Modulating insulin signaling and trafficking at the blood-brain barrier endothelium using lipid based nanoemulsions.

The compositionally distinct lipid rafts present in the plasma membrane regulate the restrictive trafficking and signal transduction in the blood-brain barrier (BBB) endothelium. Several metabolic and neurodegenerative diseases are associated with lipid homeostasis disruption within the BBB endothelium. Here, we hypothesized that the delivery of lipid triglyceride based nanoemulsions containing unsaturated fatty acids (UFAs) provides a novel non-pharmacological approach to modulate lipid raft integrity and rectify the aberrant trafficking and signal transduction. The current study has shown that soybean oil nanoemulsions (SNEs) altered the morphology of lipid rafts that are stained by Alex Fluor 647 labelled cholera toxin (AF647-CTX) in polarized human cerebral microvascular endothelial (hCMEC/D3) cell monolayers. Moreover, western blot and flow cytometry analysis showed that SNEs containing polyunsaturated fatty acids (PUFAs) increased phospo-AKT (p-AKT) expression, a marker for the stimulation of metabolic arm of insulin signaling, and insulin uptake in hCMEC/D3 monolayers. However, olive oil nanoemulsions (ONEs) containing monounsaturated fatty acids (MUFAs) had no detectable impact on lipid raft integrity, AKT phosphorylation, or insulin uptake. These findings provided direct evidence that SNEs containing PUFAs can upregulate insulin-pAKT pathway, facilitate insulin trafficking at the BBB, and potentially address cerebrovascular dysfunction in metabolic and neurodegenerative diseases.

High-Density Lipoprotein Mimetic Peptide 4F Efficiently Crosses the Blood-Brain Barrier and Modulates Amyloid-ß Distribution between Brain and Plasma.

Treatments to elevate high-density lipoprotein (HDL) levels in plasma have decreased cerebrovascular amyloid -ß (Aß) deposition and mitigated cognitive decline in Alzheimer disease (AD) transgenic mice. Since the major protein component of HDL particles, apolipoprotein A-I (ApoA-I), has very low permeability at the blood-brain barrier (BBB), we investigated 4F, an 18-amino-acid ApoA-I/HDL mimetic peptide, as a therapeutic alternative. Specifically, we examined the BBB permeability of 4F and its effects on [125I]Aß trafficking from brain to blood and from blood to brain. After systemic injection in mice, the BBB permeability of [125I]4F, estimated as the permeability-surface area (PS) product, ranged between 2 and 5 × 10-6 ml/g per second in various brain regions. The PS products of [125I]4F were ∼1000-fold higher compared with those determined for [125I]ApoA-I. Moreover, systemic infusion with 4F increased the brain efflux of intracerebrally injected [125I]Aß42. Conversely, 4F infusion decreased the brain influx of systemically injected [125I]Aß42. Interestingly, 4F did not significantly alter the brain influx of [125I]Aß40. To corroborate the in vivo findings, we evaluated the effects of 4F on [125I]Aß42 transcytosis across polarized human BBB endothelial cell (hCMEC/D3) monolayers. Treatment with 4F increased the abluminal-to-luminal flux and decreased the luminal-to-abluminal flux of [125I]Aß42 across the hCMEC/D3 monolayers. Additionally, 4F decreased the endothelial accumulation of fluorescein-labeled Aß42 in the hCMEC/D3 monolayers. These findings provide a mechanistic interpretation for the reductions in brain Aß burden reported in AD mice after oral 4F administration, which represents a novel strategy for treating AD and cerebral amyloid angiopathy. SIGNIFICANCE STATEMENT: The brain permeability of the ApoA-I mimetic peptide 4F was estimated to be ∼1000-fold greater than ApoA-I after systemic injection of radiolabeled peptide/protein in mice. Further, 4F treatment increased the brain efflux of amyloid -ß and also decreased its brain influx, as evaluated in mice and in blood-brain barrier cell monolayers. Thus, 4F represents a potential therapeutic strategy to mitigate brain amyloid accumulation in cerebral amyloid angiopathy and Alzheimer disease.

Amyloid-ß Peptides Disrupt Interactions Between VAMP-2 and SNAP-25 in Neuronal Cells as Determined by FRET/FLIM.

BACKGROUND: Synaptic dysfunction prevalent in Alzheimer's disease (AD) brain is closely associated with increased accumulation of amyloid-ß (Aß) peptides in the brain parenchyma. It is widely believed that Aß peptides trigger synaptic dysfunction by interfering with the synaptic vesicular fusion and the release of neurotransmitters, primarily facilitated by the SNARE protein complexes formed by VAMP-2, SNAP-25, and syntaxin-1. However, Aß interactions with SNARE proteins to ultimately disrupt synaptic vesicular fusion are not well understood. OBJECTIVE: Our objective is to elucidate mechanisms by which Aß peptides perturb SNARE complexes. METHODS: Intensity (qualitative) and lifetime (quantitative) based measurements involving Forster (fluorescence) resonance energy transfer (FRET) followed by fluorescence lifetime imaging microscopy (FLIM) were employed to investigate the effect of Aß peptides on dynamic interactions between VAMP-2, labeled with cerulean (Cer) at the N-terminus (FRET donor), and SNAP-25 labeled with citrine (Cit) on the N-terminus (FRET acceptor). The FRET and FLIM interactions at the exocytosis locations on the pre-synaptic membrane were recorded under spontaneous and high potassium evoked conditions. Moreover, cellular accumulation of fluorescein labeled Aß (F-Aß) peptides and their co-localization with Cer-VAMP2 was investigated by confocal microscopy. RESULTS: The F-Aß40 and F-Aß42 are internalized by differentiated N2A cells, where they colocalize with Cer-VAMP2. Both Aß40 and Aß42 decrease interactions between the N-termini of Cer-VAMP2 and Cit-SNAP25 in N2A cells, as determined by FRET/FLIM. CONCLUSION: By perturbing the N-terminal interactions between VAMP-2 and SNAP-25, Aß40 and Aß42, can directly interfere with the SNARE complex formation, which is critical for the docking and fusion of synaptic vesicles.

Design, Development, and Characterization of Imiquimod-Loaded Chitosan Films for Topical Delivery.

Aldara™ (5% w/w imiquimod) topical cream is approved by the US FDA for the treatment of superficial basal cell carcinoma. However, the cream formulation suffers from dose variability, low drug availability due to the incomplete release, and poor patient compliance. To achieve sustained and complete release of imiquimod, chitosan films were prepared by casting using propylene glycol as a plasticizer. Chitosan films had appropriate physicochemical characteristics for wound dressing and excellent content uniformity and maintained the original physical form of imiquimod. Films were capable of releasing a defined dose of imiquimod over a period of 7 days. The bioactivity of imiquimod was not affected by its entrapment in chitosan matrix as indicated by the results of in vitro growth inhibition assay. In addition, the film formulation showed significantly (p Ë‚ 0.05) higher drug accumulation in the skin when compared to commercial cream formulation.

Sensitive Determination of Fentanyl in Low-Volume Serum Samples by LC-MS/MS.

Fentanyl is a widely used drug in the management of pain. Present LC-MS/MS methods for analysis of fentanyl require a large volume of serum, but yet the sensitivity was at about 50 pg/mL. Here, we report a modified liquid-liquid extraction method for the analysis of fentanyl in serum. The method is very sensitive with a LLOQ of 5 pg/mL while using only 0.175 mL of serum for analysis. The separation was performed on a Zorbax XDB-C18 column (4.6 × 50 mm, 1.8 µm, 600 bar) using a mobile phase of water: acetonitrile (70:30 v/v) with 0.1% formic acid that was pumped isocratically at a flow rate of 0.5 mL per minute. The calibration curve was found to be linear over a range of 5-10,000 pg/mL. The inter-day and intra-day accuracy and precision were tested using low (20 pg/mL), medium (1000 pg/mL), and high (5000 pg/mL) quality control samples of fentanyl prepared in blank human serum and were within ± 15% of the nominal value. Fentanyl was also found to be stable in various storage and sample preparation conditions, including short-term bench-top storage (for 5 h), freeze-thaw cycling (three cycles), long-term frozen condition (4.5 months at - 70°C), and post-preparative storage (for 48 h).

High-density lipoprotein mimetic peptide 4F mitigates amyloid-ß-induced inhibition of apolipoprotein E secretion and lipidation in primary astrocytes and microglia.

The apolipoprotein E (apoE) ε4 allele is the primary genetic risk factor for late-onset Alzheimer's disease (AD). ApoE in the brain is produced primarily by astrocytes; once secreted from these cells, apoE binds lipids and forms high-density lipoprotein (HDL)-like particles. Accumulation of amyloid-ß protein (Aß) in the brain is a key hallmark of AD, and is thought to initiate a pathogenic cascade leading to neurodegeneration and dementia. The level and lipidation state of apoE affect Aß aggregation and clearance pathways. Elevated levels of plasma HDL are associated with lower risk and severity of AD; the underlying mechanisms, however, have not been fully elucidated. This study was designed to investigate the impact of an HDL mimetic peptide, 4F, on the secretion and lipidation of apoE. We found that 4F significantly increases apoE secretion and lipidation in primary human astrocytes as well as in primary mouse astrocytes and microglia. Aggregated Aß inhibits glial apoE secretion and lipidation, causing accumulation of intracellular apoE, an effect that is counteracted by co-treatment with 4F. Pharmacological and gene editing approaches show that 4F mediates its effects partially through the secretory pathway from the endoplasmic reticulum to the Golgi apparatus and requires the lipid transporter ATP-binding cassette transporter A1. We conclude that the HDL mimetic peptide 4F promotes glial apoE secretion and lipidation and mitigates the detrimental effects of Aß on proper cellular trafficking and functionality of apoE. These findings suggest that treatment with such an HDL mimetic peptide may provide therapeutic benefit in AD. Read the Editorial Highlight for this article on page 580.

Differential Effects of Alzheimer's Disease Aß40 and 42 on Endocytosis and Intraneuronal Trafficking.

Anomalous neuronal accumulation of Aß peptides was shown to affect synaptic transmission and contribute to neurodegeneration in Alzheimer's disease (AD) brain. Neuronal cells internalize amyloid beta (Aß) peptides from the brain extracellular space even under normal physiological conditions, and these endocytotic pathways go awry during AD progression. We hypothesized that exposure to toxic Aß species accumulating in AD brain contributes to perturbations in neuronal endocytosis. We have shown substantial down-regulation of KEGG endocytotic pathway genes in AD patient brain regions that accumulate Aß compared to those in non-demented individuals. While both Aß40 and Aß42 perturbed endocytosis and intracellular trafficking in neuronal cells, Aß40 had a greater effect than Aß42. Moreover, Aß40 decreased the neuronal uptake and lysosomal accumulation of Aß42, which tends to oligomerize at low lysosomal pH. Hence, Aß40 may reduce the prevalence of stable Aß42 oligomers that are closely associated with neurodegeneration and are intercellularly propagated across the vulnerable brain regions to eventually nucleate as amyloid plaques. In conclusion, elevated brain Aß levels and Aß42:40 ratio apparent in the early stages of AD could perturb intraneuronal trafficking, augment the anomalous accumulation of amyloid peptides in AD brain, and drive AD pathogenesis.

A comprehensive analysis of breast cancer microbiota and host gene expression.

The inflammatory tumoral-immune response alters the physiology of the tumor microenvironment, which may attenuate genomic instability. In addition to inducing inflammatory immune responses, several pathogenic bacteria produce genotoxins. However the extent of microbial contribution to the tumor microenvironment biology remains unknown. We utilized The Cancer Genome Atlas, (TCGA) breast cancer data to perform a novel experiment utilizing unmapped and mapped RNA sequencing read evidence to minimize laboratory costs and effort. Our objective was to characterize the microbiota and associate the microbiota with the tumor expression profiles, for 668 breast tumor tissues and 72 non-cancerous adjacent tissues. The prominent presence of Proteobacteria was increased in the tumor tissues and conversely Actinobacteria abundance increase in non-cancerous adjacent tissues. Further, geneset enrichment suggests Listeria spp to be associated with the expression profiles of genes involved with epithelial to mesenchymal transitions. Moreover, evidence suggests H. influenza may reside in the surrounding stromal material and was significantly associated with the proliferative pathways: G2M checkpoint, E2F transcription factors, and mitotic spindle assembly. In summary, further unraveling this complicated interplay should enable us to better diagnose and treat breast cancer patients.

Traffic jam at the blood-brain barrier promotes greater accumulation of Alzheimer's disease amyloid-ß proteins in the cerebral vasculature.

Amyloid-ß (Aß) deposition in the brain vasculature results in cerebral amyloid angiopathy (CAA), which occurs in about 80% of Alzheimer's disease (AD) patients. While Aß42 predominates parenchymal amyloid plaques in AD brain, Aß40 is prevalent in the cerebrovascular amyloid. Dutch mutation of Aß40 (E22Q) promotes aggressive cerebrovascular accumulation and leads to severe CAA in the mutation carriers; knowledge of how DutchAß40 drives this process more efficiently than Aß40 could reveal various pathophysiological events that promote CAA. In this study we have demonstrated that DutchAß40 shows preferential accumulation in the blood-brain-barrier (BBB) endothelial cells due to its inefficient blood-to-brain transcytosis. Consequently, DutchAß40 establishes a permeation barrier in the BBB endothelium, prevents its own clearance from the brain, and promotes the formation of amyloid deposits in the cerebral microvessels. The BBB endothelial accumulation of native Aß40 is not robust enough to exercise such a significant impact on its brain clearance. Hence, the cerebrovascular accumulation of Aß40 is slow and may require other copathologies to precipitate into CAA. In conclusion, the magnitude of Aß accumulation in the BBB endothelial cells is a critical factor that promotes CAA; hence, clearing vascular endothelium of Aß proteins may halt or even reverse CAA.

Differences in the cellular uptake and intracellular itineraries of amyloid beta proteins 40 and 42: ramifications for the Alzheimer's drug discovery.

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD), neurofibrillary tangles and parenchymal amyloid plaques, are downstream reflections of neurodegeneration caused by the intraneuronal accumulation of amyloid-ß proteins (Aß), particularly Aß42 and Aß40. While the neurotoxicity of more amyloidogenic but less abundant Aß42 is well documented, the effect of Aß40 on neurons has been understudied. The Aß40 expression in the presymptomatic AD brain is ten times greater than that of Aß42. However, the Aß40:42 ratio decreases with AD progression and coincides with increased amyloid plaque deposition in the brain. Hence, it is thought that Aß40 protects neurons from the deleterious effects of Aß42. The pathophysiological pathways involved in the neuronal uptake of Aß40 or Aß42 have not been clearly elucidated. Lack of such critical information obscures therapeutic targets and thwarts rational drug development strategies aimed at preventing neurodegeneration in AD. The current study has shown that fluorescein labeled Aß42 (F-Aß42) is internalized by neurons via dynamin dependent endocytosis and is sensitive to membrane cholesterol, whereas the neuronal uptake of F-Aß40 is energy independent and nonendocytotic. Following their uptake, both F-Aß40 and F-Aß42 did not accumulate in early/recycling endosomes; F-Aß42 but not F-Aß40 accumulated in late endosomes and in the vesicles harboring caveolin-1. Furthermore, F-Aß42 demonstrated robust accumulation in the lysosomes and damaged their integrity, whereas F-Aß40 showed only a sparse lysosomal accumulation. Such regulated trafficking along distinct pathways suggests that Aß40 and Aß42 exercise differential effects on neurons. These differences must be carefully considered in the design of a pharmacological agent intended to block the neurodegeneration triggered by Aß proteins.

Alzheimer's disease amyloid ß-protein mutations and deletions that define neuronal binding/internalization as early stage nonfibrillar/fibrillar aggregates and late stage fibrils.

Accumulation of amyloid ß-protein (Aß) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aß accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aß might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aß40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aß40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aß lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aß in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aß40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aß seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aß39E22Δ, Aß41E22Δ, and Aß42 but not Aß40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aß39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aß, therefore, results from aggregate binding to neurons, which requires H13 for Aß40 but not for E22Δ or Aß42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aß40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aß40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aß proteins and their aggregation states or fibrillar structure.

HH domain of Alzheimer's disease Abeta provides structural basis for neuronal binding in PC12 and mouse cortical/hippocampal neurons.

A key question in understanding AD is whether extracellular Abeta deposition of parenchymal amyloid plaques or intraneuronal Abeta accumulation initiates the AD process. Amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Abeta which is then released into the brain interstitial fluid. Intraneuronal Abeta accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Abeta rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Abeta40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Abeta fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Abeta prevented neuronal binding and drove Abeta to enhanced fibril formation and subsequent amyloid plaque deposition--a potential mechanism for removing toxic species of Abeta. Substitution or even masking of these Abeta histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.

Mechanism of neuronal versus endothelial cell uptake of Alzheimer's disease amyloid beta protein.

Alzheimer's disease (AD) is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of beta-amyloid (Abeta) proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Abeta proteins due to their inefficient clearance at the blood-brain-barrier (BBB), places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Abeta proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT) mouse brain slices treated with fluorescein labeled Abeta40 (F-Abeta40) demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Abeta proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH) neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Abeta40 or F-Abeta42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Abeta40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Abeta40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Abeta proteins and help explain the vulnerability of cortical and hippocampal neurons to Abeta toxicity.

In vivo targeting of antibody fragments to the nervous system for Alzheimer's disease immunotherapy and molecular imaging of amyloid plaques.

Targeting therapeutic or diagnostic proteins to the nervous system is limited by the presence of the blood-brain barrier. We report that a F(ab')(2) fragment of a monoclonal antibody against fibrillar human Abeta42 that is polyamine (p)-modified has increased permeability at the blood-brain barrier, comparable binding to the antigen, and comparable in vitro binding to amyloid plaques in Alzheimer's disease (AD) transgenic mouse brain sections. Intravenous injection of the pF(ab')(2)4.1 in the AD transgenic mouse demonstrated efficient targeting to amyloid plaques throughout the brain, whereas the unmodified fragment did not. Removal of the Fc portion of this antibody derivative will minimize the inflammatory response and cerebral hemorrhaging associated with passive immunization and provide increased therapeutic potential for treating AD. Coupling contrast agents/radioisotopes might facilitate the molecular imaging of amyloid plaques with magnetic resonance imaging/positron emission tomography. The efficient delivery of immunoglobulin G fragments may also have important applications to other neurodegenerative disorders or for the generalized targeting of nervous system antigens.