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.

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.

Medium-Term Outcomes in Severely to Critically Ill Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: The medium- and long-term effects of severe acute respiratory syndrome coronavirus 2 infection on survivors are unknown. In the current study, we assessed the medium-term effects of coronavirus disease 2019 (COVID-19) on survivors of severe disease. METHODS: This is a retrospective, case series of 200 patients hospitalized across 3 large Birmingham hospitals with severe-to-critical COVID-19 infection 4-7 months from disease onset. Patients underwent comprehensive clinical, laboratory, imaging, lung function tests (LFTs), and quality of life and cognitive assessments. RESULTS: At 4-7 months after disease onset, 63.2% of patients reported persistent breathlessness; 53.5%, significant fatigue; 37.5%, reduced mobility; and 36.8% pain. Serum markers of inflammation and organ injuries that persisted at hospital discharge had normalized on follow-up, indicating no sustained immune response causing chronic maladaptive inflammation. Chest radiographs showed complete resolution in 82.8%, and significant improvement or no change in 17.2%. LFTs revealed gas transfer abnormalities in 80.0% and abnormal spirometric values in 37.6% of patients. Compared with patients who did not experience breathlessness, those who did had significantly higher incidences of comorbid conditions and residual chest radiographic and LFT abnormalities (P < .01 to all). For all parameters assessed and persisting symptoms there were no significant differences between patients in hospital wards and those in intensive treatment units. All patients reported a significantly reduced quality of life in all domains of the EQ-5D-5L quality-of-life measures. CONCLUSIONS: A significant proportion of severely ill patients with COVID-19 still experience symptoms of breathlessness, fatigue, pain, reduced mobility, depression and reduced quality of life 4-7 months after disease onset. Symptomatic patients tend to have more residual chest radiographic and LFT abnormalities.

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.

Brain and blood biomarkers of tauopathy and neuronal injury in humans and rats with neurobehavioral syndromes following blast exposure.

Traumatic brain injury (TBI) is a risk factor for the later development of neurodegenerative diseases that may have various underlying pathologies. Chronic traumatic encephalopathy (CTE) in particular is associated with repetitive mild TBI (mTBI) and is characterized pathologically by aggregation of hyperphosphorylated tau into neurofibrillary tangles (NFTs). CTE may be suspected when behavior, cognition, and/or memory deteriorate following repetitive mTBI. Exposure to blast overpressure from improvised explosive devices (IEDs) has been implicated as a potential antecedent for CTE amongst Iraq and Afghanistan Warfighters. In this study, we identified biomarker signatures in rats exposed to repetitive low-level blast that develop chronic anxiety-related traits and in human veterans exposed to IED blasts in theater with behavioral, cognitive, and/or memory complaints. Rats exposed to repetitive low-level blasts accumulated abnormal hyperphosphorylated tau in neuronal perikarya and perivascular astroglial processes. Using positron emission tomography (PET) and the [18F]AV1451 (flortaucipir) tau ligand, we found that five of 10 veterans exhibited excessive retention of [18F]AV1451 at the white/gray matter junction in frontal, parietal, and temporal brain regions, a typical localization of CTE tauopathy. We also observed elevated levels of neurofilament light (NfL) chain protein in the plasma of veterans displaying excess [18F]AV1451 retention. These findings suggest an association linking blast injury, tauopathy, and neuronal injury. Further study is required to determine whether clinical, neuroimaging, and/or fluid biomarker signatures can improve the diagnosis of long-term neuropsychiatric sequelae of mTBI.

Presentation of the septic patient to the emergency department with respect to age and sex - a retrospective cross-sectional study.

OBJECTIVE: It is challenging to identify sepsis in the emergency department, in part due to the non-specific presentation of septic patients. Current clinical sepsis screening tools rely on vital signs but many patients present with near normal vital signs and are therefore not identified as septic. This suggests that variables, e.g. signs and symptoms, need to be included to improve sepsis detection in the emergency department. Our hypothesis was that the presentation of sepsis differs based age and sex. The potential differences in presentation could be used to apply to future sepsis screening tools. The aim was to analyze the prevalence of keywords reflecting the presentation of septic patients in the emergency department in relation to age and sex. METHOD: Retrospective cross-sectional study. Keywords reflecting sepsis presentation to the emergency department were quantified and compared between age categories and the sex. 479 patients admitted to the emergency department of Södersjukhuset, Stockholm during 2013 and discharged with an ICD-10 code consistent with sepsis were included. We adjusted for multiple comparisons by applying Bonferroni-adjusted significance levels for all comparisons. RESULT: "Pain" and "risk factors for sepsis" were significantly more common among patients younger than 65 years as compared with those 75 years and older: (n = 87/137; 63.5% vs n = 99/240; 41.3%, P-value < 0.000) and (n = 74/137; 54.0% vs 55/240; 22.9%, P-value < 0.000) respectively. "Risk factors for sepsis" was also significantly more common among patients between 65 and 74 years as compared with those 75 years and older: (n = 43/102; 42.2% vs 55/240; 22.9%, P-value < 0.000). "Pain" and "gastrointestinal symptoms" were significantly more common among women as compared with men: (n = 128/224; 57.1% vs n = 102/255; 40.0%, P-value < 0.000) and (n = 82/244; 36.6% vs n = 55/255; 21.6%, P-value < 0.000) respectively. CONCLUSION: The keywords "pain" and "risk factors for sepsis" were more common among younger patients and "pain" and "gastrointestinal symptoms" were more common among women. However, most keywords had a similar prevalence irrespective of age and sex. The results could potentially be used to augment sepsis screening tools or clinical decision tools.

Management of functional communication, swallowing, cough and related disorders: consensus recommendations for speech and language therapy.

Communication problems (eg, dysphonia, dysfluency and language and articulation disorders), swallowing disorders (dysphagia and globus), cough and upper airway symptoms, resulting from functional neurological disorder (FND), are commonly encountered by speech and language professionals. However, there are few descriptions in the literature of the most effective practical management approaches. This consensus document aims to provide recommendations for assessment and intervention that are relevant to both adults and young people. An international panel of speech and language professionals with expertise in FND were approached to take part. Participants responded individually by email to a set of key questions regarding best practice for assessment and interventions. Next, a video conference was held in which participants discussed and debated the answers to these key questions, aiming to achieve consensus on each issue. Drafts of the collated consensus recommendations were circulated until consensus was achieved. FND should be diagnosed on the basis of positive clinical features. Speech and language therapy for FND should address illness beliefs, self-directed attention and abnormal movement patterns through a process of education, symptomatic treatment and cognitive behavioural therapy within a supportive therapeutic environment. We provide specific examples of these strategies for different symptoms. Speech and language professionals have a key role in the management of people with communication and related symptoms of FND. It is intended that these expert recommendations serve as both a practical toolkit and a starting point for further research into evidence-based treatments.

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.

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.

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.

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.

Mitigating Chinese-Indian rhesus macaque (Macaca mulatta) hybridity at the California National Primate Research Center (CNPRC).

The effectiveness of abating hybridity in a rhesus breeding colony was evaluated. STR data from the 2006 to 2015 newborns were analyzed. Hybridity decreased over successive years. Birth cohorts retained high genetic variability without signs of inbreeding and differentiation. Hybridity was minimized without compromising overall genetic variability.

Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice.

Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive.

Drug access to the central nervous system in Alzheimer's disease: preclinical and clinical insights.

Alzheimer's disease (AD) is a neurodegenerative disorder, characterized by ß-amyloid plaques and hyperphosphorylated tau tangles in the brain. Alongside these pathological lesions, there have been multiple reports of physical and biochemical alterations to the blood-brain barrier (BBB) in people with AD, potentially impacting on the ability of systemically-administered drugs to reach the brain parenchyma. Though there has been much research into the identification of these BBB alterations during AD, there are very few studies that have assessed the impact of such BBB changes on the ability of therapeutic agents to traverse the BBB. Due to their increased age-associated risk of chronic disease, most people with AD are prescribed multiple concurrent medications. In people with AD, the altered nature of the BBB could impact upon the disposition and therefore pharmacological effects of a wide range of medicines. This review therefore evaluates the impact of BBB alterations in AD on CNS drug exposure, along with relevant examples of preclinical and clinical studies that address this current issue. This review highlights that the CNS exposure of drugs is likely to differ between people with AD and healthy individuals, warranting further clinical investigations and the consideration to tailor dosing regimens in people with this neurodegenerative disorder.

Development of a P2X1-purinoceptor mediated contractile response in the aged mouse prostate gland through slowing down of ATP breakdown.

AIMS: An age-related increase in prostatic smooth muscle tone is partly responsible for the lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH). Changes in the effectors of prostatic smooth muscle contraction with age may play a role in the development of these symptoms. Using a mouse model of prostate contractility, this study investigated the effect of age on the different components of contractility in the prostate gland. METHODS: The isometric force developed in response to electrical field stimulation or exogenously applied agonists by mouse prostates mounted in organ baths, was evaluated to determine the effect of age on contractile mechanisms. Changes with age in the rate of ATP breakdown and levels of the P2rx1 gene and P2X1-purinoceptor expression in mouse prostate were measured by a modified luciferin-luciferase assay, RT-PCR and western blot, respectively. RESULTS: Nerve mediated contractile responses containing a component elicited by P2X1-purinoceptors were observed in prostates taken from aged mice, but not in prostates taken from young adult mice. Furthermore, the potency of the endogenous purinoceptor agonist ATP was 50-fold greater in aged mice, whereas the potency of its stable analogue α,ß-metATP was unchanged. An age-related decrease in ATP metabolism was also observed. CONCLUSIONS: With age, a purinergic contractile response to nerve stimulation develops in the mouse prostate gland due to a decrease in the rate of ATP breakdown. This may contribute to the increase in muscular tone observed in BPH and suggests that P2X1-purinoceptors are an additional target for the treatment of BPH.

Memantine transport across the mouse blood-brain barrier is mediated by a cationic influx H+ antiporter.

Memantine (MEM) is prescribed in mono and combination therapies for treating the symptoms of moderate to severe Alzheimer's disease (AD). Despite MEM being widely prescribed with other AD and non-AD medicines, very little is known about its mechanism of transport across the blood-brain barrier (BBB), and whether the nature of this transport lends MEM to a potential for drug-drug interactions at the BBB. Therefore, the purpose of this study was to characterize the mechanisms facilitating MEM brain uptake in Swiss Outbred mice using an in situ transcardiac perfusion technique, and identify the putative transporter involved in MEM disposition into the brain. Following transcardiac perfusion of MEM with increasing concentrations, the brain uptake of MEM was observed to be saturable. Furthermore, MEM brain uptake was reduced (up to 55%) by various cationic transporter inhibitors (amantadine, quinine, tetraethylammonium, choline and carnitine) and was dependent on extracellular pH, while being independent of membrane depolarization and the presence of Na(+) in the perfusate. In addition, MEM brain uptake was observed to be sensitive to changes in intracellular pH, hence, likely to be driven by H(+)/MEM antiport mechanisms. Taken together, these findings implicate the involvement of an organic cation transporter regulated by proton antiport mechanisms in the transport of MEM across the mouse BBB, possibly the organic cation/carnitine transporter, OCTN1. These studies also clearly demonstrate the brain uptake of MEM is significantly reduced by other cationic compounds, highlighting the need to consider the possibility of drug interactions with MEM at the BBB, potentially leading to reduced brain uptake and, therefore, altered efficacy of MEM when used in patients on multidrug regimens.

Altered brain uptake of therapeutics in a triple transgenic mouse model of Alzheimer's disease.

PURPOSE: The purpose of this study was to systematically assess the impact of Alzheimer's disease (AD)-associated blood-brain barrier (BBB) alterations on the uptake of therapeutics into the brain. METHODS: The brain uptake of probe compounds was measured in 18-20 month old wild type (WT) and triple transgenic (3×TG) AD mice using an in situ transcardiac perfusion technique. These results were mechanistically correlated with immunohistochemical and molecular studies. RESULTS: The brain uptake of the paracellular marker, [(14)C] sucrose, did not differ between WT and 3×TG mice. The brain uptake of passively diffusing markers, [(3)H] diazepam and [(3)H] propranolol, decreased 54-60% in 3×TG mice, consistent with a 33.5% increase in the thickness of the cerebrovascular basement membrane in 3×TG mice. Despite a 42.4% reduction in P-gp expression in isolated brain microvessels from a sub-population of 3×TG mice (relative to WT mice), the brain uptake of P-gp substrates ([(3)H] digoxin, [(3)H] loperamide and [(3)H] verapamil) was not different between genotypes, likely due to a compensatory thickening in the cerebrovascular basement membrane counteracting any reduced efflux of these lipophilic substrates. CONCLUSION: These studies systematically assessed the impact of AD on BBB drug transport in a relevant animal model, and have demonstrated a reduction in the brain uptake of passively-absorbed molecules in this mouse model of AD.

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.