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.

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.

Positive Allosteric Modulation of the Muscarinic M1 Receptor Improves Efficacy of Antipsychotics in Mouse Glutamatergic Deficit Models of Behavior.

Current antipsychotics are effective in treating the positive symptoms associated with schizophrenia, but they remain suboptimal in targeting cognitive dysfunction. Recent studies have suggested that positive allosteric modulation of the M1 muscarinic acetylcholine receptor (mAChR) may provide a novel means of improving cognition. However, very little is known about the potential of combination therapies in extending coverage across schizophrenic symptom domains. This study investigated the effect of the M1 mAChR positive allosteric modulator BQCA [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid], alone or in combination with haloperidol (a first-generation antipsychotic), clozapine (a second-generation atypical antipsychotic), or aripiprazole (a third-generation atypical antipsychotic), in reversing deficits in sensorimotor gating and spatial memory induced by the N-methyl-d-aspartate receptor antagonist, MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]. Sensorimotor gating and spatial memory induction are two models that represent aspects of schizophrenia modeled in rodents. In prepulse inhibition (an operational measure of sensorimotor gating), BQCA alone had minimal effects but exhibited different levels of efficacy in reversing MK-801-induced prepulse inhibition disruptions when combined with a subeffective dose of each of the three (currently prescribed) antipsychotics. Furthermore, the combined effect of BQCA and clozapine was absent in M1-/- mice. Interestingly, although BQCA alone had no effect in reversing MK-801-induced memory impairments in a Y-maze spatial test, we observed a reversal upon the combination of BQCA with atypical antipsychotics, but not with haloperidol. These findings provide proof of concept that a judicious combination of existing antipsychotics with a selective M1 mAChR positive allosteric modulator can extend antipsychotic efficacy in glutamatergic deficit models of behavior.

Blockade of Microglial Kv1.3 Potassium Channels by the Peptide HsTX1[R14A] Attenuates Lipopolysaccharide-mediated Neuroinflammation.

The expression of voltage-gated potassium Kv1.3 channels is increased in activated microglia, with non-selective blockade reported to attenuate microglial-mediated neuroinflammation. In this study, we evaluated the impact of a potent and selective peptidic blocker of Kv1.3 channels, HsTX1[R14A], on microglial-mediated neuroinflammation in vitro and in vivo. Treatment with both 0.1 and 1 µg/mL lipopolysaccharide (LPS) significantly (p < 0.05) increased Kv1.3 abundance on the surface of BV-2 microglia in association with increased levels of mRNA for tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The increased transcription of TNF-α and IL-6 was significantly attenuated (by 24.9 and 20.2%, respectively) by HsTX1[R14A] (100 nM). The concomitant increase in TNF-α and IL-6 release from BV-2 microglia was significantly attenuated by HsTX1[R14A] by 10.7 and 12.6%, respectively. In LPS-treated primary mouse microglia, the levels of TNF-α and nitric oxide were also attenuated by HsTX1[R14A] (26.1 and 20.4%, respectively). In an LPS-induced mouse model of neuroinflammation, both an immediate and delayed subcutaneous dose of HsTX1[R14A] (2 mg/kg) significantly reduced plasma and brain levels of the pro-inflammatory mediators TNF-α, IL-1ß and IL-6, with no impact on the anti-inflammatory IL-10. These results demonstrate that HsTX1[R14A] is a promising therapeutic candidate for the treatment of diseases with a neuroinflammatory component.

The effect of angiotensin AT1A inactivation on innate and learned fear responses in mice and its relationship to blood pressure.

Angiotensin AT1 receptors are implicated in behavioral and physiological processes associated with fear and stress. However, the precise role of AT1 receptors in modulating fear-related behavior and its relation to their physiological effects remains unclear. Here, we examined innate and learned fear responses and their relationship to cardiovascular arousal in AT1A receptor knockout (AT1A-/-) mice. Using synchronized video and blood pressure telemetry, we found that, in a novel test environment, AT1A-/- mice showed reduced neophobia but a similar rise in blood pressure, as compared to AT1A+/+ mice. In response to a discrete threat, footshock, both flight behavior and cardiovascular arousal were decreased in AT1A-/- mice. Reduced flight behavior was also observed in AT1A-/- mice in the elevated T-maze test. During fear conditioning, the immediate freezing response to the first shock, but not the rate of freezing acquisition was decreased in AT1A-/- mice. Likewise, AT1A-/- mice showed reduced freezing and pressor responses to the first re-exposure, but normal rate of freezing extinction over subsequent trials. Similarly, in the elevated T-maze, the rates of avoidance acquisition and escape learning remained unchanged in AT1A-/- mice. Finally, after re-exposure, AT1A-/- mice displayed altered c-Fos expression, compared to AT1A+/+ mice, in the hypothalamus and periaqueductal gray but not in fear-related limbic-cortical areas, nor in medullary nuclei that convey visceral afferent information. We conclude that AT1A receptor knockout reduces innate fear responses, without affecting learning efficiency in mice. These effects are dissociable from cardiovascular effects and likely reflect altered neurotransmission in hypothalamic-midbrain defense regions.

6-Phenylpyrimidin-4-ones as Positive Allosteric Modulators at the M1 mAChR: The Determinants of Allosteric Activity.

Targeting allosteric sites of the M1 muscarinic acetylcholine receptor (mAChR) is an enticing approach to overcome the lack of receptor subtype selectivity observed with orthosteric ligands. This is a promising strategy for obtaining novel therapeutics to treat cognitive deficits observed in Alzheimer's disease and schizophrenia, while reducing the peripheral side effects such as seen in the current treatment regimes, which are non-subtype selective. We previously described compound 2, the first positive allosteric modulator (PAM) of the M1 mAChR based on a 6-phenylpyrimidin-4-one scaffold, which has been further developed in this study. Herein, we present the synthesis, characterization, and pharmacological evaluation of a series of 6-phenylpyrimidin-4-ones with modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant. Selected compounds, BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were further profiled in terms of their allosteric affinity, cooperativity with acetylcholine (ACh), and intrinsic efficacy. Additionally, 2 and 9i were tested in mouse primary cortical neurons, displaying various degrees of intrinsic agonism and potentiation of the acetylcholine response. Overall, the results suggest that the pendant moiety is important for allosteric binding affinity and the direct agonistic efficacy of the 6-phenylpyrimidin-4-one based M1 mAChR PAMs.