isolation-of-cerebral-capillaries-from-fresh-human-brain-tissue

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

University of Kentucky

The blood-brain barrier (BBB) is the critical structure for preventing human immunodeficiency virus (HIV) trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1-infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMECs). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMECs. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions on Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients.

Caveolin-1 regulates human immunodeficiency virus-1 Tat-induced alterations of tight junction protein expression via modulation of the Ras signaling.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors which down-regulate inflammatory signaling pathways. Therefore, we hypothesized that alterations of PPAR functions can contribute to human immunodeficiency virus-1 (HIV-1)-induced dysfunction of brain endothelial cells. Indeed, treatment with HIV-1 transactivator of transcription (Tat) protein decreased PPAR transactivation in brain endothelial cells. We next stably over-expressed PPARalpha and PPARgamma in a newly developed cell line of human brain endothelial cells (hCMEC/D3 cells). Tat-induced up-regulation of inflammatory mediators, such as interleukin (IL)-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin were markedly attenuated in hCMEC/D3 over-expressing PPARalpha or PPARgamma. These results were confirmed in CCL2 and E-selectin promoter activity studies. Similar protective effects were observed in hCMEC/D3 after activation of PPARgamma by exogenous PPAR agonists (dPGJ(2) and rosiglitazone). PPAR over-expression also prevented Tat-induced binding activity and transactivation of nuclear factor-kappaB. Importantly, increased PPAR activity attenuated induction of IL-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin in hCMEC/D3 cells co-cultured with HIV-1-infected Jurkat cells. The protective effects of PPAR over-expression were reversed by the antagonists of PPARalpha (MK886) or PPARgamma (GW9662). The present data suggest that targeting PPAR signaling may provide a novel therapeutic approach to attenuate HIV-1-induced local inflammatory responses in brain endothelial cells.

PPARalpha and PPARgamma effectively protect against HIV-induced inflammatory responses in brain endothelial cells.

The Rho signaling has an essential function in human immunodeficiency virus (HIV)-1-mediated disruption of the integrity of the blood-brain barrier (BBB). However, it is unknown how membrane domains, such as lipid rafts, can influence HIV-1-mediated activation of the Rho pathway and how these processes can affect the expression of the efflux transporters at the BBB level. This study is focused on the function of HIV-1 protein Tat in activation of the Rho signaling and upregulation of P-glycoprotein (P-gp) in human brain endothelial cells. Treatment with Tat markedly elevated GTP-RhoA levels and the potential downstream effectors, such as myosin phosphatase target subunit 1 and myosin light chain. In addition, Tat upregulated expression and promoter activity of P-gp as well as its efflux function. Inhibition of the Rho signaling cascade effectively blocked P-gp overexpression at the level of promoter activity. Disruption of lipid rafts by depletion of membrane cholesterol by methyl-beta-cyclodextrin, but not caveolin-1 silencing, also abolished Tat-mediated RhoA activation and P-gp upregulation. The present data indicate the critical function of intact lipid rafts and the Rho signaling in HIV-1-mediated upregulation of P-gp and potential development of drug resistance in brain endothelial cells.

Intact lipid rafts regulate HIV-1 Tat protein-induced activation of the Rho signaling and upregulation of P-glycoprotein in brain endothelial cells.

HIV-1-infected brains are characterized by increased amyloid deposition. To study the influence of HIV-1 on amyloid beta (Abeta) homeostasis at the blood-brain barrier (BBB) level, we employed a model of brain microvascular endothelial cells exposed to HIV-1 in the presence or absence of Abeta. HIV-1 markedly increased endogenous Abeta levels and elevated accumulation of exogenous Abeta. Simvastatin, the HMG-CoA reductase inhibitor, blocked these effects. We next evaluated the effects of HIV-1 and/or simvastatin on expression of the receptor for lipoprotein related protein (LRP1) and the receptor for advanced glycation end products (RAGE), known to regulate Abeta transport across the BBB. LRP1 expression was not affected by HIV-1; however, it was increased by simvastatin. Importantly, simvastatin attenuated HIV-1-induced RAGE expression. These results suggest that HIV-1 may directly contribute to Abeta accumulation at the BBB level. In addition, statins may protect against increased Abeta levels associated with HIV-1 infection in the brain.

HIV-1-induced amyloid beta accumulation in brain endothelial cells is attenuated by simvastatin.

The human immunodeficiency virus (HIV)-specific protein trans-activator of transcription (Tat) can contribute to the dysfunction of brain endothelial cells and HIV trafficking into the brain by disrupting tight junction (TJ) integrity at the blood-brain barrier (BBB) level. Specific TJ proteins, such as zonula occludens (ZO) proteins, localize not only at the cell-cell borders but are also present in the nuclei. The objective of the present study was to evaluate the mechanisms and significance of Tat-induced nuclear localization of ZO-1. Treatment of a brain endothelial cell line (hCMEC/D3 cells) with Tat resulted in a decrease in total levels of ZO-1 but significantly upregulated ZO-1 protein expression in the nuclei. In addition, exposure to Tat stimulated Rho signaling and induced phosphorylation and activity of transcription factor cAMP response element-binding protein (CREB), binding sites that have been identified in the proximal region of the ZO-1 promoter. Interestingly, inhibition of the Rho cascade protected against Tat-induced upregulation of ZO-1 in the nuclei and activation of CREB. Depletion of CREB by infection of cells with specific shRNA lentiviral particles attenuated both Tat-induced Rho signaling and nuclear targeting of ZO-1. A decrease in CREB levels also attenuated Tat-induced endothelial and BBB hyperpermeability as well as transendothelial migration of monocytic cells. The role of CREB in Tat-mediated alterations of ZO-1 was confirmed in brain microvessels in mice with CREB shRNA lentiviral particles injected into the cerebral circulation. The present results indicate the crucial role of Rho signaling and CREB in modulation of nuclear localization of ZO-1 and maintaining the integrity of endothelial monolayers.

HIV-1 Tat triggers nuclear localization of ZO-1 via Rho signaling and cAMP response element-binding protein activation.

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1(-/-);Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.

Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels.

Autophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling cascades and requires proteases, acidic compartments, and membrane fusion. To assess the physiological role of platelet autophagy, we generated a mouse strain with a megakaryocyte- and platelet-specific deletion of Atg7, an enzyme required for LC3II production. Ex vivo analysis of platelets from these mice shows modest defects in aggregation and granule cargo packaging. Although these mice have normal platelet numbers and size distributions, they exhibit a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Our results demonstrate that autophagy occurs in platelets and is important for hemostasis and thrombosis. 

Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis.

Failure to clear amyloid-β (Aβ) from the brain is in part responsible for Aβ brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aβ across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp) in the luminal plasma membrane of the brain capillary endothelium. P-gp is reduced at the blood-brain barrier in AD, which has been shown to be associated with Aβ brain accumulation. However, the mechanism responsible for P-gp reduction in AD is not well understood. Here we focused on identifying critical mechanistic steps involved in reducing P-gp in AD. We exposed isolated rat brain capillaries to 100 nm Aβ40, Aβ40, aggregated Aβ40, and Aβ42. We observed that only Aβ40 triggered reduction of P-gp protein expression and transport activity levels; this occurred in a dose- and time-dependent manner. To identify the steps involved in Aβ-mediated P-gp reduction, we inhibited protein ubiquitination, protein trafficking, and the ubiquitin-proteasome system, and monitored P-gp protein expression, transport activity, and P-gp-ubiquitin levels. Thus, exposing brain capillaries to Aβ40 triggers ubiquitination, internalization, and proteasomal degradation of P-gp. These findings may provide potential therapeutic targets within the blood-brain barrier to limit P-gp degradation in AD and improve Aβ brain clearance.

Aβ40 Reduces P-Glycoprotein at the Blood-Brain Barrier through the Ubiquitin-Proteasome Pathway.

Neurovascular integrity plays an important role in protecting cognitive and mental health in aging. Lifestyle interventions that sustain neurovascular integrity may thus be critical on preserving brain functions in aging and reducing the risk for age-related neurodegenerative disorders. Here we show that caloric restriction (CR) had an early effect on neurovascular enhancements, and played a critical role in preserving vascular, cognitive and mental health in aging. In particular, we found that CR significantly enhanced cerebral blood flow (CBF) and blood-brain barrier function in young mice at 5-6 months of age. The neurovascular enhancements were associated with reduced mammalian target of rapamycin expression, elevated endothelial nitric oxide synthase signaling, and increased ketone bodies utilization. With age, CR decelerated the rate of decline in CBF. The preserved CBF in hippocampus and frontal cortex were highly correlated with preserved memory and learning, and reduced anxiety, of the aging mice treated with CR (18-20 months of age). Our results suggest that dietary intervention started in the early stage (e.g., young adults) may benefit cognitive and mental reserve in aging. Understanding nutritional effects on neurovascular functions may have profound implications in human brain aging and age-related neurodegenerative disorders.

Caloric restriction preserves memory and reduces anxiety of aging mice with early enhancement of neurovascular functions.

A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood-brain barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood-brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood-brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood-brain barrier. This approach can be extended to other blood-brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.

P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy.

Abdominal aortic aneurysm (AAA) has high mortality rate when ruptured, but currently, there is no proven pharmacological therapy for AAA because of our poor understanding of its pathogenesis. The current study explored a novel role of smooth muscle cell (SMC) BMAL1 (brain and muscle Arnt-like protein-1)-a transcription factor known to regulate circadian rhythm-in AAA development.

Deletion of BMAL1 in Smooth Muscle Cells Protects Mice From Abdominal Aortic Aneurysms.

One characteristic of Alzheimer's disease (AD) is excessive accumulation of amyloid-β (Aβ) in the brain. Aβ brain accumulation is, in part, due to a reduction in Aβ clearance from the brain across the blood-brain barrier. One key element that contributes to Aβ brain clearance is P-glycoprotein (P-gp) that transports Aβ from brain to blood. In AD, P-gp protein expression and transport activity levels are significantly reduced, which impairs Aβ brain clearance. The mechanism responsible for reduced P-gp expression and activity levels is poorly understood. We recently demonstrated that Aβ triggers P-gp degradation through the ubiquitin-proteasome pathway. Consistent with these data, we show here that ubiquitinated P-gp levels in brain capillaries isolated from brain samples of AD patients are increased compared to capillaries isolated from brain tissue of cognitive normal individuals. We extended this line of research to  studies using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576) that were treated with PYR41, a cell-permeable, irreversible inhibitor of the ubiquitin-activating enzyme E1. Our data show that inhibiting P-gp ubiquitination protects the transporter from degradation, and immunoprecipitation experiments confirmed that PYR41 prevented P-gp ubiquitination. We further found that PYR41 treatment prevented reduction of P-gp protein expression and transport activity levels and substantially lowered Aβ brain levels in hAPP mice. Together, our findings provide  proof that the ubiquitin-proteasome system mediates reduction of blood-brain barrier P-gp in AD and that inhibiting P-gp ubiquitination prevents P-gp degradation and lowers Aβ brain levels. Thus, targeting the ubiquitin-proteasome system may provide a novel therapeutic approach to protect blood-brain barrier P-gp from degradation in AD and other Aβ-based pathologies.

Article	Year
Caveolin-1 regulates human immunodeficiency virus-1 Tat-induced alterations of tight junction protein expression via modulation of the Ras signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 18667611	2008
PPARalpha and PPARgamma effectively protect against HIV-induced inflammatory responses in brain endothelial cells. Journal of neurochemistry\| PubMed ID: 18710415	2008
Intact lipid rafts regulate HIV-1 Tat protein-induced activation of the Rho signaling and upregulation of P-glycoprotein in brain endothelial cells. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism\| PubMed ID: 19794400	2010
HIV-1-induced amyloid beta accumulation in brain endothelial cells is attenuated by simvastatin. Molecular and cellular neurosciences\| PubMed ID: 19944163	2010
HIV-1 Tat triggers nuclear localization of ZO-1 via Rho signaling and cAMP response element-binding protein activation. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 22219277	2012
Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels. The Journal of biological chemistry\| PubMed ID: 25086043	2014
Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. Blood\| PubMed ID: 26209658	2015
Aβ40 Reduces P-Glycoprotein at the Blood-Brain Barrier through the Ubiquitin-Proteasome Pathway. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 26865616	2016
Caloric restriction preserves memory and reduces anxiety of aging mice with early enhancement of neurovascular functions. Aging\| PubMed ID: 27829242	2016
P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy. Molecular pharmaceutics\| PubMed ID: 28195743	2017
Deletion of BMAL1 in Smooth Muscle Cells Protects Mice From Abdominal Aortic Aneurysms. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 29437576	2018
Preventing P-gp Ubiquitination Lowers Aβ Brain Levels in an Alzheimer's Disease Mouse Model. Frontiers in aging neuroscience\| PubMed ID: 29997495	2018

Yu Zhong

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