Effect of Apolipoprotein E isoforms on the Abundance and Function of P-glycoprotein in Human Brain Microvascular Endothelial Cells.

BACKGROUND: Individuals with Alzheimer's disease (AD) often require many medications; however, these medications are dosed using regimens recommended for individuals without AD. This is despite reduced abundance and function of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in AD, which can impact brain exposure of drugs. The fundamental mechanisms leading to reduced P-gp abundance in sporadic AD remain unknown; however, it is known that the apolipoprotein E (apoE) gene has the strongest genetic link to sporadic AD development, and apoE isoforms can differentially alter BBB function. The aim of this study was to assess if apoE affects P-gp abundance and function in an isoform-dependent manner using a human cerebral microvascular endothelial cell (hCMEC/D3) model. METHODS: This study assessed the impact of apoE isoforms on P-gp abundance (by western blot) and function (by rhodamine 123 (R123) uptake) in hCMEC/D3 cells. Cells were exposed to recombinant apoE3 and apoE4 at 2 - 10 µg/mL over 24 - 72 hours. hCMEC/D3 cells were also exposed for 72 hours to astrocyte-conditioned media (ACM) from astrocytes expressing humanised apoE isoforms. RESULTS: P-gp abundance in hCMEC/D3 cells was not altered by recombinant apoE4 relative to recombinant apoE3, nor did ACM containing human apoE isoforms alter P-gp abundance. R123 accumulation in hCMEC/D3 cells was also unchanged with recombinant apoE isoform treatments, suggesting no change to P-gp function, despite both abundance and function being altered by positive controls SR12813 (5 µM) and PSC 833 (5 µM), respectively. CONCLUSIONS: Different apoE isoforms have no direct influence on P-gp abundance or function within this model, and further in vivo studies would be required to address whether P-gp abundance or function are reduced in sporadic AD in an apoE isoform-specific manner.

Cu(ATSM) Increases P-Glycoprotein Expression and Function at the Blood-Brain Barrier in C57BL6/J Mice.

P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), is critical in preventing brain access to substrate drugs and effluxing amyloid beta (Aß), a contributor to Alzheimer's disease (AD). Strategies to regulate P-gp expression therefore may impact central nervous system (CNS) drug delivery and brain Aß levels. As we have demonstrated that the copper complex copper diacetyl bis(4-methyl-3-thiosemicarbazone) (Cu(ATSM)) increases P-gp expression and function in human brain endothelial cells, the present study assessed the impact of Cu(ATSM) on expression and function of P-gp in mouse brain endothelial cells (mBECs) and capillaries in vivo, as well as in peripheral organs. Isolated mBECs treated with Cu(ATSM) (100 nM for 24 h) exhibited a 1.6-fold increase in P-gp expression and a 20% reduction in accumulation of the P-gp substrate rhodamine 123. Oral administration of Cu(ATSM) (30 mg/kg/day) for 28 days led to a 1.5 & 1.3-fold increase in brain microvascular and hepatic expression of P-gp, respectively, and a 20% reduction in BBB transport of [3H]-digoxin. A metallomic analysis showed a 3.5 and 19.9-fold increase in Cu levels in brain microvessels and livers of Cu(ATSM)-treated mice. Our findings demonstrate that Cu(ATSM) increases P-gp expression and function at the BBB in vivo, with implications for CNS drug delivery and clearance of Aß in AD.

Fatty Acid-Binding Protein 4 is Essential for the Inflammatory and Metabolic Response of Microglia to Lipopolysaccharide.

Prolonged activation of microglia leads to excessive release of proinflammatory mediators, which are detrimental to brain health. Therefore, there are significant efforts to identify pathways mediating microglial activation. Recent studies have demonstrated that fatty acid-binding protein 4 (FABP4), a lipid binding protein, is a critical player in macrophage-mediated inflammation. Given that we have previously identified FABP4 in microglia, the aim of this study was to assess whether FABP4 activity contributed to inflammation, metabolism and immune function (i.e. immunometabolism) in immortalised mouse microglia (BV-2 cells) using the proinflammatory stimulus lipopolysaccharide (LPS) to induce general microglial activation. Microglial FABP4 expression was significantly increased following exposure to LPS, an outcome associated with a significant increase in microglial proliferation rate. LPS-stimulated BV-2 microglia demonstrated a significant increase in the production of reactive oxygen species (ROS) and tumour necrosis factor-alpha (TNF-α), phosphorylation of c-Jun N-terminal kinase (JNK), increased expression of Toll-like receptor 4 (TLR4), and reduced expression of uncoupling protein 2 (UCP2), all of which were reversed following FABP4 genetic silencing and chemical inhibition with BMS309403. The oxidation rate of 3H-oleic acid and microglial uptake of 3H-2-deoxy-D-glucose were modulated with LPS activation, processes which were restored with genetic and chemical inhibition of FABP4. This is the first study to report on the critical role of FABP4 in mediating the deleterious effects of LPS on microglial immunometabolism, suggesting that FABP4 may present as a novel therapeutic target to alleviate microglia-mediated neuroinflammation, a commonly reported factor in multiple neurodegenerative diseases.

Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling.

PURPOSE: The ATP-binding cassette (ABC) transport protein ABCG2 (also known as breast cancer resistance protein (BCRP)) is expressed at the luminal face of the blood-brain barrier (BBB), where it limits the brain uptake of a number of therapeutic drugs. We recently reported that the ABC efflux transporter P-glycoprotein (P-gp) was downregulated in human immortalised brain endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC). The aim of the present study, therefore, was to assess whether BCRP expression is also affected by FAC and identify any signalling mechanisms involved. METHODS: ABCG2 mRNA was assessed by RT-qPCR. Protein levels of BCRP, phosphorylated extracellular-regulated kinases 1 and 2 (p-ERK1/2) and total ERK 1/2 were assessed by Western blot. Reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate. RESULTS: Treatment of hCMEC/D3 cells with FAC (250 µM, 72 h) significantly reduced ABCG2 mRNA levels (32.2 ± 3.7%) without a concomitant reduction in BCRP protein expression. ABCG2 mRNA levels were restored to control levels when co-treated with the antioxidant N-acetylcysteine (NAC), suggesting the effect of FAC was mediated by a ROS-sensitive pathway. We also found that FAC-treatment was associated with increased levels of p-ERK1/2, suggesting involvement of the ERK1/2 signalling pathway in the observed ABCG2 mRNA downregulation. The ERK1/2 signalling pathway inhibitor U0126 restored p-ERK1/2 levels and partially attenuated the FAC-induced reduction in ABCG2 mRNA. CONCLUSIONS: This study suggests that FAC-induced downregulation of ABCG2 mRNA is driven by ROS and ERK1/2 signalling, mechanisms which may be exploited to modulate BCRP expression at the BBB.

Copper bis(thiosemicarbazone) complexes modulate P-glycoprotein expression and function in human brain microvascular endothelial cells.

P-glycoprotein (P-gp) is an efflux transporter at the blood-brain barrier (BBB) that hinders brain access of substrate drugs and clears endogenous molecules such as amyloid beta (Aß) from the brain. As biometals such as copper (Cu) modulate many neuronal signalling pathways linked to P-gp regulation, it was hypothesised that the bis(thiosemicarbazone) (BTSC) Cu-releasing complex, copper II glyoxal bis(4-methyl-3-thiosemicarbazone) (CuII [GTSM]), would enhance P-gp expression and function at the BBB, while copper II diacetyl bis(4-methyl-3-thiosemicarbazone) (CuII [ATSM]), which only releases Cu under hypoxic conditions, would not modulate P-gp expression. Following treatment with 25-250 nM CuII (BTSC)s for 8-48 h, expression of P-gp mRNA and protein in human brain endothelial (hCMEC/D3) cells was assessed by RT-qPCR and Western blot, respectively. P-gp function was assessed by measuring accumulation of the fluorescent P-gp substrate, rhodamine 123 and intracellular Cu levels were quantified by inductively coupled plasma mass spectrometry. Interestingly, CuII (ATSM) significantly enhanced P-gp expression and function 2-fold and 1.3-fold, respectively, whereas CuII (GTSM) reduced P-gp expression 0.5-fold and function by 200%. As both compounds increased intracellular Cu levels, the effect of different BTSC backbones, independent of Cu, on P-gp expression was assessed. However, only the Cu-ATSM complex enhanced P-gp expression and this was mediated partly through activation (1.4-fold) of the extracellular signal-regulated kinase 1 and 2, an outcome that was significantly attenuated in the presence of an inhibitor of the mitogen-activated protein kinase regulatory pathway. Our findings suggest that CuII (ATSM) and CuII (GTSM) have the potential to modulate the expression and function of P-gp at the BBB to impact brain drug delivery and clearance of Aß.

Development of a Vertically Integrated Pharmacy Degree.

Whilst curriculum revision is commonplace, whole degree transformation is less so. In this paper we discuss the rationale, design and implementation of a unique pharmacy program by a research-intensive faculty. The new Monash pharmacy curriculum, which had its first intake in 2017, was built using a range of key innovations that aimed to produce graduates that demonstrate key conceptual understanding and all the skills required to deliver world-best patient outcomes. The key elements of the re-design are outlined and include the process and principles developed, as well as key features such as a student-centred individualised program of development arranged around specific, authentic tasks for each skill and earlier enhanced experiential placements where students become proficient in entrustable professional activities. It is hoped the dissemination of this process, as well as the lessons learnt in the process, will be useful to others looking to transform a health curriculum.

Effect of Changing From Closed-Book to Formulary-Allowed Examinations.

Objective. To determine whether allowing final-year Bachelor of Pharmacy students to use a medicines formulary during examinations modified their learning behaviors and performance, and to investigate students' perceptions about having this resource available during examinations.Methods. Student performance and examination difficulty (as measured by classification of examination questions as high or low according to Bloom's taxonomy of learning) in second semester examinations (formulary allowed) was compared with first semester examinations (closed book) in successive years. Students completed a survey regarding their study and examination approaches and experiences after both semesters.Results. Examinations in semester two had more questions rated higher on Bloom's taxonomy than did examinations in semester one. Data were collected from student surveys for closed book examinations (response rate of 25% and 19% in 2015 and 2016, respectively) and open book examinations (response rate of 22% and 15% in 2015 and 2016, respectively). Students' study approaches, hours studied per week, and anxiety (all self-reported) did not differ between semesters one and two, but students in semester two spent more time studying with a formulary compared with students in semester one. Qualitative analysis of student comments revealed students preferred the formulary-allowed examinations over the closed-book examinations. The majority of students (68%) agreed with the statement: "Knowing that I will have access to the AMH [Australian Medicines Handbook] during the exams allowed me to pay more attention to higher level skills such as analysis and evaluation."Conclusion. When students were allowed to use a formulary for examinations, they studied more using their formulary prior to the examination. Students performed similarly on examinations with a greater proportion of questions addressing higher levels of Bloom's taxonomy and on closed-book examinations that were comparatively less cognitively challenging.

Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells.

PURPOSE: P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of ß-amyloid, which accumulates in the Alzheimer's disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown. METHODS: P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 µM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [3H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay. RESULTS: FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [3H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation. CONCLUSIONS: These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain ß-amyloid clearance.

A Combination of Clioquinol, Zinc and Copper Increases the Abundance and Function of Breast Cancer Resistance Protein in Human Brain Microvascular Endothelial Cells.

Modulating the abundance of the blood-brain barrier (BBB) efflux transporter breast cancer resistance protein (BCRP) has the potential to impact brain levels of drugs and endogenous substrates. Studies have demonstrated that the metal ionophore clioquinol (CQ) increases BBB abundance of P-glycoprotein (P-gp), an effect associated with increased endothelial cell levels of Cu2+. This study therefore assessed whether human brain endothelial (hCMEC/D3) cell abundance and function of BCRP is modulated by CQ. hCMEC/D3 cells were treated with CQ, Zn2+ and Cu2+ (CZC) (0.5 µM, 0.5 µM, 0.1 µM, respectively) for 24 h and BCRP mRNA and protein abundance was determined by Western blot and qPCR, respectively. After a series of optimisation studies assessing specificity of bodipy prazosin (BP) and Ko143 as a substrate and inhibitor of BCRP, respectively, the impact of CZC on BP uptake was assessed. While CZC did not increase mRNA expression of BCRP, BCRP abundance was increased 1.8 ± 0.1-fold; this was associated with a 68.1 ± 3.3% reduction in accumulation of BP in hCMEC/D3 cells. This is the first study to demonstrate that augmenting metal ion availability enhances protein abundance and function of BCRP at the BBB, which may be exploited to modulate CNS access of therapeutics and endogenous substrates.

Development and validation of a LC-MS/MS assay for quantifying the uptake of docosahexaenoic acid-d5 into mouse microglia.

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid with the capacity to reduce the proinflammatory response of activated microglia that occurs during neuroinflammation. The uptake of DHA into microglia is essential for it to exert its neuroprotective effects. However, quantifying the uptake of DHA into microglia is complicated by the presence of endogenous DHA interfering with any quantification technique. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was therefore developed and validated in order to assess the microglial uptake of docosahexaenoic acid-d5 (DHA-d5) as a surrogate for DHA. Using a mobile phase consisting of 90 % (v/v) acetonitrile and 10 % (v/v) water containing 2 mM ammonium acetate, a flow rate of 0.3 mL/min, and MS/MS detection in the negative ionization mode, DHA-d5 was detected at m/z transitions of 332.1/228.3/234.2, with good linearity between chromatographic area under the curve (AUC) and DHA-d5 mass (R2 = 0.999) over the range of 0.0063-0.1 ng. The precision and accuracy values for the quality control samples (0.0063, 0.025, and 0.1 ng) were less than 9.3 % and 96.6-109.8 %, respectively, and a comparison of DHA-d5 AUC when prepared in PBS or in microglial cell lysate demonstrated no significant difference between quantification of these quality control samples. Utilizing this quantification approach (with preparation of DHA-d5 calibration standards in PBS), the uptake of DHA-d5 into BV-2 microglial cells over a 15 min period was assessed, following the spiking of DHA-d5 at 50 ng/mL. Following protein normalization using a BCA protein assay, a rapid and linear uptake of DHA-d5 into BV-2 cells was observed in the first 2 min, after which a plateau in uptake was observed, in line with that reported for DHA uptake in other cell types. This novel LC-MS/MS technique can now be exploited to unravel the processes involved in microglial uptake of DHA, insights that may be used to maximize the anti-inflammatory effects of DHA in neuroinflammation.

The Effects of Clioquinol on P-glycoprotein Expression and Biometal Distribution in the Mouse Brain Microvasculature.

Previous studies have demonstrated that the ionophore clioquinol (CQ), in conjunction with the biometals copper and zinc, increases the expression of P-glycoprotein (P-gp) in human cerebral microvascular endothelial (hCMEC/D3) cells. As P-gp expression and function at the blood-brain barrier (BBB) is of great interest regarding CNS drug access and endogenous toxin trafficking (e.g., amyloid beta), the present study assessed the in vivo translation of these previous in vitro findings. Swiss outbred mice received an 11-day treatment of CQ (30 mg/kg) by oral gavage, after which brain microvessel-enriched fractions (MEFs) and surrounding interfaces (subcortical brain tissue and plasma) were extracted. P-gp expression was quantified in the MEF, and biometal concentrations in all 3 compartments were assessed via inductively coupled plasma mass spectrometry. CQ treatment did not modify the expression of P-gp, nor copper or zinc concentrations in the brain MEF under this treatment regime. Metallomic analysis revealed, however, that CQ reduced potassium and magnesium levels in the brain MEF and also lowered brain iron levels. This study has shown that under this dosing regimen, CQ does not increase BBB P-gp expression in Swiss outbred mice, but that CQ facilitates redistribution of certain metal ions within the brain MEF, plasma, and brain parenchyma.

Development of a self-report instrument for measuring in-class student engagement reveals that pretending to engage is a significant unrecognized problem.

Student engagement during classes includes behavioural, cognitive and emotional components, and is a pre-requisite for successful active learning environments. A novel approach to measuring student engagement was developed, involving triangulation of real-time student-self report, observation by trained observers and heart rate measurement. The self-report instrument was evaluated in four separate cohorts (n = 123) at Monash University and the University of North Carolina. The six item self-report demonstrated good reliability (Cronbach's alpha values ranged from 0.7-0.81). The self-report showed predictive validity in that small group activities were rated as significantly more engaging than didactic lecturing. Additionally, there was significant inter-instructor variability and within-class variability, indicating good discrimination between classroom activities. This self-report may prove useful to academic teaching staff in evaluating and refining their active learning activities. Independent observation was not found to correlate with student self-report, due in part to students who were pretending to engage being rated as engaged by an observer. Strikingly, students reported that they were pretending to engage for 23% of class time, even for highly regarded instructors. Individual participants were rated as engaged for 42 of the 46 intervals for which they reported that they had "pretended to engage", indicating that the two observers were unable to detect disengagement during periods in which students pretended to engage. Instructors should be aware that student cues such as eye contact and nodding may indicate pretending to engage. One particular self-report item; "I tried a new approach or way of thinking about the content", correlated positively with heart rates, and a controlled study reproduced this finding during two activities that required students to try a new approach to understanding a concept. Agreement with this item also correlated with superior performance on two in-class written assessment tasks (n = 101, p<0.01). Further use of this tool and related educational research may be useful to identify in-class activities that are engaging and likely to lead to improved student attainment of learning outcomes.

Cognitive benefits of lithium chloride in APP/PS1 mice are associated with enhanced brain clearance of ß-amyloid.

Epidemiological evidence suggests that people with bipolar disorder prescribed lithium exhibit a lower risk of Alzheimer's disease (AD) relative to those prescribed other mood-stabilizing medicines. Lithium chloride (LiCl) reduces brain ß-amyloid (Aß) levels, and the brain clearance of Aß is reduced in AD. Therefore, the purpose of this study was to assess whether the cognitive benefits of LiCl are associated with enhanced brain clearance of exogenously-administered Aß. The brain clearance of intracerebroventricularly (icv) administered 125I-Aß42 was assessed in male Swiss outbred mice administered daily oral NaCl or LiCl (300 mg/kg for 21 days). LiCl exhibited a 31% increase in the brain clearance of 125I-Aß42 over 10 min, which was associated with a 1.6-fold increase in brain microvascular expression of the blood-brain barrier efflux transporter low density lipoprotein receptor-related protein 1 (LRP1) and increased cerebrospinal fluid (CSF) bulk-flow. 8-month-old female wild type (WT) and APP/PS1 mice were also administered daily NaCl or LiCl for 21 days, which was followed by cognitive assessment by novel object recognition and water maze, and measurement of soluble Aß42, plaque-associated Aß42, and brain efflux of 125I-Aß42. LiCl treatment restored the long-term spatial memory deficit observed in APP/PS1 mice as assessed by the water maze (back to similar levels of escape latency as WT mice), but the short-term memory deficit remained unaffected by LiCl treatment. While LiCl did not affect plaque-associated Aß42, soluble Aß42 levels were reduced by 49.9% in APP/PS1 mice receiving LiCl. The brain clearance of 125I-Aß42 decreased by 27.8% in APP/PS1 mice, relative to WT mice, however, LiCl treatment restored brain 125I-Aß42 clearance in APP/PS1 mice to a rate similar to that observed in WT mice. These findings suggest that the cognitive benefits and brain Aß42 lowering effects of LiCl are associated with enhanced brain clearance of Aß42, possibly via brain microvascular LRP1 upregulation and increased CSF bulk-flow, identifying a novel mechanism of protection by LiCl for the treatment of AD.

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

PURPOSE: Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. METHODS: Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. RESULTS: A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 µM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). CONCLUSIONS: Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

Reduced blood-brain barrier expression of fatty acid-binding protein 5 is associated with increased vulnerability of APP/PS1 mice to cognitive deficits from low omega-3 fatty acid diets.

Lower levels of the cognitively beneficial docosahexaenoic acid (DHA) are often observed in Alzheimer's disease (AD) brains. Brain DHA levels are regulated by the blood-brain barrier (BBB) transport of plasma-derived DHA, a process facilitated by fatty acid-binding protein 5 (FABP5). This study reports a 42.1 ± 12.6% decrease in the BBB transport of 14 C-DHA in 8-month-old AD transgenic mice (APPswe,PSEN1∆E9) relative to wild-type mice, associated with a 34.5 ± 6.7% reduction in FABP5 expression in isolated brain capillaries of AD mice. Furthermore, short-term spatial and recognition memory deficits were observed in AD mice on a 6-month n-3 fatty acid-depleted diet, but not in AD mice on control diet. This intervention led to a dramatic reduction (41.5 ± 11.9%) of brain DHA levels in AD mice. This study demonstrates FABP5 deficiency and impaired DHA transport at the BBB are associated with increased vulnerability to cognitive deficits in mice fed an n-3 fatty acid-depleted diet, in line with our previous studies demonstrating a crucial role of FABP5 in BBB transport of DHA and cognitive function.

Neurovascular Alterations in Alzheimer's Disease: Transporter Expression Profiles and CNS Drug Access.

Despite a century of steady and incremental progress toward understanding the underlying biochemical mechanisms, Alzheimer's disease (AD) remains a complicated and enigmatic disease, and greater insight will be necessary before substantive clinical success is realised. Over the last decade in particular, a large body of work has highlighted the cerebral microvasculature as an anatomical region with an increasingly apparent role in the pathogenesis of AD. The causative interplay and temporal cascade that manifest between the brain vasculature and the wider disease progression of AD are not yet fully understood, and further inquiry is required to properly characterise these relationships. The purpose of this review is to highlight the recent advancements in research implicating neurovascular factors in AD, at both the molecular and anatomical levels. We begin with a brief introduction of the biochemical and genetic aspects of AD, before reviewing the essential concepts of the blood-brain barrier (BBB) and the neurovascular unit (NVU). In detail, we then examine the evidence demonstrating involvement of BBB dysfunction in AD pathogenesis, highlighting the importance of neurovascular components in AD. Lastly, we include within this review research that focuses on how altered properties of the BBB in AD impact upon CNS exposure of therapeutic agents and the potential clinical impact that this may have on people with this disease.

Assessing the Impact of Lithium Chloride on the Expression of P-Glycoprotein at the Blood-Brain Barrier.

In addition to extruding drugs from the brain, P-glycoprotein (P-gp) at the blood-brain barrier (BBB) facilitates the brain-to-blood clearance of beta-amyloid (Aß) and is down-regulated in Alzheimer's disease. Studies suggest that the mood-stabilizing drug lithium exerts a protective effect against Alzheimer's disease. Although the mechanisms underlying this effect are not fully understood, evidence suggests that lithium chloride (LiCl) increases P-gp expression in vitro, albeit at concentrations substantially outside the therapeutic window. Therefore, we investigated the effects of pharmacologically-relevant concentrations of LiCl on P-gp expression using in vitro and in vivo approaches. Swiss outbred mice administered LiCl (300 mg/kg/day, 21 days) showed no change in brain microvascular P-gp protein expression. Furthermore, P-gp transcript and protein levels were unaltered by LiCl (1.25-5 mM, 24 h) in human immortalized brain endothelial cells, while both gene and protein expression were significantly enhanced by the P-gp up-regulator, SR12813 by 1.5-fold and 2.0-fold, respectively. P-gp efflux function was also unaffected by LiCl in vitro, by measuring accumulation of the fluorescent P-gp substrate rhodamine-123. This suggests therefore that LiCl is unlikely to affect the BBB efflux of Aß or other P-gp substrates at pharmacologically-relevant concentrations, suggesting that the Aß-lowering effects of LiCl are unrelated to elevated BBB P-gp expression.

Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells.

An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 µM rifampicin and 5 µM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.

Student Engagement with a Flipped Classroom Teaching Design Affects Pharmacology Examination Performance in a Manner Dependent on Question Type.

Objective: To investigate the relationship between student engagement with the key elements of a flipped classroom approach (preparation and attendance), their attitudes to learning, including strategy development, and their performance on two types of examination questions (knowledge recall and providing rational predictions when faced with novel scenarios). Methods. This study correlated student engagement with the flipped classroom and student disposition to learning with student ability to solve novel scenarios in examinations. Results. Students who both prepared for and attended classes performed significantly better on examination questions that required analysis of novel scenarios compared to students who did not prepare and missed classes. However, there was no difference for both groups of students on examination questions that required knowledge and comprehension. Student motivation and use of strategies correlated with higher examination scores on questions requiring novel scenario analysis. Conclusion. There is a synergistic relationship between class preparation and attendance. The combination of preparation and attendance was positively correlated to assessment type; the relationship was apparent for questions requiring students to solve novel problems but not for questions requiring knowledge or comprehension.

Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier Regulates Endogenous Brain Docosahexaenoic Acid Levels and Cognitive Function.

Fatty acid-binding protein 5 (FABP5) at the blood-brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5+/+ and FABP5-/- mice using a battery of memory paradigms. FABP5-/- mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood-brain barrier transport of DHA was assessed by measuring 14C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5+/+ and FABP5-/- mice. In line with a crucial role of FABP5 in the brain uptake of DHA, 14C-DHA uptake into brain endothelial cells and brain capillaries of FABP5-/- mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5+/+ mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5-/- mice are associated with reduced CNS access of DHA. SIGNIFICANCE STATEMENT: Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5-/- mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role for FABP5 in the maintenance of cognitive function via regulating the brain uptake of DHA, and suggests that upregulation of FABP5 in neurodegenerative diseases, where brain DHA levels are possibly diminished (e.g., Alzheimer's disease), may provide a novel therapeutic approach for restoring cognitive function.