Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-β42 by LRP1-dependent apolipoprotein E isoform-specific mechanism

Background

Clearance at the blood-brain barrier (BBB) plays an important role in removal of Alzheimer’s amyloid-β (Aβ) toxin from brain both in humans and animal models. Apolipoprotein E (apoE), the major genetic risk factor for AD, disrupts Aβ clearance at the BBB. The cellular and molecular mechanisms, however, still remain unclear, particularly whether the BBB-associated brain capillary pericytes can contribute to removal of aggregated Aβ from brain capillaries, and whether removal of Aβ aggregates by pericytes requires apoE, and if so, is Aβ clearance on pericytes apoE isoform-specific.

Methods

We performed immunostaining for Aβ and pericyte biomarkers on brain capillaries (<?6 μm in diameter) on tissue sections derived from AD patients and age-matched controls, and APP^Swe/0 mice and littermate controls. Human Cy3-Aβ42 uptake by pericytes was studied on freshly isolated brain slices from control mice, pericyte LRP1-deficient mice (Lrp^lox/lox;Cspg4-Cre) and littermate controls. Clearance of aggregated Aβ42 by mouse pericytes was studied on multi-spot glass slides under different experimental conditions including pharmacologic and/or genetic inhibition of the low density lipoprotein receptor related protein 1 (LRP1), an apoE receptor, and/or silencing mouse endogenous Apoe in the presence and absence of human astrocyte-derived lipidated apoE3 or apoE4. Student’s t-test and one-way ANOVA followed by Bonferroni's post-hoc test were used for statistical analysis.

Results

First, we found that 35% and 60% of brain capillary pericytes accumulate Aβ in AD patients and 8.5-month-old APP^Sw/0 mice, respectively, compared to negligible uptake in controls. Cy3-Aβ42 species were abundantly taken up by pericytes on cultured mouse brain slices via LRP1, as shown by both pharmacologic and genetic inhibition of LRP1 in pericytes. Mouse pericytes vigorously cleared aggregated Cy3-Aβ42 from multi-spot glass slides via LRP1, which was inhibited by pharmacologic and/or genetic knockdown of mouse endogenous apoE. Human astrocyte-derived lipidated apoE3, but not apoE4, normalized Aβ42 clearance by mouse pericytes with silenced mouse apoE.

Conclusions

Our data suggest that BBB-associated pericytes clear Aβ aggregates via an LRP1/apoE isoform-specific mechanism. These data support the role of LRP1/apoE interactions on pericytes as a potential therapeutic target for controlling Aβ clearance in AD.

     

Pericytes Regulate Vascular Basement Membrane Remodeling and Govern Neutrophil Extravasation during Inflammation

During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.     

Characterization of microvascular cell cultures from normotensive and hypertensive rat brains: pericyte-endothelial cell interactions in vitro

We used specific markers and fluorescence microscopy to identify and characterize cerebrovascular cells. Cultures were derived from brain microvessels isolated from normotensive (Wistar Kyoto, WKY) and spontaneously hypertensive (SHR) rat brains prior to, coincident with and following the onset of chronic hypertension. Endothelial cells were characterized using di-acyl LDL and non-muscle isoactin-specific antibodies. Cerebrovascular pericytes were identified with the anti-muscle and non-muscle actin antibody staining. Using this combination of cell culture and fluorescence localization, we have been able to demonstrate that brain pericytes are tightly associated with the endothelial cells of the hypertensive-prone and hypertensive cell cultures, but not with the normotensive endothelial cultures. While the endothelial-pericyte ratio in the hypertensive-prone microvascular cultures was between 5:1 and 10:1, the number of pericytes associated with the hypertensive rat brain cultures increased two to five times (2:1-1:1). Muscle and non-muscle actin antibody staining localized the spindle-shaped pericytes of the hypertensive microvascular colonies. Pericytes were found overlaying and encircling the endothelial cells. Normotensive pericytes were not endothelial-associated. Whereas the hypertensive pericyte is devoid of stress fibers, the normotensive pericyte is a larger, spread-out cell possessing numerous stress fibers rich in muscle and non-muscle actin. These results provide the first evidence that the etiology and inception of cerebrovascular disease may be pericyte-related and suggest that pericyte contraction could play a pivotal role in regulating the flow of blood within the brain microcirculation.     

Homocysteine, another risk factor for Alzheimer disease, impairs apolipoprotein E3 function

Apolipoprotein E (apoE) ε4 and hyperhomocysteinemia are risk factors for Alzheimer disease (AD). The dimerization of apoE3 by disulfide bonds between cysteine residues enhances apoE3 function to generate HDL. Because homocysteine (Hcy) harbors a thiol group, we examined whether Hcy interferes with the dimerization of apoE3 and thereby impairs apoE3 function. We found that Hcy inhibits the dimerization of apoE3 and reduces apoE3-mediated HDL generation to a level similar to that by apoE4, whereas Hcy does not affect apoE4 function. Western blot analysis of cerebrospinal fluid showed that the ratio of apoE3 dimers was significantly lower in the samples from the patients with hyperhomocysteinemia than in those that from control subjects. Hyperhomocysteinemia induced by subcutaneous injection of Hcy to apoE3 knock-in mice decreased the level of the apoE3 dimer in the brain homogenate. Because apoE-HDL plays a role in amyloid β-protein clearance, these results suggest that two different risk factors, apoE4 and hyperhomocysteinemia, may share a common mechanism that accelerates the pathogenesis of AD in terms of reduced HDL generation.     

SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-β1 activity

Pericytes migrate to nascent vessels and promote vessel stability. Recently, we reported that secreted protein acidic and rich in cysteine (SPARC)-deficient mice exhibited decreased pericyte-associated vessels in an orthotopic model of pancreatic cancer, suggesting that SPARC influences pericyte behavior. In this paper, we report that SPARC promotes pericyte migration by regulating the function of endoglin, a TGF-β1 accessory receptor. Primary SPARC-deficient pericytes exhibited increased basal TGF-β1 activity and decreased cell migration, an effect blocked by inhibiting TGF-β1. Furthermore, TGF-β-mediated inhibition of pericyte migration was dependent on endoglin and αV integrin. SPARC interacted directly with endoglin and reduced endoglin interaction with αV integrin. SPARC deficiency resulted in endoglin-mediated blockade of pericyte migration, aberrant association of endoglin in focal complexes, an increase in αV integrins present in endoglin immunoprecipitates, and enhanced αV integrin-mediated activation of TGF-β. These results demonstrate that SPARC promotes pericyte migration by diminishing TGF-β activity and identify a novel function for endoglin in controlling pericyte behavior.     

NKCC1 promotes EMT-like process in GBM via RhoA and Rac1 signaling pathways

Glioblastoma is the most common and lethal primary intracranial tumor. As the key regulator of tumor cell volume, sodium-potassium-chloride cotransporter 1 (NKCC1) expression increases along with the malignancy of the glioma, and NKCC1 has been implicated in glioblastoma invasion. However, little is known about the role of NKCC1 in the epithelial-mesenchymal transition-like process in gliomas. We noticed that aberrantly elevated expression of NKCC1 leads to changes in the shape, polarity, and adhesion of cells in glioma. Here, we investigated whether NKCC1 promotes an epithelial–mesenchymal transition (EMT)-like process in gliomas via the RhoA and Rac1 signaling pathways. Pharmacological inhibition and knockdown of NKCC1 both decrease the expressions of mesenchymal markers, such as N-cadherin, vimentin, and snail, whereas these treatments increase the expression of the epithelial marker E-cadherin. These findings indicate that NKCC1 promotes an EMT-like process in gliomas. The underlying mechanism is the facilitation of the binding of Rac1 and RhoA to GTP by NKCC1, which results in a significant enhancement of the EMT-like process. Specific inhibition or knockdown of NKCC1 both attenuate activated Rac1 and RhoA, and the pharmacological inhibitions of Rac1 and RhoA both impair the invasion and migration abilities of gliomas. Furthermore, we illustrated that NKCC1 knockdown abolished the dissemination and spread of glioma cells in a nude mouse intracranial model. These findings suggest that elevated NKCC1 activity acts in the regulation of an EMT-like process in gliomas, and thus provides a novel therapeutic strategy for targeting the invasiveness of gliomas, which might help to inhibit the spread of malignant intracranial tumors.     

Activation of AMP-activated protein kinase is essential for lysophosphatidic acid-induced cell migration in ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-β3 (PLC-β3) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-β3, or (CaMKKβ) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.     

A Novel In Vitro Model to Study Pericytes in the Neurovascular Unit of the Developing Cortex

Cortical function is impaired in various disorders of the central nervous system including Alzheimer’s disease, autism and schizophrenia. Some of these disorders are speculated to be associated with insults in early brain development. Pericytes have been shown to regulate neurovascular integrity in development, health and disease. Hence, precisely controlled mechanisms must have evolved in evolution to operate pericyte proliferation, repair and cell fate within the neurovascular unit (NVU). It is well established that pericyte deficiency leads to NVU injury resulting in cognitive decline and neuroinflammation in cortical layers. However, little is known about the role of pericytes in pathophysiological processes of the developing cortex. Here we introduce an in vitro model that enables to precisely study pericytes in the immature cortex and show that moderate inflammation and hypoxia result in caspase-3 mediated pericyte loss. Using heterozygous EYFP-NG2 mouse mutants we performed live imaging of pericytes for several days in vitro. In addition we show that pericytes maintain their capacity to proliferate which may allow cell-based therapies like reprogramming of pericytes into induced neuronal cells in the presented approach.     

Apolipoprotein E Is a Ligand for Triggering Receptor Expressed on Myeloid Cells 2 (TREM2)

Several heterozygous missense mutations in the triggering receptor expressed on myeloid cells 2 (TREM2) have recently been linked to risk for a number of neurological disorders including Alzheimer disease (AD), Parkinson disease, and frontotemporal dementia. These discoveries have re-ignited interest in the role of neuroinflammation in the pathogenesis of neurodegenerative diseases. TREM2 is highly expressed in microglia, the resident immune cells of the central nervous system. Along with its adaptor protein, DAP12, TREM2 regulates inflammatory cytokine release and phagocytosis of apoptotic neurons. Here, we report apolipoprotein E (apoE) as a novel ligand for TREM2. Using a biochemical assay, we demonstrated high-affinity binding of apoE to human TREM2. The functional significance of this binding was highlighted by increased phagocytosis of apoE-bound apoptotic N2a cells by primary microglia in a manner that depends on TREM2 expression. Moreover, when the AD-associated TREM2-R47H mutant was used in biochemical assays, apoE binding was vastly reduced. Our data demonstrate that apoE-TREM2 interaction in microglia plays critical roles in modulating phagocytosis of apoE-bound apoptotic neurons and establish a critical link between two proteins whose genes are strongly linked to the risk for AD.     

Apolipoprotein E regulates the integrity of tight junctions in an isoform-dependent manner in an in vitro blood-brain barrier model

Apolipoprotein E (apoE) is a major apolipoprotein in the brain. The ε4 allele of apoE is a major risk factor for Alzheimer disease, and apoE deficiency in mice leads to blood-brain barrier (BBB) leakage. However, the effect of apoE isoforms on BBB properties are as yet unknown. Here, using an in vitro BBB model consisting of brain endothelial cells and pericytes prepared from wild-type (WT) mice, and primary astrocytes prepared from human apoE3- and apoE4-knock-in mice, we show that the barrier function of tight junctions (TJs) was impaired when the BBB was reconstituted with primary astrocytes from apoE4-knock-in mice (apoE4-BBB model). The phosphorylation of occludin at Thr residues and the activation of protein kinase C (PKC)η in mBECs were attenuated in the apoE4-BBB model compared with those in the apoE3-BBB model. The differential effects of apoE isoforms on the activation of PKCη, the phosphorylation of occludin at Thr residues, and TJ integrity were abolished following the treatment with an anti-low density lipoprotein receptor-related protein 1 (LRP1) antibody or a LRP1 antagonist receptor-associated protein. Consistent with the results of in vitro studies, BBB permeability was higher in apoE4-knock-in mice than in apoE3-knock-in mice. Our studies provide evidence that TJ integrity in BBB is regulated by apoE in an isoform-dependent manner.     

Generation and characterization of rgs5 mutant mice

Regulators of G-protein signaling (RGS) are involved in a wide variety of functions, including olfaction, vision, and cell migration. RGS5 has a perivascular expression pattern and was recently identified as a marker for brain pericytes. This suggests a role for RGS5 in vascular development and pericyte biology. We have created a mouse line which lacks the rgs5 gene and replaced it with a green fluorescent protein (GFP) reporter (rgs5(GFP/GFP)). The mice are viable and fertile and display no obvious developmental defects, and the vasculature appears to develop normally with proper pericyte coverage. Also, no differences were observed in the vasculature under pathological conditions, such as tumor growth and oxygen-induced retinopathy. The GFP expression in pericytes of rgs5(GFP) mice allows detection and sorting of these cells, thereby providing a valuable novel tool for pericyte research.     

Preferential interactions between ApoE-containing lipoproteins and Aβ revealed by a detection method that combines size exclusion chromatography with non-reducing gel-shift

The association between apolipoprotein E (apoE) and amyloid-β peptide (Aβ) may significantly impact the function of both proteins, thus affecting the etiology of Alzheimer's disease (AD). However, apoE/Aβ interactions remain fundamentally defined by the stringency of the detection method. Here we use size exclusion chromatography (SEC) as a non-stringent approach to the detection of apoE/Aβ interactions in solution, specifically apoE and both endogenous and exogenous Aβ from plasma, CSF and astrocyte conditioned media. By SEC analysis, Aβ association with plasma and CNS lipoproteins is apoE-dependent. While endogenous Aβ elutes to specific human plasma lipoproteins distinct from those containing apoE, it is the apoE-containing lipoproteins that absorb excess amounts of exogenous Aβ40. In human CSF, apoE, endogenous Aβ and phospholipid elute in an almost identical profile, as do apoE, exogenous Aβ and phospholipid from astrocyte conditioned media. Combining SEC fractionation with subsequent analysis for SDS-stable apoE/Aβ complex reveals that apoE-containing astrocyte lipoproteins exhibit the most robust interactions with Aβ. Thus, standardization of the methods for detecting apoE/Aβ complex is necessary to determine its functional significance in the neuropathology characteristic of AD. Importantly, a systematic understanding of the role of apoE-containing plasma and CNS lipoproteins in Aβ homeostasis could potentially contribute to identifying a plasma biomarker currently over-looked because it has multiple components.     

Alzheimer amyloid-beta peptide forms denaturant-resistant complex with type epsilon 3 but not type epsilon 4 isoform of native apolipoprotein E.

BACKGROUND: The apolipoprotein E (apoE) type epsilon 4 isoform specifies increased cerebral and cerebrovascular accumulation of amyloid-beta protein (A beta) and contributes to the genetic susceptibility underlying a large proportion (approximately 60%) of typical, sporadic Alzheimer disease. Unfortunately, in vitro biochemical studies of direct apoE isoform-specific interactions with A beta have been inconsistent, perhaps due to the use by different research groups of apoE isoform preparations in different conformational states (purified denatured versus native). MATERIALS AND METHODS: In the current study, we have investigated the possibility that synthetic A beta(1-40) preferentially associates with native apoE of either the type epsilon 3 or the type epsilon 4 isoform. RESULTS: Here, we demonstrate the preferential association of synthetic A beta(1-40) with native apoE epsilon 3. The complex between apoE epsilon 3 and A beta(1-40) could not be disrupted by sodium dodecyl sulfate. In a parallel assay, no denaturant-resistant association of A beta(1-40) with apoE epsilon 4 was detectable. CONCLUSIONS: These results support the notion that the apoE epsilon 4 isoform may foster beta-amyloidogenesis because apoE epsilon 4 is inefficient in forming complexes with A beta.     

The axonal repellent Slit2 inhibits pericyte migration: potential implications in angiogenesis

The Slit family of secreted proteins acts through the Roundabout (Robo) receptors to repel axonal migration during central nervous system development. Emerging evidence shows that Slit/Robo interactions also play a role in angiogenesis. The effect of Robo signaling on endothelial cells has been shown to be context-dependent. However, the role of Slit/Robo in pericytes has been largely unexplored. The aim of this study was to determine the effect of Slit2 on primary human pericytes and to address the underlying mechanisms, including the receptors potentially implicated. We demonstrate that both Robo1 and Robo4 are expressed by human pericytes. In the presence of their ligand Slit2, spontaneous and PDGF-induced migration of pericytes was impaired. This antimigratory activity of Slit-2 correlated with the inhibition of actin-based protrusive structures. Interestingly, human pericyte interaction with immobilized Slit2 was inhibited in the presence of anti-Robo1 and anti-Robo4 blocking antibodies, suggesting the implication of both receptors. These results add new insights into the role of Slit proteins during the angiogenic process that relies on the directional migration not only of endothelial cells but also of pericytes.     

Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging

Pericytes play a key role in the development of cerebral microcirculation. The exact role of pericytes in the neurovascular unit in the adult brain and during brain aging remains, however, elusive. Using adult viable pericyte-deficient mice, we show that pericyte loss leads to brain vascular damage by two parallel pathways: (1) reduction in brain microcirculation causing diminished brain capillary perfusion, cerebral blood flow, and cerebral blood flow responses to brain activation that ultimately mediates chronic perfusion stress and hypoxia, and (2) blood-brain barrier breakdown associated with brain accumulation of serum proteins and several vasculotoxic and/or neurotoxic macromolecules ultimately leading to secondary neuronal degenerative changes. We show that age-dependent vascular damage in pericyte-deficient mice precedes neuronal degenerative changes, learning and memory impairment, and the neuroinflammatory response. Thus, pericytes control key neurovascular functions that are necessary for proper neuronal structure and function, and pericyte loss results in a progressive age-dependent vascular-mediated neurodegeneration.     

In situ analysis of microvascular pericytes in hypertensive rat brains

We used immunofluorescence microscopy and isoactin-specific antibodies to characterize the pattern and prevalence of pericytes within the brain microcirculation. Blood pressures of normotensive, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were measured prior to sacrifice and pressure-perfusion fixation. WKY and SHR brains were subdivided into ten major regions prior to ultracryomicrotomy. Sections 0.3-0.5 micron wide were treated with 10-40 micrograms/ml affinity-purified antibodies to the muscle and non-muscle actin isoforms. These localization studies show that there are four times the number of pericyte-rich capillaries in the SHR motor cortex compared to WKY counterparts (59.9 vs. 15.3%). In contrast, the sensory cortex of both rat strains is deficient in muscle actin staining surrounding the capillaries. The most striking difference in pericyte presence and muscle actin antibody staining between the SHR and WKY was observed in the tegmentum of the brainstem. There is nearly a one-to-one coincidence observed in pericyte and capillary profiles present within thin, frozen sections of the SHR midbrain. SHR pons capillaries were also pericyte-enriched. WKY analyses of plastic embedded thin sections confirmed the presence of pericytes and their filament-enriched processes encircling the capillaries of the hypertensive brains. These results suggest that pericytes may play important roles in hypertension and cerebrovascular disease processes.     

Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.     

Migration of turkey muscle satellite cells is enhanced by the syndecan-4 cytoplasmic domain through the activation of RhoA

Syndecan-4 (S4) is a cell membrane-associated heparan sulfate proteoglycan that forms oligomers in muscle satellite cells. The S4 oligomers activate protein kinase Cα (PKCα) through the S4 cytoplasmic domain and may regulate the activation of ras homolog gene family member A (RhoA), a signal transduction molecule down-stream of PKCα which is thought to influence cell migration. However, little is known about the function of the S4 cytoplasmic domain in satellite cell migration and RhoA activation. The objective of the current study was to determine the function of S4 and its cytoplasmic domain in cell migration and RhoA activation. To study the objective, clones of S4 and S4 without the cytoplasmic domain (S4C) were used in overexpression studies, and small interference RNAs targeting S4 or RhoA were used in knockdown studies. Satellite cell migration was increased by S4 overexpression, but decreased by the knockdown or deletion of the S4 cytoplasmic domain. The RhoA protein was activated by the overexpression of S4, but not with the deletion of the S4 cytoplasmic domain. The treatment of Rho activator II or the knockdown of RhoA also modulated satellite cell migration. Finally, co-transfection (S4 overexpression and RhoA knockdown) and rescue (the knockdown of S4 and the treatment with Rho activator II) studies demonstrated that S4-mediated satellite cell migration was regulated through the activation of RhoA. The cytoplasmic domain of S4 is required for cell migration and RhoA activation which will affect muscle fiber formation.