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Background: Age is the most common risk factor for Alzheimer’s disease (AD), a neurodegenerative disorder characterized by the hallmarks of toxic amyloid-β (Aβ) plaques and hyperphosphorylated tau tangles. Moreover, sub-physiological brain insulin levels have emerged as a pathological manifestation of AD. Objective: Identify age-related changes in the plasma disposition and blood-brain barrier (BBB) trafficking of Aβ peptides and insulin in mice. Methods: Upon systemic injection of 125I-Aβ40, 125I-Aβ42, or 125I-insulin, the plasma pharmacokinetics and brain influx were assessed in wild-type (WT) or AD transgenic (APP/PS1) mice at various ages. Additionally, publicly available single-cell RNA-Seq data [GSE129788] was employed to investigate pathways regulating BBB transport in WT mice at different ages. Results: The brain influx of 125I-Aβ40, estimated as the permeability-surface area product, decreased with age, accompanied by an increase in plasma AUC. In contrast, the brain influx of 125I-Aβ42 increased with age, accompanied by a decrease in plasma AUC. The age-dependent changes observed in WT mice were accelerated in APP/PS1 mice. As seen with 125I-Aβ40, the brain influx of 125I-insulin decreased with age in WT mice, accompanied by an increase in plasma AUC. This finding was further supported by dynamic single-photon emission computed tomography (SPECT/CT) imaging studies. RAGE and PI3K/AKT signaling pathways at the BBB, which are implicated in Aβ and insulin transcytosis, respectively, were upregulated with age in WT mice, indicating BBB insulin resistance. Conclusion: Aging differentially affects the plasma pharmacokinetics and brain influx of Aβ isoforms and insulin in a manner that could potentially augment AD risk.

Disruptions in glucose uptake and metabolism in the brain are implicated in metabolic disorders and Alzheimer’s disease (AD). Toxic soluble amyloid-beta (sAß) peptides accumulating in the brain and plasma of AD patients were suggested to promote blood-brain barrier (BBB) dysfunction, brain hypometabolism, and cognitive decline. Exposure to sAß peptides is reported to interfere with glucose metabolism in the brain parenchyma, although their effects on the BBB have not been fully characterized. Our data showed that the brain uptake of glucose surrogate, [18F]-fluorodeoxyglucose (18FDG), was reduced significantly in APP/PS1 transgenic mice (overproduce Aß) compared to wild-type (WT) mice. In addition, the influx rate of18FDG was also decreased in both Aß40 and Aß42 pre-infused mice compared to control mice. Glucose is primarily delivered from blood into the brain via glucose transporter 1 (GLUT1). The confocal microscopy experiment showed that Aß40 and Aß42 peptides significantly decreased GLUT1 expression in polarized human cerebral microvascular endothelial cell (hCMEC/D3) monolayers. Insulin-AKT pathway has been observed to induce glucose uptake via regulating the expression of TXNIP, the only α-arrestin protein known to bind to thioredoxin. We found that Aß40 and Aß42 peptides decreased p-AKT and increased TXNIP expression in the hCMEC/D3 cell monolayers. MK2206, a kinase inhibitor of AKT, was used to confirm that inhibition of insulin/AKT pathway reduced GLUT1 expression in an insulin-independent manner in the hCMEC/D3 cell monolayers. These results suggest that inhibitory effects of sAß on GLUT1 expression are mediated by inhibition of the insulin/AKT pathway. The role of TXNIP on endothelial GLUT1 expression was investigated using resveratrol, which has been reported to downregulate TXNIP overexpression. Consistently, resveratrol treatment led to a significant increase in GLUT1 expression in the hCMEC/D3 cell monolayers. Furthermore, by co-incubation of resveratrol and sAß peptides in hCMEC/D3 cell monolayers, we found that resveratrol rectified the aberrant TXNIP expression caused by sAß peptides. Together, these findings provide novel evidence that toxic sAß peptide exposure inhibits glucose transport at the BBB by decreasing GLUT1 expression via the insulin/Akt/TXNIP axis.

Blood-brain barrier (BBB) is a critical portal regulating the bidirectional transport of amyloid beta (Aβ) proteins between blood and brain. Disrupted trafficking at the BBB may not only promote the build-up of Aβ plaques in the brain parenchyma, but also facilitate Aβ accumulation within the BBB endothelium, which aggravates BBB dysfunction. Soluble Aβ42:Aβ40 ratios in plasma and cerebrospinal fluid have been reported to decrease during Alzheimer’s disease (AD) progression. Our previous publications demonstrated that trafficking of Aβ42 and Aβ40 at the BBB is distinct and is disrupted under various pathophysiological conditions. However, the intracellular mechanisms that allow BBB endothelium to differentially handle Aβ40 and Aβ42 have not been clearly elucidated. In this study, we identified mechanisms of fluorescently labeled Aβ (F-Aβ) endocytosis in polarized human cerebral microvascular endothelial (hCMEC/D3) cell monolayers using pharmacological inhibition and siRNA knock-down approaches. Further, intracellular transit of F-Aβ following endocytosis was tracked using live cell imaging. Our studies demonstrated that both F-Aβ peptides were internalized by BBB endothelial cells via energy, dynamin and actin dependent endocytosis. Interestingly, endocytosis of F-Aβ40 is found to be clathrin-mediated, whereas F-Aβ42 endocytosis is caveolae-mediated. Following endocytosis, both isoforms were sorted by the endo-lysosomal system. While Aβ42 was shown to accumulate more in the lysosome which could lead to its higher degradation and/or aggregation at lower lysosomal pH, Aβ40 demonstrated robust accumulation in recycling endosomes which may facilitate its transcytosis across the BBB. These results provide a mechanistic insight into the selective ability of BBB endothelium to transport Aβ40 versus Aβ42. This knowledge contributes to the understanding of molecular pathways underlying Aβ accumulation in the BBB endothelium and associated cerebrovascular dysfunction as well as amyloid deposition in the brain parenchyma which are implicated in AD pathogenesis.

Plasma pharmacokinetic (PK) data is required as an input function for graphical analysis (e.g., Patlak plot) of single positron emission computed tomography/computed tomography (SPECT/CT) and positron emission tomography/CT (PET/CT) data to evaluate tissue influx rate of radiotracers. Dynamic heart imaging data is often used as a surrogate of plasma PK. However, accumulation of radiolabel (representing both intact and degraded tracer) in the heart tissue may interfere with accurate prediction of plasma PK from the heart data. Therefore, we developed a compartmental model, which involves forcing functions to describe intact and degraded radiolabeled proteins in plasma and their accumulation in heart tissue, to deconvolve plasma PK of125I-amyloid beta 40 (125I-Aβ40) and125I-insulin from their dynamic heart imaging data. The three-compartment model was shown to adequately describe the plasma concentration-time profile of intact/degraded proteins and the heart radioactivity time data obtained from SPECT/CT imaging for both tracers. The model was successfully applied to deconvolve the plasma PK of both tracers from their naïve datasets of dynamic heart imaging. In agreement with our previous observations made by conventional serial plasma sampling, the deconvolved plasma PK of125I-Aβ40and125I-insulin in young mice exhibited lower area under the curve (AUC) than the aged mice. Further, Patlak plot parameters (Ki) extracted using deconvolved plasma PK as input function successfully recapitulated age-dependent blood-to-brain influx kinetics changes for both125I-Aβ40and125I-insulin. Therefore, the compartment model developed in this study provides a novel approach to deconvolve plasma PK of radiotracers from their noninvasive dynamic heart imaging. This method facilitates the application of preclinical SPECT or PET imaging data to characterize distribution kinetics of tracers where simultaneous plasma sampling is not feasible.

At the stroke of the New Year 2020, COVID-19, a zoonotic disease that would turn into a global pandemic, was identified in the Chinese city of Wuhan. Although unique in its transmission and virulence, COVID-19 is similar to zoonotic diseases, including other SARS variants (e.g., SARS-CoV) and MERS, in exhibiting severe flu-like symptoms and acute respiratory distress. Even at the molecular level, many parallels have been identified between SARS and COVID-19 so much so that the COVID-19 virus has been named SARS-CoV-2. These similarities have provided several opportunities to treat COVID-19 patients using clinical approaches that were proven to be effective against SARS. Importantly, the identification of similarities in how SARS-CoV and SARS-CoV-2 access the host, replicate, and trigger life-threatening pathological conditions have revealed opportunities to repurpose drugs that were proven to be effective against SARS. In this article, we first provided an overview of COVID-19 etiology vis-à-vis other zoonotic diseases, particularly SARS and MERS. Then, we summarized the characteristics of droplets/aerosols emitted by COVID-19 patients and how they aid in the transmission of the virus among people. Moreover, we discussed the molecular mechanisms that enable SARS-CoV-2 to access the host and become more contagious than other betacoronaviruses such as SARS-CoV. Further, we outlined various approaches that are currently being employed to diagnose and symptomatically treat COVID-19 in the clinic. Finally, we reviewed various approaches and technologies employed to develop vaccines against COVID-19 and summarized the attempts to repurpose various classes of drugs and novel therapeutic approaches.

Blood-brain barrier (BBB) endothelial cells lining the cerebral microvasculature maintain dynamic equilibrium between soluble amyloid-β (Aβ) levels in the brain and plasma. The BBB dysfunction prevalent in Alzheimer disease contributes to the dysregulation of plasma and brain Aβ and leads to the perturbation of the ratio between Aβ42 and Aβ40, the two most prevalent Aβ isoforms in patients with Alzheimer disease. We hypothesize that BBB endothelium distinguishes between Aβ40 and Aβ42, distinctly modulates their trafficking kinetics between plasma and brain, and thereby contributes to the maintenance of healthy Aβ42/Aβ40 ratios. To test this hypothesis, we investigated Aβ40 and Aβ42 trafficking kinetics in hCMEC/D3 monolayers (human BBB cell culture model) in vitro as well as in mice in vivo. Although the rates of uptake of fluorescein-labeled Aβ40 and Aβ42 (F-Aβ40 and F-Aβ42) were not significantly different on the abluminal side, the luminal uptake rate of F-Aβ42 was substantially higher than F-Aβ40. Since higher plasma Aβ levels were shown to aggravate BBB dysfunction and trigger cerebrovascular disease, we systematically investigated the dynamic interactions of luminal [125I]Aβ peptides and their trafficking kinetics at BBB using single-photon emission computed tomography/computed tomography imaging in mice. Quantitative modeling of the dynamic imaging data thus obtained showed that the rate of uptake of toxic [125I]Aβ42 and its subsequent BBB transcytosis is significantly higher than [125I]Aβ40. It is likely that the molecular mechanisms underlying these kinetic differences are differentially affected in Alzheimer and cerebrovascular diseases, impact plasma and brain levels of Aβ40 and Aβ42, engender shifts in the Aβ42/Aβ40 ratio, and unleash downstream toxic effects. SIGNIFICANCE STATEMENT: Dissecting the binding and uptake kinetics of Aβ40 and Aβ42 at the BBB endothelium will facilitate the estimation of Aβ40 versus Aβ42 exposure to the BBB endothelium and allow assessment of the risk of BBB dysfunction by monitoring Aβ42 and Aβ40 levels in plasma. This knowledge, in turn, will aid in elucidating the role of these predominant Aβ isoforms in aggravating BBB dysfunction and cerebrovascular disease.

Treatments to elevate high-density lipoprotein (HDL) levels in plasma have decreased cerebrovascular amyloid -β (Aβ) 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 [(125)I]Aβ trafficking from brain to blood and from blood to brain. After systemic injection in mice, the BBB permeability of [(125)I]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 [(125)I]4F were ∼1000-fold higher compared with those determined for [(125)I]ApoA-I. Moreover, systemic infusion with 4F increased the brain efflux of intracerebrally injected [(125)I]Aβ42. Conversely, 4F infusion decreased the brain influx of systemically injected [(125)I]Aβ42. Interestingly, 4F did not significantly alter the brain influx of [(125)I]Aβ40. To corroborate the in vivo findings, we evaluated the effects of 4F on [(125)I]Aβ42 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 [(125)I]Aβ42 across the hCMEC/D3 monolayers. Additionally, 4F decreased the endothelial accumulation of fluorescein-labeled Aβ42 in the hCMEC/D3 monolayers. These findings provide a mechanistic interpretation for the reductions in brain Aβ 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 -β 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.

Elevated exposure to toxic amyloid beta (Aβ) peptides and consequent blood–brain barrier (BBB) dysfunction are believed to promote vasculopathy in Alzheimer’s disease (AD). However, the accumulation kinetics of different Aβ isoforms within the BBB endothelium and how it drives BBB dysfunction are not clearly characterized. Using single positron emission computed tomography (SPECT)-computed tomography (CT) dynamic imaging coupled with population pharmacokinetic modeling, we investigated the accumulation kinetics of Aβ40 and Aβ42 in the BBB endothelium. Brain clearance was quantified after intracerebral administration of (125)I-Aβ, and BBB-mediated transport was shown to account for 54% of (125)I-Aβ40 total clearance. A brain influx study demonstrated lower values of both maximal rate (V(max)) and Michaelis constant (K(m)) for (125)I-Aβ42 compared to (125)I-Aβ40. Validated by a transcytosis study in polarized human BBB endothelial cell (hCMEC/D3) monolayers, model simulations demonstrated impaired exocytosis was responsible for inefficient permeability and enhanced accumulation of Aβ42 in the BBB endothelium. Further, both isoforms were shown to disrupt the exocytosis machinery of BBB endothelial cells so that a vicious cycle could be generated. The validated model was able to capture changes in Aβ steady-state levels in plasma as well as the brain during AD progression and allowed us to predict the kinetics of Aβ accumulation in the BBB endothelium.

Recent studies suggest that apolipoprotein A-I (ApoA-I), the major protein constituent of high-density lipoprotein particles, plays a critical role in preserving cerebrovascular integrity and reducing Alzheimer’s risk. ApoA-I present in brain is thought to be primarily derived from the peripheral circulation. Although plasma-to-brain delivery of ApoA-I is claimed to be handled by the blood-cerebrospinal fluid barrier (BCSFB), a contribution by the blood-brain barrier (BBB), which serves as a major portal for protein delivery to brain, cannot be ruled out. In this study, we assessed the permeability–surface area product (PS) of radioiodinated ApoA-I ((125)I-ApoA-I) in various brain regions of wild-type rats after an intravenous bolus injection. The PS value at the cortex, caudate putamen, hippocampus, thalamus, brain stem, and cerebellum was found to be 0.39, 0.28, 0.28, 0.36, 0.69, and 0.76 (ml/g per second × 10(−6)), respectively. Solutes delivered into brain via the BCSFB are expected to show greater accumulation in the thalamus due to its periventricular location. The modest permeability for (125)I-ApoA-I into the thalamus relative to other regions suggests that BCSFB transport accounts for only a portion of total brain uptake and thus BBB transport cannot be ruled out. In addition, we show that Alexa Flour 647-labeled ApoA-I (AF647-ApoA-I) undergoes clathrin-independent and cholesterol-mediated endocytosis in transformed human cerebral microvascular endothelial cells (hCMEC/D3). Further, Z-series confocal images of the hCMEC/D3 monolayers and Western blot detection of intact ApoA-I on the abluminal side demonstrated AF647-ApoA-I transcytosis across the endothelium. These findings implicate the BBB as a significant portal for ApoA-I delivery into brain.

Scopolamine is an anticholinergic alkaloid that is widely used in the form of a transdermal system to manage nausea associated with motion sickness. Currently available methods to quantify scopolamine require large sample volumes and involve cumbersome sample preparation. In this work, a simple method for the rapid separation and sensitive quantification of scopolamine in human serum was developed. Scopolamine was extracted from 0.5 mL of human serum using solid-phase extraction. The extracted samples were injected onto Zorbax XDB-C18 column (4.6 × 50 mm, 1.8 μm, and 600 bar) on an Agilent 1200 series HPLC. The chromatographic separation involved gradient elution with water and acetonitrile containing 0.1% v/v formic acid as a mobile phase. The samples were quantified in positive ion mode using a TSQ Quantum triple quadrupole mass spectrometer. The assay was validated and found to be linear over a concentration range of 5–5000 pg/mL. The total assay precision and accuracy was 6.3% and 96%, respectively. The lower limit of quantification (LLOQ) of the assay was 5 pg/mL. The assay was used in a human pharmacokinetic study to measure the concentration of scopolamine in serum after an administering scopolamine as transdermal delivery system or as an intravenous bolus dose.

Accumulation of amyloid beta (Aβ) peptides in the cerebral vasculature, referred to as cerebral amyloid angiopathy (CAA), is widely observed in Alzheimer's disease (AD) brain and was shown to accelerate cognitive decline. There is no effective method for detecting cerebrovascular amyloid (CVA) and treat CAA. The targeted nanoparticles developed in this study effectively migrated from the blood flow to the vascular endothelium as determined by using quartz crystal microbalance with dissipation monitoring (QCM-D) technology. We also improved the stability, and blood–brain barrier (BBB) transcytosis of targeted nanoparticles by coating them with a cationic BBB penetrating peptide (K16ApoE). The K16ApoE-Targeted nanoparticles demonstrated specific targeting of vasculotropic DutchAβ40 peptide accumulated in the cerebral vasculature. Moreover, K16ApoE-Targeted nanoparticles demonstrated significantly greater uptake into brain and provided specific MRI contrast to detect brain amyloid plaques.

BackgroundCritical functions of the blood-brain barrier (BBB), including cerebral blood flow and vascular response, are regulated by insulin signaling pathways. Therefore, endothelial insulin resistance could lead to vascular dysfunction, which is associated with neurodegenerative diseases such as Alzheimer’s disease (AD).ObjectiveThe objective of the current study is to map the dynamics of insulin-responsive pathways in polarized human cerebral microvascular endothelial cells (hCMEC/D3) cell monolayers, a widely used BBB cell culture model, to identify molecular mechanisms underlying BBB dysfunction in AD.MethodsRNA-Sequencing (RNA-Seq) was performed on hCMEC/D3 cell monolayers with and without insulin treatment at various time points. The Short Time-series Expression Miner (STEM) method was used to identify clusters of genes with distinct and representative patterns. Functional annotation and pathway analysis of the genes from top clusters were conducted using the Webgestalt and Ingenuity Pathway Analysis (IPA) software, respectively.ResultsQuantitative expression differences of 19,971 genes between the insulin-treated and control monolayers at five-time points were determined. STEM software identified 11 clusters with 3061 genes across that displayed various temporal patterns. Gene ontology enrichment analysis performed using the top 5 clusters demonstrated that these genes were enriched in various biological processes associated with AD pathophysiology. The IPA analyses revealed that signaling pathways exacerbating AD pathology such as inflammation were downregulated after insulin treatment (clusters 1 to 3). In contrast, pathways attenuating AD pathology were upregulated, including synaptogenesis and BBB repairment (clusters 4 and 5).ConclusionsThese findings unravel the dynamics of insulin action on the BBB endothelium and inform about downstream signaling cascades that potentially regulate neurovascular unit (NVU) functions that are disrupted in AD.

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized primarily by motor and non-motor gastrointestinal (GI) deficits. GI symptoms’ including compromised intestinal barrier function often accompanies altered gut microbiota composition and motor deficits in PD. Therefore, in this study, we set to investigate the role of gut microbiota and epithelial barrier dysfunction on motor symptom generation using a rotenone-induced mouse model of PD. We found that while six weeks of 10 mg/kg of chronic rotenone administration by oral gavage resulted in loss of tyrosine hydroxylase (TH) neurons in both germ-free (GF) and conventionally raised (CR) mice, the decrease in motor strength and coordination was observed only in CR mice. Chronic rotenone treatment did not disrupt intestinal permeability in GF mice but resulted in a significant change in gut microbiota composition and an increase in intestinal permeability in CR mice. These results highlight the potential role of gut microbiota in regulating barrier dysfunction and motor deficits in PD.

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.

Dense nanolipid fluid (DNLF) dispersions are highly concentrated aqueous dispersions of lipid nanocarriers (LNCs) with more than 1015 lipid particles per cubic centimeter. Descriptions of dense nanolipid fluid dispersions in the scientific literature are rare, and they have not been used to encapsulate drugs. In this paper we describe the synthesis of DNLF dispersions comprising ibuprofen using a recently described twin-screw extrusion process. We report that such dispersions are stable, bind ibuprofen tightly and yet provide high transdermal drug permeation. Ibuprofen DNLF dispersions prepared according to the present study provide up to five times greater flux of the pharmacologically active S-ibuprofen isomer through human skin than a commercially available racemic ibuprofen emulsion product. We demonstrate scaling up the twin-screw extrusion method to pilot production for a stable, highly permeating ibuprofen DNLF composition based on excipients approved by the US FDA for use in topical products as a key step towards development of a commercially viable, FDA approvable topical ibuprofen medicine to treat osteoarthritis, which has never before been accomplished.

Background: Synaptic dysfunction prevalent in Alzheimer’s disease (AD) brain is closely associated with increased accumulation of amyloid-β (Aβ) peptides in the brain parenchyma. It is widely believed that Aβ 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, Aβ interactions with SNARE proteins to ultimately disrupt synaptic vesicular fusion are not well understood. Objective: Our objective is to elucidate mechanisms by which Aβ 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 Aβ 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 Aβ (F-Aβ) peptides and their co-localization with Cer-VAMP2 was investigated by confocal microscopy. Results: The F-Aβ40 and F-Aβ42 are internalized by differentiated N2A cells, where they colocalize with Cer-VAMP2. Both Aβ40 and Aβ42 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, Aβ40 and Aβ42, can directly interfere with the SNARE complex formation, which is critical for the docking and fusion of synaptic vesicles.

Blood-brain barrier (BBB) endothelial cells lining the cerebral microvasculature maintain dynamic equilibrium between soluble amyloid

To estimate strength of a scopolamine transdermal delivery system (TDS) in vivo, using residual drug vs. pharmacokinetic analyses with the goal of scientifically supporting a single and robust method for use across the dosage form and ultimately facilitate the development of more consistent and clinically meaningful labeling. A two-arm, open-label, crossover pharmacokinetic study was completed in 26 volunteers. Serum samples were collected and residual scopolamine was extracted from worn TDS. Delivery extent and rate were estimated by (1) numeric deconvolution and (2) steady-state serum concentration determined from graphical and non-compartmental analyses. In residual drug analyses, mean ± SD scopolamine release rate was 0.015 ± 0.002 mg/h (11% RSD), vs. 0.016 ± 0.006 mg/h (35% RSD) from numeric deconvolution, 0.015 ± 0.005 mg/h (34% RSD) from graphical analysis, and 0.015 ± 0.007 mg/h (44% RSD) from non-compartmental analysis. In residual drug analyses, total drug released was 1.09 ± 0.11 mg (10% RSD), vs. 1.12 ± 0.40 mg (35% RSD) from numeric deconvolution, 1.07 ± 0.35 mg (33% RSD) from graphical analysis, and 1.07 ± 0.45 (42% RSD) from non-compartmental analysis. Extent and rate of scopolamine release were comparable by both approaches, but pharmacokinetic analysis demonstrated greater inter-subject variability.

The apolipoprotein E (apoE) e4 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-β protein (Aβ) 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 Aβ 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 Aβ 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 Aβ 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.

Anomalous neuronal accumulation of Aβ peptides was shown to affect synaptic transmission and contribute to neurodegeneration in Alzheimer's disease (AD) brain. Neuronal cells internalize amyloid beta (Aβ) 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 Aβ 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 Aβ compared to those in non-demented individuals. While both Aβ40 and Aβ42 perturbed endocytosis and intracellular trafficking in neuronal cells, Aβ40 had a greater effect than Aβ42. Moreover, Aβ40 decreased the neuronal uptake and lysosomal accumulation of Aβ42, which tends to oligomerize at low lysosomal pH. Hence, Aβ40 may reduce the prevalence of stable Aβ42 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 Aβ levels and Aβ42:40 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.

Impaired brain clearance of amyloid-beta peptides (Aβ) 40 and 42 across the blood–brain barrier (BBB) is believed to be one of the pathways responsible for Alzheimer’s disease (AD) pathogenesis. Hyperinsulinemia prevalent in type II diabetes was shown to damage cerebral vasculature and increase Aβ accumulation in AD brain. However, there is no clarity on how aberrations in peripheral insulin levels affect Aβ accumulation in the brain. This study describes, for the first time, an intricate relation between plasma insulin and Aβ transport at the BBB. Upon peripheral insulin administration in wild-type mice: the plasma clearance of Aβ40 increased, but Aβ42 clearance reduced; the plasma-to-brain influx of Aβ40 increased, and that of Aβ42 reduced; and the clearance of intracerebrally injected Aβ40 decreased, whereas Aβ42 clearance increased. In hCMEC/D3 monolayers (in vitro BBB model) exposed to insulin, the luminal uptake and luminal-to-abluminal permeability of Aβ40 increased and that of Aβ42 reduced; the abluminal-to-luminal permeability of Aβ40 decreased, whereas Aβ42 permeability increased. Moreover, Aβ cellular trafficking machinery was altered. In summary, Aβ40 and Aβ42 demonstrated distinct distribution kinetics in plasma and brain compartments, and insulin differentially modulated their distribution. Cerebrovascular disease and metabolic disorders may disrupt this intricate homeostasis and aggravate AD pathology.

See Lowe et al. (doi:10.1093/brain/awy226) for a scientific commentary on this article. Alzheimer’s disease targets discrete neural systems, but the mechanisms underlying this regionally selective vulnerability remain unknown. Grothe et al. report that Alzheimer’s disease pathology specifically affects brain regions with certain molecular properties, and that the biological pathways underlying this vulnerability differ for amyloid accumulation versus neurodegeneration., Amyloid deposition and neurofibrillary degeneration in Alzheimer’s disease specifically affect discrete neuronal systems, but the underlying mechanisms that render some brain regions more vulnerable to Alzheimer’s disease pathology than others remain largely unknown. Here we studied molecular properties underlying these distinct regional vulnerabilities by analysing Alzheimer’s disease-typical neuroimaging patterns of amyloid deposition and neurodegeneration in relation to regional gene expression profiles of the human brain. Graded patterns of brain-wide vulnerability to amyloid deposition and neurodegeneration in Alzheimer’s disease were estimated by contrasting multimodal amyloid-sensitive PET and structural MRI data between patients with Alzheimer’s disease dementia (n = 76) and healthy controls (n = 126) enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Regional gene expression profiles were derived from brain-wide microarray measurements provided by the Allen brain atlas of the adult human brain transcriptome. In a hypothesis-driven analysis focusing on the genes coding for the amyloid precursor (APP) and tau proteins (MAPT), regional expression levels of APP were positively correlated with the severity of regional amyloid deposition (r = 0.44, P = 0.009), but not neurodegeneration (r = 0.01, P = 0.96), whereas the opposite pattern was observed for MAPT (neurodegeneration: r = 0.46, P = 0.006; amyloid: r = 0.08, P = 0.65). Using explorative gene set enrichment analysis, amyloid-vulnerable regions were found to be characterized by relatively low expression levels of gene sets implicated in protein synthesis and mitochondrial respiration. By contrast, neurodegeneration-vulnerable regions were characterized by relatively high expression levels of gene sets broadly implicated in neural plasticity, with biological functions ranging from neurite outgrowth and synaptic contact over intracellular signalling cascades to proteoglycan metabolism. At the individual gene level this data-driven analysis further corroborated the association between neurodegeneration and MAPT expression, and additionally identified associations with known tau kinases (CDK5, MAPK1/ERK2) alongside components of their intracellular (Ras-ERK) activation pathways. Sensitivity analyses showed that these pathology-specific imaging-genetic associations were largely robust against changes in some of the methodological parameters, including variation in the brain donor sample used for estimating regional gene expression profiles, and local variations in the Alzheimer’s disease-typical imaging patterns when these were derived from an independent patient cohort (BioFINDER study). These findings highlight that the regionally selective vulnerability to Alzheimer’s disease pathology relates to specific molecular-functional properties of the affected neural systems, and that the implicated biochemical pathways largely differ for amyloid accumulation versus neurodegeneration. The data provide novel insights into the complex pathophysiological mechanisms of Alzheimer’s disease and point to pathology-specific treatment targets that warrant further exploration in independent studies.

Aberrant insulin signaling constitutes an early change in Alzheimer's disease (AD). Insulin receptors (IR) and low-density lipoprotein receptor-related protein-1 (LRP-1) are expressed in brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). There, insulin may regulate the function of LRP-1 in Aβ clearance from the brain. Changes in IR-β and LRP-1 and insulin signaling at the BBB in AD are not well understood. Herein, we identified a reduction in cerebral and cerebrovascular IR-β levels in 9-month-old male and female 3XTg-AD (PS1M146V, APPSwe, and tauP301L) as compared to NTg mice, which is important in insulin mediated signaling responses. Reduced cerebral IR-β levels corresponded to impaired insulin signaling and LRP-1 levels in brain. Reduced cerebral and cerebrovascular IR-β and LRP-1 levels in 3XTg-AD mice correlated with elevated levels of autophagy marker LC3B. In both genotypes, high-fat diet (HFD) feeding decreased cerebral and hepatic LRP-1 expression and elevated cerebral Aβ burden without affecting cerebrovascular LRP-1 and IR-β levels. In vitro studies using primary porcine (p)BCEC revealed that Aβ peptides 1–40 or 1–42 (240 nM) reduced cellular levels and interaction of LRP-1 and IR-β thereby perturbing insulin-mediated signaling. Further mechanistic investigation revealed that Aβ treatment accelerated the autophagy-lysosomal degradation of IR-β and LRP-1 in pBCEC. LRP-1 silencing in pBCEC decreased IR-β levels through post-translational pathways further deteriorating insulin-mediated responses at the BBB. Our findings indicate that LRP-1 proves important for insulin signaling at the BBB. Cerebral Aβ burden in AD may accelerate LRP-1 and IR-β degradation in BCEC thereby contributing to impaired cerebral and cerebromicrovascular insulin effects.

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 ˂ 0.05) higher drug accumulation in the skin when compared to commercial cream formulation.

Background Microbial conversion of dietary substrate to bioactive molecules represents a potential regulatory mechanism by which gut microbes can alter intestinal physiology. Tryptamine, a monoamin...

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).

Tryptamine, a tryptophan-derived monoamine similar to 5-hydroxytryptamine (5-HT), is produced by gut bacteria and is abundant in human and rodent feces. However, the physiologic effect of tryptamine in the gastrointestinal (GI) tract remains unknown. Here, we show that the biological effects of tryptamine are mediated through 5-HT(4) receptor (5-HT(4)R), a G-protein coupled receptor (GPCR) uniquely expressed in the colonic epithelium. Tryptamine increases ionic flux across the colonic epithelium and increases fluid secretion in colonoids from germ free (GF) and humanized (ex-GF colonized with human stool) mice consistent with increased intestinal secretion. The secretory effect of tryptamine is dependent on 5-HT(4)R activation and is blocked by 5-HT(4)R antagonist and absent in 5-HT(4)R(−/−) mice. GF mice colonized by Bacteroides thetaiotaomicron engineered to produce tryptamine exhibit accelerated gastrointestinal transit. Our study demonstrates an aspect of host physiology under control of a bacterial metabolite that can be exploited as a therapeutic modality.

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.

Cerebral amyloid angiopathy (CAA) results from the accumulation of Aβ proteins primarily within the media and adventitia of small arteries and capillaries of the cortex and leptomeninges. CAA affects a majority of Alzheimer's disease (AD) patients and is associated with a rapid decline in cognitive reserve. Unfortunately, there is no pre-mortem diagnosis available for CAA. Furthermore, treatment options are few and relatively ineffective. To combat this issue, we have designed nanovehicles (nanoparticles-IgG4.1) capable of targeting cerebrovascular amyloid (CVA) and serving as early diagnostic and therapeutic agents. These nanovehicles were loaded with Gadolinium (Gd) based (Magnevist(®)) magnetic resonance imaging contrast agents or single photon emission computed tomography (SPECT) agents, such as (125)I. In addition, the nanovehicles carry either anti-inflammatory and anti-amyloidogenic agents such as curcumin or immunosuppressants such as dexamethasone, which were previously shown to reduce cerebrovascular inflammation. Owing to the anti-amyloid antibody (IgG4.1) grafted on the surface, the nanovehicles are capable of specifically targeting CVA deposits. The nanovehicles effectively marginate from the blood flow to the vascular wall as determined by using quartz crystal microbalance with dissipation monitoring (QCM-D) technology. They demonstrate excellent distribution to the brain vasculature and target CVA, thus providing MRI and SPECT contrast specific to the CVA in the brain. In addition, they also display the potential to carry therapeutic agents to reduce cerebrovascular inflammation associated with CAA, which is believed to trigger hemorrhage in CAA patients.

Recurrence of bacterial vaginosis is attributed to the inability of various formulations to disrupt bacterial biofilms. A negatively charged polysaccharide matrix coats the bacterial communities in the biofilm and restricts the penetration of antibiotics. Therefore, bacteria in the deeper segments of the biofilm persist and perpetuate the infection. In this study, we have tested the efficacy of two bioadhesive polymers, cationic chitosan and anionic polycarbophil, to disrupt Pseudomonas aeruginosa biofilms grown in the Center for Disease Control bioreactor as well as on the 96-well plates. The biofilms were treated with various concentrations of polycarbophil and chitosan at pH 4 or 6. Biofilm integrity following various treatments was evaluated by crystal violet stain and laser confocal microscopy employing Syto9 (live-cell stain) and propidium iodide (dead-cell stain). These studies demonstrated that chitosan gel disrupts the P. aeruginosa biofilm more effectively than does polycarbophil; and this effect is independent of the pH and charge densities on either polymers.

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-β proteins (Aβ), particularly Aβ42 and Aβ40. While the neurotoxicity of more amyloidogenic but less abundant Aβ42 is well documented, the effect of Aβ40 on neurons has been understudied. The Aβ40 expression in the presymptomatic AD brain is ten times greater than that of Aβ42. However, the Aβ40:42 ratio decreases with AD progression and coincides with increased amyloid plaque deposition in the brain. Hence, it is thought that Aβ40 protects neurons from the deleterious effects of Aβ42. The pathophysiological pathways involved in the neuronal uptake of Aβ40 or Aβ42 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 Aβ42 (F-Aβ42) is internalized by neurons via dynamin dependent endocytosis and is sensitive to membrane cholesterol, whereas the neuronal uptake of F-Aβ40 is energy independent and nonendocytotic. Following their uptake, both F-Aβ40 and F-Aβ42 did not accumulate in early/recycling endosomes; F-Aβ42 but not F-Aβ40 accumulated in late endosomes and in the vesicles harboring caveolin-1. Furthermore, F-Aβ42 demonstrated robust accumulation in the lysosomes and damaged their integrity, whereas F-Aβ40 showed only a sparse lysosomal accumulation. Such regulated trafficking along distinct pathways suggests that Aβ40 and Aβ42 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 Aβ proteins.

This study investigates physicochemical processes responsible for the biphasic transdermal flux profiles of melatonin in the presence of saturated fatty alcohols (SFAL) and unsaturated fatty alcohols (USFAL). The first phase melatonin flux (J(1st)) in the presence of USFAL enhancers increased with increase in the number of double bonds and reached a limiting value with two double bonds in the molecule. In case of SFAL enhancers, J(1st) increased with enhancer chain length and log formulation/skin partition coefficients (log Ps), which were calculated using the solubility parameters of various formulation components. But, melatonin flux in the second phase decreased with increase in the enhancer chain length and log P values. On the other hand, the transepidermal water loss (TEWL) from the SFAL treated skin increased drastically in the second phase and correlated with log P value of the enhancer. High TEWL value, indicative of a severely disrupted SC, may help the polar formulation components to accumulate in the SC. As a consequence, the SC polarity could change significantly and reduce the partitioning of lipophilic enhancer and/or melatonin in the second phase. This study demonstrated that an optimal level of barrier disruption enhances the transdermal permeation of drugs, whereas, a drastic barrier disruption impedes transdermal transport.

A novel magnetic resonance (MR) imaging contrast agent based on a derivative of human amyloid beta (Abeta) peptide, Gd[N-4ab/Q-4ab]Abeta 30, was previously shown to cross the blood-brain barrier (BBB) and bind to amyloid plaques in Alzheimer's disease (AD) transgenic mouse (APP/PS1) brain. We now report extensive plasma and brain pharmacokinetics of this contrast agent in wild-type (WT) and in APP/PS1 mice along with a quantitative summary of various physiological factors that govern its efficacy. Upon i.v. bolus administration, (125)I-Gd[N-4ab/Q-4ab]Abeta 30 was rapidly eliminated from the plasma following a three-exponential disposition, which is saturable at higher concentrations. Nevertheless, the contrast agent exhibited rapid and nonsaturable absorption at the BBB. The brain pharmacokinetic profile of (125)I-Gd[N-4ab/Q-4ab]Abeta 30 showed a rapid absorption phase followed by a slower elimination phase. No significant differences were observed in the plasma or brain kinetics of WT and APP/PS1 animals. Emulsion autoradiography studies conducted on WT and APP/PS1 mouse brain after an i.v. bolus administration of (125)I-Gd[N-4ab/Q-4ab]Abeta 30 in vivo confirmed the brain pharmacokinetic data and also demonstrated the preferential localization of the contrast agent on the plaques for an extended period of time. These attributes of the contrast agent are extremely useful in providing an excellent signal/noise ratio during longer MR scans, which may be essential for obtaining a high resolution image. In conclusion, this study documents the successful plaque targeting of Gd[N-4ab/Q-4ab]Abeta 30 and provides crucial pharmacokinetic information to determine the dose, mode of administration, and scan times for future in vivo MR imaging of amyloid plaques in AD transgenic mice.

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.

Blood-brain barrier (BBB) is a monolayer of endothelial cells that line brain capillaries. The BBB protects brain by blocking the entry of harmful substances from blood and shielding the brain from peripheral fluctuations in hormones, fatty acids, and electrolytes. In addition, the BBB effectively clears brain metabolites and serves as a major conduit for the delivery of crucial nutrients and growth factors needed for proper brain function. Owing to these critical responsibilities, any functional and structural impairment of the BBB may result in severe pathophysiological consequences in the brain. BBB dysfunction is implicated in several neurodegenerative disorders including Alzheimer's disease (Carmeliet and De Strooper, 2012), Parkinson's disease (Kortekaas et al., 2005), and cerebrovascular diseases (Yang and Rosenberg, 2011) such as cerebral amyloid angiopathy, stroke, and vascular dementia. Hence, the research community has been actively investigating the cerebrovascular contributions to neurological diseases with major emphasis on the BBB. The success of these efforts is heavily dependent upon the availability of reliable in vitro as well as in vivo BBB models. Polarized monolayers of human cerebrovascular endothelial cells (hCMEC/D3) described in the current work serves as one such in vitro model that can be easily cultured and manipulated in the lab (Poller et al., 2008; Vu et al., 2009; Weksler et al., 2013). The barrier properties and the expression of several classes of receptors, transporters, and enzymes in hCMEC/D3 cells have been previously investigated (Urich et al., 2012; Lopez-Ramirez et al., 2013, 2014; Bamji-Mirza et al., 2014; Ilina et al., 2015; Naik et al., 2015; Sajja and Cucullo, 2015). Thus, far, the genomic data for hCMEC/D3 cell lines have been generated using array-based approaches (Lopez-Ramirez et al., 2013). However, a comprehensive transcriptomic landscape of hCMEC/D3 cells, which is required for investigating molecular mechanisms using sophisticated computational biology approaches, is not currently available. Next-generation sequencing technology unveils the full potential of systems biology approaches to resolve cellular and molecular interaction networks that regulate the functional integrity of the BBB. Such a panoramic view of the interaction networks could enable us to isolate key players regulating a physiological process and investigate how they are affected in various diseases. To our knowledge, we are the first group to generate deep RNA sequencing and microRNA sequencing of a human BBB cell line. This data report describes BBBomics hub as a comprehensive portal for BBB transcriptomics data, obtained by sequencing mRNA (mRNA-seq) and microRNA (miRNA-seq) of polarized hCMEC/D3 cell monolayers. This data encompasses coding (gene expression, alternate splice forms, expressed single nucleotide variants -eSNVs) and non-coding (microRNA, LincRNA, circular RNA) counts that are easily accessible through BBBomics hub database. We also superimposed the RNA-seq coding data on 285 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which include canonical, non-canonical, and/or atypical pathways retrievable using BBBomics hub. The data is easily accessible and freely available at http://bioinformaticstools.mayo.edu/bbbomics/.

Hydroxyzine and triprolidine have both been reported to reach the CNS following nasal administration. The objective of this study was to investigate their in vitro permeation across bovine olfactory mucosa in order to further characterize the biological and physicochemical parameters that influence direct nose-to-brain transport. In vitro experiments were conducted using Sweetana-Grass (Navicyte) vertical diffusion cells to evaluate the effect of directionality, donor concentration and pH on the permeation of hydroxyzine and triprolidine across excised bovine olfactory mucosa. These studies demonstrated that the Jm-s (mucosal-submucosal flux) and Js-m (submucosal-mucosal flux) of hydroxyzine and triprolidine across the olfactory mucosa were linearly dependent upon the donor concentration without any evidence of saturable transport. Hydroxyzine inhibited the efflux of P-gp substrates like etoposide and chlorpheniramine across the olfactory mucosa. Both hydroxyzine and triprolidine reduced the net flux (Js-m-Jm-s) of etoposide with IC50 values of 39.2 and 130.6 microM, respectively. The lipophilicty of these compounds, coupled with their ability to inhibit P-gp, enable them to freely permeate across the olfactory mucosa. Despite the presence of a number of protective barriers such as efflux transporters and metabolizing enzymes in the olfactory system, lipophilic compounds such as hydroxyzine and triprolidine can access the CNS primarily by passive diffusion when administered via the nasal cavity.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, characterized by the degeneration of upper and lower motor neurons (MNs). Central nervous system features include a loss of Betz cells and other pyramidal cells from sensorimotor cortex. The intrinsic mechanism underlying this selective motor neuron loss has not been identified. A recent in vitro study has provided evidence of a novel programmed cell death (PCD) pathway that is unique to spinal cord MNs and is exacerbated by superoxide dismutase (SOD) mutations. This PCD pathway is triggered through the Fas receptor and involves the apoptosis signal-regulating kinase 1 (ASK1), the p38 MAP kinase, and the neuronal form of nitric oxide synthase (nNOS). Previously, we found significant increases in the numbers of ventral horn MNs immunopositive for these enzymes in the spinal cords of mutant SOD transgenic (G93A) mice as early as 60 days of age, suggesting that this pathway may be active in vivo. Since the upper MNs of ALS patients and G93A mice are also known to degenerate, the purpose of the present study was to investigate the possible activation of this PCD pathway in the MNs of the sensorimotor cortex of G93A transgenic mice. Compared to non-transgenic littermates, the G93A mice showed significant increases in the numbers of MNs immunopositive for the active (phosphorylated) forms of ASK1, p38, MKK3/6 (the known activator of p38), and also active caspase-3, as early as 60 days of age. Another stress-activated protein kinase, c-Jun N-terminal kinase (JNK), commonly activated in other neurodegenerative disorders such as Alzheimer's disease, showed no increases in G93A mice at any age. These results suggest that, not only has a PCD pathway been activated in the cortical MNs, but one that may be unique to ALS. Moreover, these findings suggest that earlier diagnosis and therapeutic intervention may be possible for successful treatment of ALS. Consequently, these enzymes may provide the biochemical markers to enable earlier diagnosis of ALS and molecular targets for the development of new therapeutic compounds.