ) composition and blood-brain barrier (BBB) integrity in brain endothelium. In Mep1b-transfected mouse brain endothelial cells (bEnd.3), we observed a reduction of the TJ protein claudin-5, decreased transendothelial electrical resistance (TEER) and an elevated permeability to paracellular diffusion marker [14C]-inulin. Analysis of global Mep1b knock-out (Mep1b-/-) mice showed increased TJ protein expression (claudin-5, occludin, ZO-1) in cerebral microvessels and increased TEER in cultivated primary mouse brain endothelial compared to wild-type (wt) mice. Furthermore, we investigated the IgG levels in cerebrospinal fluid (CSF) and the brain water content as additional permeability markers and detected lower IgG levels and reduced brain water content in Mep1b-/- mice compared to wt mice. Showing opposing features in overexpression and knock-out, we conclude that Mep1b plays a role in regulating brain endothelial TJ-proteins and therefore affecting BBB tightness in vitro and in vivo.
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Project description:The inflammatory response plays a pivotal role in Blood-Brain Barrier (BBB) destruction following ischemic brain injury. Enhanced leukocyte adhesion to vascular endothelial cells is an essential event in the inflammatory process. TMEM16A, a newly discovered protein regulating calcium-activated chloride channels, is widely expressed in eukaryotes. Recent studies have suggested that upregulated expression of TMEM16A is associated with the occurrence and development of many diseases. However, the role of TMEM16A in regulating BBB integrity after ischemic stroke has not been fully investigated. In this study, we found that TMEM16A is mainly expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. Caccinh-A01, an TMEM16A inhibitor that reduced its upregulation, attenuated brain infarct size and neurological deficits after ischemic stroke. ICAM-1 and MPO expression and BBB permeability were decreased after TMEM16A inhibitor administration. In addition, TMEM16A silencing rescued oxygen-glucose deprivation/reoxygenation (OGD/R)-induced transendothelial permeability in vitro accompanied by decreased ICAM-1 expression and leukocyte adhesion. Furthermore, our mechanistic study showed that TMEM16A knockdown alleviated NF-κB activation and nuclear translocation, indicating that TMEM16A knockdown downregulated OGD/R-induced ICAM-1 expression in an NF-κB-dependent manner. Finally, NF-κB inhibitor treatment also alleviated OGD/ R-induced BBB permeability, confirming that activated NF-κB and increased ICAM-1 are essential factors involved in ischemia-induced BBB damage. Thus, our research provides a promising treatment strategy against BBB destruction after ischemic stroke, and TMEM16A may become a potential target for the treatment of ischemic stroke.
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Project description:Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple. In vitro models of BBB are simpler, however they have limited functionality and relevance to disease processes. To address these limitations, we developed a 3-dimensional (3D) model of BBB on a microfluidic platform. We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants. Moreover, we verified the localization at endothelial cell boundaries of ZO-1 and VE-Cadherin, two components of tight and adherens junctions. To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic conditions and measured the protective function of antioxidant and ROCK-inhibitor treatments. Overall, our 3D BBB model provides a robust platform, adequate for detailed functional studies of BBB and for the screening of BBB-targeting drugs in neurological diseases.
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Project description:Breakdown of the blood-brain barrier (BBB) and increased immune cell trafficking into the central nervous system (CNS) are hallmarks of the pathogenesis of multiple sclerosis (MS). Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is expressed on cells of the vascular compartment and regulates vascular integrity and immune cell trafficking. Involvement of PECAM-1 in MS pathogenesis has been suggested by the detection of increased levels of soluble PECAM-1 (sPECAM-1) in the serum and CSF of MS patients. Here, we report profound upregulation of cell-bound PECAM-1 in initial (pre-phagocytic) white matter as well as active cortical gray matter MS lesions. Using a human in vitro BBB model we observed that PECAM-1 is not essential for the transmigration of human CD4+ T-cell subsets (Th1, Th1*, Th2, and Th17) across the BBB. Employing an additional in vitro BBB model based on primary mouse brain microvascular endothelial cells (pMBMECs) we show that the lack of endothelial PECAM-1 impairs BBB properties as shown by reduced transendothelial electrical resistance (TEER) and increases permeability for small molecular tracers. Investigating T-cell migration across the BBB under physiological flow by in vitro live cell imaging revealed that absence of PECAM-1 in pMBMECs did not influence arrest, polarization, and crawling of effector/memory CD4+ T cells on the pMBMECs. Absence of endothelial PECAM-1 also did not affect the number of T cells able to cross the pMBMEC monolayer under flow, but surprisingly favored transcellular over paracellular T-cell diapedesis. Taken together, our data demonstrate that PECAM-1 is critically involved in regulating BBB permeability and although not required for T-cell diapedesis itself, its presence or absence influences the cellular route of T-cell diapedesis across the BBB. Upregulated expression of cell-bound PECAM-1 in human MS lesions may thus reflect vascular repair mechanisms aiming to restore BBB integrity and paracellular T-cell migration across the BBB as it occurs during CNS immune surveillance.
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