Inheritance of the apoE4 allele (4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of 4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of 2/2, 3/3, and 4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; 2/2 Ͼ3/3 Ͼ4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the 3/4 mouse brains. ApoE4 represented 30 -40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between 3/3 and 3/4 mice, implying that the reduced levels of total apoE in 3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from 3/4 human astrocytoma or 3/3, 4/4 and 3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from 4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by 4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or A␤ clearance.

Adsorption of t h e pure milk proteins, p-casein and B-lactoglobulin, at the air/water interface has been studied using the technique of specular neutron reflectance. By fitting appropriate models of the protein films to the reflectivity data the detailed structures of the interfaces have been determined. The effects of protein concentration, substrate pH, film ageing and the presence of calcium ions on the film structures have been investigated.At neutral pH both P-casein and P-lactoglobulin form a monolayer at the air/water boundary that can be divided into a protein-rich, hydrophobic, inner layer closest to the interface, and a more diffuse, hydrophilic, outer layer extending into t h e bulk aqueous phase. The adsorbed amount of protein follows the adsorption isotherms determined by other methods. Ageing h a s little effect on P-casein layers, but with B-lactoglobulin there is an increase in the adsorbed amount of protein with time and the two-layer model of the film configuration tends to become less distinct such that a one-layer model form is equally appropriate. The rate and extent of the changes occurring as a function of the age of the surface depend on the pH of the substrate solution. The adsorbed amount of both proteins increases as the pH is lowered towards their respective isoelectric points. Models are proposed to account for t h e changes in layer structure occurring during these processes. The presence of calcium ions modifies the structure of P-casein layers, reducing the hydrophilic layer thickness and the adsorbed amount of protein.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-D-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca 2ϩ assays (EC 50 ϭ 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 M) and competed for binding of3 H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) ϭ 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED ϭ 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED ϭ 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED ϭ 1 mg/kg i.p.) and reduced impulsivity in the fivechoice serial reaction time test (MED ϭ 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.The metabotropic glutamate receptor (mGlu) receptor family includes eight G-protein-coupled receptor (GPCR) subtypes classified on the basis of structural homology, Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.136580.ABBREVIATIONS: mGlu, metabotropic glutamate receptor; GPCR, G-protein-coupled receptor; PAM, positive allosteric modulator; CPPHA, 2, NMDA, MPEP,

2؉oscillations arising through activation of the metabotropic glutamate receptor mGluR5a expressed in Chinese hamster ovary cells and find that these oscillations are largely insensitive to agonist concentration. Using an inducible receptor expression system and a non-competitive antagonist, in conjunction with the translocation of eGFP-PH PLC␦ to monitor inositol 1,4,5-trisphosphate (InsP 3 ) oscillations in single cells, we show that mGluR5a density determines the frequency of these oscillations. The predominant underlying mechanism resulted from a negative feedback loop whereby protein kinase C (PKC) inhibited InsP 3 generation. Down-regulation of PKC by prolonged exposure to phorbol ester revealed a second form of Ca

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