A. S. Lossinsky

Human Immunodeficiency Virus Type 1 Enters Brain Microvascular Endothelia by Macropinocytosis Dependent on Lipid Rafts and the Mitogen-Activated Protein Kinase Signaling Pathway

ABSTRACT Brain microvascular endothelial cells (BMVECs) present an incomplete barrier to human immunodeficiency virus type 1 (HIV-1) neuroinvasion. In order to clarify the mechanisms of HIV-1 invasion, we have examined HIV-1 uptake and transcellular penetration in an in vitro BMVEC model. No evidence of productive infection was observed by luciferase, PCR, and reverse transcriptase assays. Approximately 1% of viral RNA and 1% of infectious virus penetrated the BMVEC barrier without disruption of tight junctions. The virus upregulated ICAM-1 on plasma membranes and in cytoplasmic vesiculotubular structures. HIV-1 virions were entangled by microvilli and were taken into cytoplasmic vesicles through surface invaginations without fusion of the virus envelope with the plasma membrane. Subsequently, the cytoplasmic vesicles fused with lysosomes, the virions were lysed, and the vesicles diminished in size. Upon cell entry, HIV-1 colocalized with cholera toxin B, which targets lipid raft-associated GM1 ganglioside. Cholesterol-extracting agents, cyclodextrin and nystatin, and polyanion heparin significantly inhibited virus entry. Anti-CD4 had no effect and the chemokine AOP-RANTES had only a slight inhibitory effect on virus entry. HIV-1 activated the mitogen-activated protein kinase (MAPK) pathway, and inhibition of MAPK/Erk kinase inhibited virus entry. Entry was also blocked by dimethylamiloride, indicating that HIV-1 enters endothelial cells by macropinocytosis. Therefore, HIV-1 penetrates BMVECs in ICAM-1-lined macropinosomes by a mechanism involving lipid rafts, MAPK signaling, and glycosylaminoglycans, while CD4 and chemokine receptors play limited roles in this process.

Ineffective phagocytosis of amyloid-β by macrophages of Alzheimer's disease patients

The defective clearance of amyloid-beta (Abeta) in the brain of Alzheimers disease (AD) patients is unexplained. The immunohistochemical studies of the frontal lobe and hippocampus show perivascular and intraplaque infiltration by blood-borne macrophages containing intracellular Abeta but only inefficient clearance of beta deposits. Neurons and neuronal nuclei, respectively, express interleukin-1beta and the chemokine RANTES, which could induce the inflammatory cell infiltration. To clarify the pathophysiology ofbeta clearance, we examined Abeta phagocytosis by monocytes and macrophages isolated from the blood of age-matched patients and controls. Control monocytes display excellent differentiation into macrophages and intracellular phagocytosis of Abeta followed by beta degradation or export. AD monocytes show poor differentiation and only surface uptake of Abeta and suffer apoptosis. HLA DR and cyclooxygenase-2 are abnormally expressed on neutrophils and monocytes of AD patients. AD patients have higher levels of intracellular cytokines compared to controls. Thus Abeta clearance is not restricted to brain microglia and involves systemic innate immune responses. In AD, however, macrophage phagocytosis is defective, which may elicit compensatory response by the adaptive immune system.

Sites of egress of inflammatory cells and horseradish peroxidase transport across the blood-brain barrier in a murine model of chronic relapsing experimental allergic encephalomyelitis

SummaryResults are reported of experiments designed to focus at attachment sites of inflammatory cells (ICs) on the luminal surface of brain endothelial cells (ECs) and on the mechanisms of horseradish peroxidase (HRP) transport across the altered blood-brain barrier (BBB) in a murine model of chronic relapsing experimental allergic encephalomyelitis. Cationized ferritin (CF) served as a marker for evaluting the electrostatic nature of brain microblood vessels (MBVs) on the plasma membranes of ICs or normal mouse peripheral white blood cells and erythrocytes. SJL/J mice demonstrating clinical illness were given HRP or CF, in vivo or in situ, respectively. Light microscopy and conventional transmission electron microscopy of cerebellum or thoracic and lumbar spinal cord regions demonstrated HRP leakage most pronounced in MBVs with perivascular infiltrates. HRP traversed across the ECs via numerous vesicles and tubular profiles located mostly in the parajunctional regions, while EC junctions appeared closed. Scanning electron microscopy demonstrated that IC attachment was primarily at parajunctional sites on the EC surface. We also observed increased microvillar projections extending from the EC surface into the lumen. CF demonstrated a patchy decoration on both the luminal EC surface and IC membranes but did not label uncoated invaginating membrane pits or tubular structures. Our data indicate that the points of attachment of the ICs on the EC surface may reflect specific receptor sites where the ICs eventually gain entrance into CNS across the BBB during brain inflammation.

Cytochemical localization of ouabain-sensitive, K+-dependent p-nitro-phenylphosphatase (transport ATPase) in the mouse central and peripheral nervous systems

Enzyme activity, representing the sites of K+-dependent p-nitrophenylphosphatase (K+-pNPPase), a component of the transport adenosine triphosphatase (Na+,K+-ATPase) system, has been localized at the ultrastructural in both cerebral cortex and in sciatic nerve of the mouse. Normal mice and animals with mechanically injured blood-brain barrier (BBB) were used. In the cerebral cortex, positive reaction was found in synapses, plasmalemma of neurites (axons and dendrites), in endothelial cell of microblood vessels and in the plasmalemma of mesothelial cells of the pia mater. In the sciatic nerve, a strong reaction was present in the nodes of Ranvier, with weaker reaction in the internodal areas of the axolemma. In the endothelial cells of normal blood vessels, the reaction product was localized on the luminal and abluminal, or only on the abluminal plasmalemma. After damage of BBB, numerous invaginations, pits and pinocytic vesicles showing positive reaction in their limiting membrane appeared in the endothelial cells.

Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels.

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.

Complete cerebral ischemia with short-term survival in rat induced by cardiac arrest. II: Extracellular and intracellular accumulation of apolipoproteins E and J in the brain

The distribution of apolipoprotein E (apo E) and apolipoprotein J (apo J) was investigated immunocytochemically in rats at various time intervals after 10 min global cerebral ischemia (GCI) induced by cardiac arrest. Strong apo E and weaker apo J immunoreactivity was found extracellularly in multiple deposits located close to the microvessels. These deposits appeared 3 h after GCI and were present, but not in all the animals, at all time intervals studied post-GCL. In some rats, apo E immunoreactivity was also found in small necrotic foci. Widespread, neuronal apo E immunostaining appeared 6 h post-GCI. However, the strongest neuronal apo E immunoreactivity was found 7 days post-GCI in those neurons, most often observed in the CA1 hippocampal region, exhibiting signs of ischemic cell damage. These ischemically damaged neurons displayed weaker immunoreactivity to apo J, despite its increase in the response to GCI in the various brain regions examined. Our data show that mechanisms operating in ischemia are able to supply large amounts of apo E and apo J to the brain tissue and suggest involvement of both apo E and apo J in a complex series of events occurring in the ischemic brain. Perivascular deposits of apo E/apo J colocalized with amyloid beta protein precursor epitopes that have been disclosed by us previously in this model. Whether this phenomenon is limited to postischemic brain tissue, or can be encountered also in other pathological conditions will require further elaboration.

Localization of Alkaline Phosphatase Activity in Endothelia of Developing and Mature Mouse Blood-Brain Barrier

The relationship between alkaline phosphatase (AP) activity and maturation of blood-brain barrier (BBB) in mouse brain was studied ultracytochemically. The permeability of micro-blood vessels (MBVs) to intravenously injected horseradish peroxidase was considered to be a criterion of BBB maturation, which occurs between the 12th and 24th day of mouse life. This process coincides with the appearance of cytochemically detectable AP activity in luminal plasma membrane (PM) of the endothelial cell (EC) of capillaries or in both luminal and abluminal PM in arterioles. No reaction appears in ECs of venules. These observations indicate that the development of BBB function is accompanied by the formation of enzymatic barrier in MBVs endothelia. AP constitutes one of the components of this barrier. Comparative study indicates that in various MBVs of non-BBB type, except in skeletal muscle, no AP activity is detected in similar incubation conditions.

Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy

Neuronal accumulation of oligomeric amyloid-β (Αβ) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Aβ stress to neurons by immigration into the brain and phagocytosis of Αβ. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Αβ by AD and normal subjects’ macrophages. Both AD and normal macrophages were inhibited in Αβ export across the blood-brain barrier due to adherence of Aβ-engorged macrophages to the endothelial layer. In comparison to normal subjects’ macrophages, AD macrophages ingested and cleared less Αβ, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Αβ. Confocal microscopy of stained AD brain sections revealed oligomeric Aβ in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Aβ in plaques and microvessel walls. After incubation with AD brain sections, normal subjects’ monocytes intruded into neurons and uploaded oligomeric Aβ. In conclusion, in patients with AD, macrophages appear to shuttle Aβ from neurons to vessels where their apoptosis may release fibrillar Aβ, contributing to cerebral amyloid angiopathy.

Complete cerebral ischemia with short-term survival in rats induced by cardiac arrest. I. Extracellular accumulation of Alzheimer's β-amyloid protein precursor in the brain

The distribution of beta-amyloid protein precursor (APP) was investigated immunocytochemically in rats subjected to global cerebral ischemia (GCI) induced by cardiac arrest. Rats underwent 10 min of GCI with 3, 6, and 12 h and 2 and 7 days of survival. APP immunostaining was found extracellular and intracellularly. Multiple extracellular APP immunoreactive deposits around and close to the vessels appeared as soon as 3 h after GCI. Extracellular accumulation of APP occurred frequently in the hippocampus, cerebral and cerebellar cortex, basal ganglia and thalamus and rarely in the brain stem. These deposits were labelled with antibodies against the N-terminal, beta-amyloid peptide, and C-terminal domains of APP. Our data suggests that either proteolytically cleaved fragments of the full-length APP or the entire APP molecule accumulates extracellularly after GCI. This findings may not only implicate the participation of APP in postischemic tissue damage but also suggest the involvement of pathomechanisms operating in ischemia in Alzheimers disease pathology.

Cocaine increases human immunodeficiency virus type 1 neuroinvasion through remodeling brain microvascular endothelial cells

Cocaine is a suspected cofactor in human immunodeficiency virus (HIV)-associated dementia but cocaine’s effects are not clear. Herein the authors describe investigations of the mechanisms by which cocaine increases HIV-1 invasion through brain microvascular endothelial cells (BMVECs). Cocaine binds to a site on BMVECs, which is not a biogenic amine transporter, a binding site for estrogen, or a muscarinic receptor and for which benztropine and tamoxifen have the highest affinity. Cocaine treatment of BMVECs disrupts intercellular junctions and induces cell ruffling, which could account for their increased permeability and decreased electrical resistance. HIV-1 enters BMVECs by macropinocytosis and is transported to lysosomes and inactivated. In cocaine-treated BMVECs, the virus enters and persists in large cytoplasmic “lakes.” Cocaine exposure of BMVECs up-regulates transcription of genes important in cytoskeleton organization, signal transduction, cell swelling, vesicular trafficking, and cell adhesion. The toxicity of cocaine for the blood-brain barrier may lead to increased virus neuroinvasion and neurovascular complications of cocaine abuse.