Aging reduces calreticulin expression and alters spontaneous calcium signals in astrocytic endfeet of the mouse dorsolateral striatum

Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases risk for neurodegeneration. Among the various cells participating in BBB and NVU function, spontaneous Ca2+ signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with changes in spontaneous Ca2+ signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed a genetically encoded Ca2+ indicator, Lck-GCaMP6f in DLS astrocytes of young (3-4 month) and aging (20-24 month) mice. Compared to young mice, endfeet in the DLS of aging mice demonstrated a decrease in calreticulin (CALR) expression, and dramatic alterations in the dynamics of endfoot membrane-associated and mitochondrial Ca2+ signals. While young mice required both extracellular and endoplasmic reticulum (ER) Ca2+ sources for generating endfoot Ca2+ signals, aging mice showed exclusive dependence on ER Ca2+. These data suggest that aging is associated with significant changes in Ca2+ buffers and Ca2+ signals within astrocytic endfeet, which has important implications for understanding mechanisms involved in aging-related impairment of the BBB and NVU.

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Screening for Interacting Proteins with Peptide Biomarker of Blood–Brain Barrier Alteration under Inflammatory Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms22094725 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4725

Author(s):

Karina Vargas-Sanchez ◽

Monica Losada-Barragán ◽

Maria Mogilevskaya ◽

Susana Novoa-Herrán ◽

Yehidi Medina ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Specific Interaction ◽

Brain Barrier ◽

Mass Spectrometry Analysis ◽

Peptide Ligand ◽

Inflammatory Conditions ◽

Blood Brain ◽

Potential Biomarker

Neurodegenerative diseases are characterized by increased permeability of the blood–brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1β-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope–receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit β-1. Among them, only laminin subunit β-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit β-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.

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Time-dependent dual effect of NLRP3 inflammasome in brain ischemia

10.1101/2020.10.08.332007 ◽

2020 ◽

Author(s):

Alejandra Palomino-Antolin ◽

Paloma Narros-Fernández ◽

Víctor Farré-Alins ◽

Javier Sevilla-Montero ◽

Celine Decouty-Pérez ◽

...

Keyword(s):

Brain Injury ◽

Blood Brain Barrier ◽

Inflammatory Cytokines ◽

Nlrp3 Inflammasome ◽

Neurovascular Unit ◽

Time Dependent ◽

Brain Barrier ◽

Dependent Manner ◽

Dual Effect ◽

Blood Brain

AbstractBackground and purposePost-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit.MethodsWT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining.ResultsWe identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration.ConclusionsThese findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.

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Gastrodia-Uncaria Water Extract Inhibits Endoplasmic Reticulum Stress and Matrix Metalloproteinase in Protecting against Cerebral Ischemia

10.1101/2021.07.21.452303 ◽

2021 ◽

Author(s):

Angus Y Choi ◽

Jia Wen Xian ◽

Sum Yi Ma ◽

Zhixiu Lin ◽

Chun Wai Chan

Keyword(s):

Endoplasmic Reticulum ◽

Cerebral Ischemia ◽

Endoplasmic Reticulum Stress ◽

Blood Brain Barrier ◽

Neuroprotective Effect ◽

Water Extract ◽

Neuronal Cell ◽

Brain Barrier ◽

Brain Diseases ◽

Blood Brain

Stroke is the second leading cause of death in worldwide, in which cerebral ischemia accounts for 87% of all cases. The building up of endoplasmic reticulum stress in cerebral ischemia contributes to the disruption of blood brain barrier and neuronal cell death. The only FDA-approved drug, recombinant tissue plasminogen activator, is still of limited use due to the narrow window period and lack of neuroprotective effect. Therefore, it is necessary to explore alternative treatment on cerebral ischemia. Tianma-Gouteng decoction is a traditional Chinese Medicine prescription used to treat brain diseases in China. In this study, we investigated the neuroprotective effect of a water extract consisting of Gastrodia elata and Uncaria rhynchophylla, which are the two main herbs in the decoction. Cerebral ischemia was induced in rats using middle cerebral artery occlusion. GUW-treated rats have significantly reduced infarction volume and recovered neurological functions. The number of protein aggregates and caspase-12 positive cells were significantly inhibited. In vitro oxygen-glucose deprivation / reoxygenation stroke model demonstrated that the unfolded protein response proteins GRP78 and PDI were upregulated by GUW. Less ubiquitin puncta and normalized ubiquitin distribution indicated the reduction in endoplasmic reticulum stress. Furthermore, a lower Evan blue signal and MMPsense signal was observed, suggesting that GUW may preserve the blood brain barrier integrity through inhibiting MMP activity. Taken together, this suggested that GUW protected ischemic neurons and the blood brain barrier through inhibiting endoplasmic reticulum stress.

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Targeting microRNAs to Regulate the Integrity of the Blood–Brain Barrier

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.673415 ◽

2021 ◽

Vol 9 ◽

Author(s):

Juntao Wang ◽

Fang Xu ◽

Xiaoming Zhu ◽

Xianghua Li ◽

Yankun Li ◽

...

Keyword(s):

Blood Brain Barrier ◽

Neurovascular Unit ◽

Cerebrovascular Diseases ◽

Brain Parenchyma ◽

Brain Barrier ◽

Brain Diseases ◽

Blood Brain ◽

Recent Developments ◽

Important Regulator

The blood–brain barrier (BBB) is a highly specialized neurovascular unit that protects the brain from potentially harmful substances. In addition, the BBB also engages in the exchange of essential nutrients between the vasculature and brain parenchyma, which is critical for brain homeostasis. Brain diseases, including neurological disorders and cerebrovascular diseases, are often associated with disrupted BBB integrity, evidenced by increased permeability. Therefore, defining the mechanisms underlying the regulation of BBB integrity is crucial for the development of novel therapeutics targeting brain diseases. MicroRNAs (miRNA), a type of small non-coding RNAs, are emerging as an important regulator of BBB integrity. Here we review recent developments related to the role of miRNAs in regulating BBB integrity.

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Human pluripotent stem cell–derived brain pericyte–like cells induce blood-brain barrier properties

Science Advances ◽

10.1126/sciadv.aau7375 ◽

2019 ◽

Vol 5 (3) ◽

pp. eaau7375 ◽

Cited By ~ 40

Author(s):

Matthew J. Stebbins ◽

Benjamin D. Gastfriend ◽

Scott G. Canfield ◽

Ming-Song Lee ◽

Drew Richards ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Barrier Properties ◽

Cell Types ◽

Brain Barrier ◽

Human Model ◽

Blood Brain ◽

Brain Pericytes

Brain pericytes play important roles in the formation and maintenance of the neurovascular unit (NVU), and their dysfunction has been implicated in central nervous system disorders. While human pluripotent stem cells (hPSCs) have been used to model other NVU cell types, including brain microvascular endothelial cells (BMECs), astrocytes, and neurons, hPSC-derived brain pericyte–like cells have not been integrated into these models. In this study, we generated neural crest stem cells (NCSCs), the embryonic precursor to forebrain pericytes, from hPSCs and subsequently differentiated NCSCs to brain pericyte–like cells. These cells closely resembled primary human brain pericytes and self-assembled with endothelial cells. The brain pericyte–like cells induced blood-brain barrier properties in BMECs, including barrier enhancement and reduced transcytosis. Last, brain pericyte–like cells were incorporated with iPSC-derived BMECs, astrocytes, and neurons to form an isogenic human model that should prove useful for the study of the NVU.

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Neurovascular unit dysfunction with blood-brain barrier hyperpermeability contributes to major depressive disorder: a review of clinical and experimental evidence

Journal of Neuroinflammation ◽

10.1186/1742-2094-10-142 ◽

2013 ◽

Vol 10 (1) ◽

Cited By ~ 83

Author(s):

Souhel Najjar ◽

Daniel M Pearlman ◽

Orrin Devinsky ◽

Amanda Najjar ◽

David Zagzag

Keyword(s):

Major Depressive Disorder ◽

Depressive Disorder ◽

Experimental Evidence ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Brain Barrier ◽

Major Depressive ◽

Blood Brain

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Lipid Reshaping and Lipophagy Are Induced in a Modeled Ischemia-Reperfusion Injury of Blood Brain Barrier

International Journal of Molecular Sciences ◽

10.3390/ijms20153752 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3752 ◽

Cited By ~ 3

Author(s):

Elena Lonati ◽

Paola Antonia Corsetto ◽

Gigliola Montorfano ◽

Stefania Zava ◽

Tatiana Carrozzini ◽

...

Keyword(s):

Oxidative Stress ◽

Blood Brain Barrier ◽

Ischemia Reperfusion Injury ◽

Neutral Lipids ◽

Ischemia Reperfusion ◽

Neurovascular Unit ◽

Standard Condition ◽

Brain Barrier ◽

Inflammatory Status ◽

Blood Brain

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia.

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Blood-Brain Barrier Damage in Ischemic Stroke and Its Regulation by Endothelial Mechanotransduction

Frontiers in Physiology ◽

10.3389/fphys.2020.605398 ◽

2020 ◽

Vol 11 ◽

Author(s):

Keqing Nian ◽

Ian C. Harding ◽

Ira M. Herman ◽

Eno E. Ebong

Keyword(s):

Ischemic Stroke ◽

Blood Brain Barrier ◽

Therapeutic Potential ◽

Current Knowledge ◽

Neurovascular Unit ◽

The United States ◽

Brain Barrier ◽

Endothelial Glycocalyx ◽

Altered Expression ◽

Blood Brain

Ischemic stroke, a major cause of mortality in the United States, often contributes to disruption of the blood-brain barrier (BBB). The BBB along with its supportive cells, collectively referred to as the “neurovascular unit,” is the brain’s multicellular microvasculature that bi-directionally regulates the transport of blood, ions, oxygen, and cells from the circulation into the brain. It is thus vital for the maintenance of central nervous system homeostasis. BBB disruption, which is associated with the altered expression of tight junction proteins and BBB transporters, is believed to exacerbate brain injury caused by ischemic stroke and limits the therapeutic potential of current clinical therapies, such as recombinant tissue plasminogen activator. Accumulating evidence suggests that endothelial mechanobiology, the conversion of mechanical forces into biochemical signals, helps regulate function of the peripheral vasculature and may similarly maintain BBB integrity. For example, the endothelial glycocalyx (GCX), a glycoprotein-proteoglycan layer extending into the lumen of bloods vessel, is abundantly expressed on endothelial cells of the BBB and has been shown to regulate BBB permeability. In this review, we will focus on our understanding of the mechanisms underlying BBB damage after ischemic stroke, highlighting current and potential future novel pharmacological strategies for BBB protection and recovery. Finally, we will address the current knowledge of endothelial mechanotransduction in BBB maintenance, specifically focusing on a potential role of the endothelial GCX.

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Neurological Diseases in Relation to the Blood–Brain Barrier

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2011.197 ◽

2012 ◽

Vol 32 (7) ◽

pp. 1139-1151 ◽

Cited By ~ 226

Author(s):

Gary A Rosenberg

Keyword(s):

Free Radicals ◽

Blood Brain Barrier ◽

Neurological Diseases ◽

Neurovascular Unit ◽

Brain Barrier ◽

Cellular Damage ◽

Brain Diseases ◽

Acute Injury ◽

Blood Brain ◽

The Brain

Disruption of the blood–brain barrier (BBB) has an important part in cellular damage in neurological diseases, including acute and chronic cerebral ischemia, brain trauma, multiple sclerosis, brain tumors, and brain infections. The neurovascular unit (NVU) forms the interface between the blood and brain tissues. During an injury, the cascade of molecular events ends in the final common pathway for BBB disruption by free radicals and proteases, which attack membranes and degrade the tight junction proteins in endothelial cells. Free radicals of oxygen and nitrogen and the proteases, matrix metalloproteinases and cyclooxgyenases, are important in the early and delayed BBB disruption as the neuroinflammatory response progresses. Opening of the BBB occurs in neurodegenerative diseases and contributes to the cognitive changes. In addition to the importance of the NVU in acute injury, angiogenesis contributes to the recovery process. The challenges to treatment of the brain diseases involve not only facilitating drug entry into the brain, but also understanding the timing of the molecular cascades to block the early NVU injury without interfering with recovery. This review will describe the molecular and cellular events associated with NVU disruption and potential strategies directed toward restoring its integrity.

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