Defective Blood-brain Barrier Research Articles

Unraveling the link between neuropathy target esterase NTE/SWS, lysosomal storage diseases, inflammation, abnormal fatty acid metabolism, and leaky brain barrier.

Mutations in Drosophila Swiss cheese (SWS) gene or its vertebrate orthologue neuropathy target esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.

Astrocyte Involvement in Blood-Brain Barrier Function: A Critical Update Highlighting Novel, Complex, Neurovascular Interactions.

Neurological disorders have been linked to a defective blood-brain barrier (BBB), with dysfunctions triggered by stage-specific disease mechanisms, some of these being generated through interactions in the neurovascular unit (NVU). Advanced knowledge of molecular and signaling mechanisms in the NVU and the emergence of improved experimental models allow BBB permeability prediction and the development of new brain-targeted therapies. As NVU constituents, astrocytes are the most numerous glial cells, characterized by a heterogeneity that occurs as a result of developmental and context-based gene expression profiles and the differential expression of non-coding ribonucleic acids (RNAs). Due to their heterogeneity and dynamic responses to different signals, astrocytes may have a beneficial or detrimental role in the BBB's barrier function, with deep effects on the pathophysiology of (and on the progression of) central nervous system diseases. The implication of astrocytic-derived extracellular vesicles in pathological mechanisms, due to their ability to pass the BBB, must also be considered. The molecular mechanisms of astrocytes' interaction with endothelial cells at the BBB level are considered promising therapeutic targets in different neurological conditions. Nevertheless, a personalized and well-founded approach must be addressed, due to the temporal and spatial heterogeneity of reactive astrogliosis states during disease.

20 kDa isoform of connexin-43 augments spatial reorganization of the brain endothelial junctional complex and lesion leakage in cerebral cavernous malformation type-3

Cerebral cavernous malformation type-3 (CCM3) is a type of brain vascular malformation caused by mutations in programmed cell death protein-10 (PDCD10). It is characterized by early life occurrence of hemorrhagic stroke and profound blood-brain barrier defects. The pathogenic mechanisms responsible for microvascular hyperpermeability and lesion progression in CCM3 are still largely unknown. The current study examined brain endothelial barrier structural defects formed in the absence of CCM3 in vivo and in vitro that may lead to CCM3 lesion leakage. We found significant upregulation of a 20 kDa isoform of connexin 43 (GJA1–20 k) in brain endothelial cells (BEC) in both non-leaky and leaky lesions, as well as in an in vitro CCM3 knockdown model (CCM3KD-BEC). Morphological, biochemical, FRET, and FRAP analyses of CCM3KD-BEC found GJA1–20 k regulates full-length GJA1 biogenesis, prompting uncontrolled gap junction growth. Furthermore, by binding to a tight junction scaffolding protein, ZO-1, GJA1–20 k interferes with Cx43/ZO-1 interactions and gap junction/tight junction crosstalk, promoting ZO-1 dissociation from tight junction complexes and diminishing claudin-5/ZO-1 interaction. As a consequence, the tight junction complex is destabilized, allowing “replacement” of tight junctions with gap junctions leading to increased brain endothelial barrier permeability. Modifying cellular levels of GJA1–20 k rescued brain endothelial barrier integrity re-establishing the spatial organization of gap and tight junctional complexes. This study highlights generation of potential defects at the CCM3-affected brain endothelial barrier which may underlie prolonged vascular leakiness.

Ferroptosis of brain microvascular endothelial cells contributes to hypoxia-induced blood-brain barrier injury.

Hypoxia is pivotal to the pathogeneses of myriad disorders, especially hypoxic cerebropathy. Much is known about the damage to the blood-brain barrier (BBB) in response to hypoxia. Studies have shown that endothelial cell death is closely linked to functional impairment of BBB. Mounting evidences have demonstrated that ferroptosis, a new pathway regulating cell death, is implicated in brain injury. However, whether ferroptosis is involved in hypoxia-induced BBB disruption remains ambiguous. Here, we utilized in vivo zebrafish and in vitro bEnd.3 cells to explore the correlation between endothelial ferroptosis and hypoxia-induced BBB damage. We found that hypoxic treatment for 45 min can induce BBB disruption by triggering down-regulation of claudin-5 (CLDN5) both in zebrafish cerebrovascluar endothelial cells and bEnd.3 cells. Besides, in vitro and in vivo studies revealed the cysteine/glutamate antiporter xCT (also known as solute carrier family 7 member 11; SLC7A11) decrease, glutathione peroxidase 4 (GPX4) and glutathione (GSH) reduction, 4-Hydroxynonenal (4-HNE) increasement, malondialdehyde (MDA) upregulation and reactive oxygen species (ROS) accumulation in hypoxia group. Further mechanism studies indicated that hypoxia-induced BBB damage might associate with microvascular endothelial cellular ferroptosis, since hypoxic exposure significantly activated the expression of ferroptosis-related genes (Ptgs2, Por, Lpcat3, Alox5, Alox12, Nfe2l2, and Ncoa4) and inhibited the expression of Slc7a11. Additionally, the application of 20 μM ferrostatin-1 (Fer-1), a ferroptosis inhibitor, could partially alleviate BBB disruption under hypoxia, suggesting that inhibition of ferroptosis might be a potential strategy for some neurological diseases with BBB defect.

131 Hedgehog Signaling Mediates Astrocytic Response and Blood-Brain Barrier Repair Following Traumatic Central Nervous System Injury

INTRODUCTION: Injury to the central nervous system (CNS) provokes a multicellular response to facilitate restoration of tissue homeostasis. Astrocytes consolidate at the lesion site to form a “glial scar” which restricts leukocyte infiltration and repairs the blood-brain barrier (BBB). However, this “scar” has been considered an impediment to regeneration and functional recovery. Therefore, uncovering molecular regulators that may be therapeutically targeted to promote pro-regenerative astrocyte phenotypes is of great interest. METHODS: Genetic reporter and conditional mutant mice were used to monitor, constitutively activate, or ablate Hh signaling following stereotactically-induced penetrating cortical or spinal cord injury. Outcomes including reactive astrocyte proliferation and polarization, leukocyte restriction, and blood-brain barrier repair were assessed via immunohistochemistry. RESULTS: Astrocytic expression of the Hh target gene Gli1 was downregulated acutely following injury but restored in the subacute phase. Constitutive activation of Hh signaling reduced astrocytic proliferation (%BrdU/EdU+ = 9.6% vs. 51.0%, p<0.01) and polarization (L/W Ratio = 2.8 vs. 1.5, p < 0.01). Additionally, in the spinal cord, constitutive activation impaired migration and integration of reactive astrocytes into the white matter lesion site. In animals with intact Hh signaling, the BBB recovered by 28 days after injury as evidenced by low levels of plasma protein and leukocyte infiltration. In contrast, conditional ablation of Hh signaling from 14-16 days after injury resulted in persistent BBB defects at 28 days. CONCLUSIONS: Our results suggest that acute downregulation of Hh signaling is required to specify reactive astrocyte properties including polarization and cell cycle re-entry. However, restoration of Hh signaling, which occurs subacutely from 10-28 days after injury and coincides with glial scar maturation, is required for leukocyte restriction and BBB repair. Understanding the temporospatial dynamics of postinjury Hh signaling is essential to modeling future therapeutic targets.

Loss of function of CMPK2 causes mitochondria deficiency and brain calcification

Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.

Single-cell transcriptomics reveals functionally specialized vascular endothelium in brain.

The blood-brain barrier (BBB) limits the entry of leukocytes and potentially harmful substances from the circulation into the central nervous system (CNS). While BBB defects are a hallmark of many neurological disorders, the cellular heterogeneity at the neurovascular interface and the mechanisms governing neuroinflammation are not fully understood. Through single cell RNA sequencing of non-neuronal cell populations of the murine cerebral cortex during development, adulthood, ageing and neuroinflammation, we identify reactive endothelial venules (REVs), a compartment of specialised post-capillary endothelial cells (ECs) that are characterized by consistent expression of cell adhesion molecules, preferential leukocyte transmigration, association with perivascular macrophage populations, and endothelial activation initiating CNS immune responses. Our results provide novel insights into the heterogeneity of the cerebral vasculature and a useful resource for the molecular alterations associated with neuroinflammation and ageing.

Evaluation of Changes in Peripheric Biomarkers Related to Blood Brain Barrier Damage in Patients with Schizophrenia and Their Correlation with Symptoms.

ObjectiveThe aim of the study was to evaluate the levels of peripheric biomarkers that have been associated with blood brain barrier (BBB) damage in healthy controls and two groups of patients with schizophrenia, those who received typical-atypical antipsychotics and those who received only atypical antipsychotics. Additionally, we sought relationships between these biomarkers and schizophrenia symptoms.MethodsThis study was conducted with the inclusion of 41 healthy volunteers and 75 patients with schizophrenia. The biomarkers measured to evaluate BBB injury were as follows spectrin breakdown product 145 (SBDP145), spectrin breakdown product 150 (SBDP150), ubiquitin carboxy terminal hydrolase L1 (UCHL1), ubiquitin ligase cullin-3 (cullin), occludin and claudin, which were measured via ELISA. Symptoms of patients with schizophrenia were evaluated with the Scale for the Assessment of Positive Symptoms (SAPS), Scale for the Assessment of Negative Symptoms, the Clinical Global Impression Scale (CGI), and the general assessment of functionality (GAF).ResultsCompared to controls, SBDP145 (p = 0.022) and cullin (p = 0.046) levels were significantly higher in patients with schizophrenia receiving atypical antipsychotic treatment. SBDP150 levels were lower in the combination treatment group compared to the control group (p = 0.022). Claudin (p = 0.804), occludin (p = 0.058) and UCHL1 (p = 0.715) levels were similar among groups. In recipients of combination treatment, SBDP145 levels were found to be positively correlated with SAPS-total (r = 0.440, p = 0.036) and SAPS-delusions (r = 0.494, p = 0.017) scores.ConclusionThe relationships demonstrated in this study indicate that more comprehensive research is needed to understand whether BBB defects contribute to clinical characteristics in patients with schizophrenia.

Schizophrenia-derived hiPSC brain microvascular endothelial-like cells show impairments in angiogenesis and blood-brain barrier function.

Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. Long-standing clinical observations and molecular data have pointed to a possible vascular deficiency that could be acting synergistically with neuronal dysfunction in SZ. As SZ is a neurodevelopmental disease, the use of human-induced pluripotent stem cells (hiPSC) allows disease biology modeling while retaining the patient's unique genetic signature. Previously, we reported a VEGFA signaling impairment in SZ-hiPSC-derived neural lineages leading to decreased angiogenesis. Here, we present a functional characterization of SZ-derived brain microvascular endothelial-like cells (BEC), the counterpart of the neurovascular crosstalk, revealing an intrinsically defective blood-brain barrier (BBB) phenotype. Transcriptomic assessment of genes related to endothelial function among three control (Ctrl BEC) and five schizophrenia patients derived BEC (SZP BEC), revealed that SZP BEC have a distinctive expression pattern of angiogenic and BBB-associated genes. Functionally, SZP BEC showed a decreased angiogenic response in vitro and higher transpermeability than Ctrl BEC. Immunofluorescence staining revealed less expression and altered distribution of tight junction proteins in SZP BEC. Moreover, SZP BEC's conditioned media reduced barrier capacities in the brain microvascular endothelial cell line HCMEC/D3 and in an in vivo permeability assay in mice. Overall, our results describe an intrinsic failure of SZP BEC for proper barrier function. These findings are consistent with the hypothesis tracing schizophrenia origins to brain development and BBB dysfunction.

Endothelial Unc5B controls blood-brain barrier integrity

Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we show that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/β-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/β-catenin signaling, and β-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/β-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases.

Inactivation of Hedgehog signal transduction in adult astrocytes results in region-specific blood–brain barrier defects

In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of Gli1, a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., Science 334, 1727-1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS.

Defective Function of the Blood-Brain Barrier in a Stroke-Prone Spontaneously Hypertensive Rat: Evaluation in an In Vitro Cell Culture Model.

The blood-brain barrier (BBB) comprises three cell types: brain capillary endothelial cells (BECs), astrocytes, and pericytes. Abnormal interaction among these cells may induce BBB dysfunction and lead to cerebrovascular diseases. The stroke-prone spontaneously hypertensive rat (SHRSP) harbors a defective BBB, so we designed the present study to examine the role of these three cell types in a functional disorder of the BBB in SHRSP in order to elucidate the role of these cells in the BBB more generally. To this end, we employed a unique in vitro model of BBB, in which various combinations of the cells could be tested. The three types of cells were prepared from both SHRSPs and Wistar Kyoto rats (WKYs). They were then co-cultured in various combinations to construct in vitro BBB models. The barrier function of the models was estimated by measuring transendothelial electrical resistance and the permeability of the endothelial monolayer to sodium fluorescein. The in vitro models revealed that (1) BECs from SHRSPs had an inherent lower barrier function, (2) astrocytes of SHRSPs had an impaired ability to induce barrier function in BECs, although (3) both pericytes and astrocytes of SHRSPs and WKYs could potentiate the barrier function of BECs under co-culture conditions. Furthermore, we found that claudin-5 expression was consistently lower in models that used BECs and/or SHRSP astrocytes. These results suggested that defective interaction among BBB cells-especially BECs and astrocytes-was responsible for a functional disorder of the BBB in SHRSPs.

Inflammation-induced endothelial to mesenchymal transition promotes brain endothelial cell dysfunction and occurs during multiple sclerosis pathophysiology

The blood-brain barrier (BBB) has a major role in maintaining brain homeostasis through the specialized function of brain endothelial cells (BECs). Inflammation of the BECs and loss of their neuroprotective properties is associated with several neurological disorders, including the chronic neuro-inflammatory disorder multiple sclerosis (MS). Yet, the underlying mechanisms of a defective BBB in MS remain largely unknown. Endothelial to mesenchymal transition (EndoMT) is a pathophysiological process in which endothelial cells lose their specialized function and de-differentiate into mesenchymal cells. This transition is characterized by an increase in EndoMT-related transcription factors (TFs), a downregulation of brain endothelial markers, and an upregulation of mesenchymal markers accompanied by morphological changes associated with cytoskeleton reorganization. Here, we postulate that EndoMT drives BEC de-differentiation, mediates inflammation-induced human BECs dysfunction, and may play a role in MS pathophysiology. We provide evidence that stimulation of human BECs with transforming growth factor (TGF)-β1 and interleukin (IL)-1β promotes EndoMT, a process in which the TF SNAI1, a master regulator of EndoMT, plays a crucial role. We demonstrate the involvement of TGF-β activated kinase 1 (TAK1) in EndoMT induction in BECs. Finally, immunohistochemical analysis revealed EndoMT-associated alterations in the brain vasculature of human post-mortem MS brain tissues. Taken together, our novel findings provide a better understanding of the molecular mechanisms underlying BECs dysfunction during MS pathology and can be used to develop new potential therapeutic strategies to restore BBB function.

Interplay of the Norrin and Wnt7a/Wnt7b signaling systems in blood–brain barrier and blood–retina barrier development and maintenance

β-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.

Caveolin-1 in Stroke Neuropathology and Neuroprotection: A Novel Molecular Therapeutic Target for Ischemic-Related Injury.

Cardiovascular disease and associated cerebral stroke are a global epidemic attributed to genetic and epigenetic factors, such as diet, life style and an increasingly sedentary existence due to technological advances in both the developing and developed world. There are approximately 5.9 million stroke-related deaths worldwide annually. Current epidemiological data indicate that nearly 16.9 million people worldwide suffer a new or recurrent stroke yearly. In 2014 alone, 2.4% of adults in the United States (US) were estimated to experience stroke, which is the leading cause of adult disability and the fifth leading cause of death in the US There are 2 main types of stroke: Hemorrhagic (HS) and ischemic stroke (IS), with IS occurring more frequently. HS is caused by intra-cerebral hemorrhage mainly due to high blood pressure, while IS is caused by either embolic or thrombotic stroke. Both result in motor impairments, numbness or abnormal sensations, cognitive deficits, and mood disorders (e.g. depression). This review focuses on the 1) pathophysiology of stroke (neuronal cell loss, defective blood brain barrier, microglia activation, and inflammation), 2) the role of the membrane protein caveolin- 1 (Cav-1) in normal brain physiology and stroke-induced changes, and, 3) we briefly discussed the potential therapeutic role of Cav-1 in recovery following stroke.

The effect of APOE genotype on Alzheimer's disease risk is influenced by sex and docosahexaenoic acid status.

An apolipoprotein E ε4 (APOE-ε4) genotype is the strongest common genetic determinant of Alzheimer's disease (AD). The pleiotropic nature of apolipoprotein E has made elucidation of the aetiological basis difficult to establish, which is further complicated by the fact that the penetrance of the APOE-ε4 allele is modulated by sex, age, and nutrition. A greater metabolic consequence of the APOE-ε4 allele is likely to contribute to the fact that two-thirds of AD patients are female. A higher tissue status of the marine n-3 fatty acid docosahexaenoic acid (DHA) is associated with a lower AD risk. However, APOE-ε4 carriers appear less sensitive to the neurocognitive benefits, which may be due to defective blood-brain barrier transport of DHA exacerbated by aging and possibly sex. This suggests higher DHA requirements in this large population subgroup. This narrative review will consider the influence of sex and DHA in modulating APOE-ε4-mediated AD risk.

Endothelial \u03b16\u03b24 integrin protects during experimental autoimmune encephalomyelitis-induced neuroinflammation by maintaining vascular integrity and tight junction protein expression

BackgroundExtracellular matrix (ECM) proteins play critical functions regulating vascular formation and function. Laminin is a major component of the vascular basal lamina, and transgenic mice deficient in astrocyte or pericyte laminin show defective blood-brain barrier (BBB) integrity, indicating an important instructive role for laminin in cerebral blood vessels. As previous work shows that in the normal brain, vascular expression of the laminin receptor α6β4 integrin is predominantly restricted to arterioles, but induced on all vessels during neuroinflammation, it is important to define the role of this integrin in the maintenance of BBB integrity.Methodsα6β4 integrin expression was analyzed using dual immunofluorescence (dual-IF) of brain sections taken from the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To investigate the role of endothelial α6β4 integrin, transgenic mice lacking β4 integrin in endothelial cells (β4-EC-KO) and wild-type (WT) littermates were subject to EAE, and clinical score and various neuropathological parameters were examined by immunofluorescence. In addition, β4 integrin null brain endothelial cells (BECs) were examined in culture for expression of tight junction proteins using immunocytochemistry and flow cytometry.ResultsCerebrovascular expression of β4 integrin was markedly upregulated during EAE progression, such that by the acute stage of EAE (day 21), the vast majority of blood vessels expressed β4 integrin. In the EAE model, while the β4-EC-KO mice showed the same time of disease onset as the WT littermates, they developed significantly worse clinical disease over time, resulting in increased clinical score at the peak of disease and maintained elevated thereafter. Consistent with this, the β4-EC-KO mice showed enhanced levels of leukocyte infiltration and BBB breakdown and also displayed increased loss of the endothelial tight junction proteins claudin-5 and ZO-1. Under pro-inflammatory conditions, primary cultures of β4KO BECs also showed increased loss of claudin-5 and ZO-1 expression.ConclusionsTaken together, our data suggest that α6β4 integrin upregulation is an inducible protective mechanism that stabilizes the BBB during neuroinflammatory conditions.

Brain and Peripheral Atypical Inflammatory Mediators Potentiate Neuroinflammation and Neurodegeneration

Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson’s disease (PD), Alzheimer’s disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1–42 (Aβ1–42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can also enter into the peripheral system through defective BBB, recruit immune cells into the brain, and exacerbate neuroinflammation. We suggest that mast cell-associated inflammatory mediators from systemic inflammation and brain could augment neuroinflammation and neurodegeneration in the brain. This review article addresses the role of some atypical inflammatory mediators that are associated with mast cell inflammation and their activation of glial cells to induce neurodegeneration.

Dehydroepiandrosterone sulfate augments blood-brain barrier and tight junction protein expression in brain endothelial cells

Tight junctions (TJ) between brain endothelial cells are essential for formation and maintenance of the blood-brain barrier (BBB). Although loss of BBB integrity is associated with several neuropathological disorders, treatments that augment or stabilise the BBB are scarce. Here we show that physiological concentrations of dehydroepiandrosterone sulfate (DHEAS) stimulate the expression of the TJ proteins zonula occludens-1 (ZO-1) and claudin-3 in the brain-derived endothelial cell line bEnd.3 and promote TJ formation between neighbouring cells, demonstrated by augmented transendothelial resistance across cell monolayers. Silencing androgen receptor expression by siRNA does not prevent DHEAS-induced stimulation of ZO-1 expression, indicating that conversion of DHEAS into testosterone is not required for its actions. Suppression of Gnα11 expression by siRNA prevents DHEAS actions, pointing towards a G-protein-coupled receptor as being a mediator of the DHEAS effects. These results are consistent with the idea that DHEAS, acting as a hormone in its own right, supports the integrity of the BBB. The current findings might help in developing new strategies for the prevention or treatment of neurological disorders associated with BBB defects.

MiR-285–Yki/Mask double-negative feedback loop mediates blood–brain barrier integrity in Drosophila

The Hippo signaling pathway is highly conserved from Drosophila to mammals and plays a central role in maintaining organ size and tissue homeostasis. The blood-brain barrier (BBB) physiologically isolates the brain from circulating blood or the hemolymph system, and its integrity is strictly maintained to perform sophisticated neuronal functions. Until now, the underlying mechanisms of subperineurial glia (SPG) growth and BBB maintenance during development are not clear. Here, we report an miR-285-Yorkie (Yki)/Multiple Ankyrin repeats Single KH domain (Mask) double-negative feedback loop that regulates SPG growth and BBB integrity. Flies with a loss of miR-285 have a defective BBB with increased SPG ploidy and disruptive septate junctions. Mechanistically, miR-285 directly targets the Yki cofactor Mask to suppress Yki activity and down-regulates the expression of its downstream target cyclin E, a key regulator of cell cycle. Disturbance of cyclin E expression in SPG causes abnormal endoreplication, which leads to aberrant DNA ploidy and defective septate junctions. Moreover, the expression of miR-285 is increased by knockdown of yki or mask and is decreased with yki overexpression, thus forming a double-negative feedback loop. This regulatory loop is crucial for sustaining an appropriate Yki/Mask activity and cyclin E level to maintain SPG ploidy and BBB integrity. Perturbation of this signaling loop, either by dysregulated miR-285 expression or Yki activity, causes irregular SPG ploidy and BBB disruption. Furthermore, ectopic expression of miR-285 promotes canonical Hippo pathway-mediated apoptosis independent of the p53 or JNK pathway. Collectively, these results reveal an exquisite regulatory mechanism for BBB maintenance through an miR-285-Yki/Mask regulatory circuit.