In vivo hippocampal microdialysis reveals impairment of NMDA receptor–cGMP signaling in APPSW and APPSW/PS1L166P Alzheimer’s transgenic mice

Abstract

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer's disease (AD) including memory deficit, presence of ß-amyloid (Aß) oligomers, and age-associated formation of amyloid deposits. In this study we used hippocampal microdialysis to characterize the signaling of N-methyl-D-aspartic acid receptors (NMDA-Rs) in awake and behaving AD Tg mice. The NMDA-R signaling is central to hippocampal synaptic plasticity underlying memory formation and several lines of evidence implicate the role of Aß oligomers in effecting NMDA-R dysfunction. CA1 NMDA-Rs were stimulated by NMDA infused through reverse microdialysis while changes in the cyclic guanosine monophosphate (cGMP) concentration in the brain interstitial fluid (ISF) were used to determine NMDA-Rs responsiveness. While four months old wild type C57BL/6 mice mounted robust cGMP response to the NMDA challenge, the same stimulus failed to significantly change the cGMP level in four and 15 months old APPSW and four months old APPSW/PS1L166P Tg mice, which were all on C57BL/6 background. Lack of response to NMDA in AD Tg mice occurred in the absence of changes in expression levels of several synaptic proteins including synaptophysin, NR1 NMDA-R subunit and postsynaptic density protein 95, which indicates lack of profound synaptic degeneration. Aß oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5±3.6ng/g in four months old APPSW mice to 60.8±15.9ng/g in four months old APPSW/PS1L166P mice. Four months old APPSW mice had no Aß amylod plaques, while the other two AD Tg mice groups showed evidence of incipient Aß amyloid plaque formation. Our studies describes a novel approach useful to study the function of NMDA-Rs in awake and behaving AD Tg mice and demonstrate impairment of NMDA-R response in the presence of endogenously formed Aß oligomers but predating onset of Aß amyloidosis.

Modulation of amyloid precursor protein expression reduces β‐amyloid deposition in a mouse model

“…APP SW /PS1 dE9 mice 19,20 were derived from our own animal colony where they were mated with wild-type (WT) C57BL/6 mice (Charles River Laboratories, Wilmington, MA) to maintain the line. Offsprings carrying APP SW /PS1 dE9 transgene were identified using MyiQ2 RT-PCR system (Bio-Rad Hercules, CA) as previously described 11,21 . Given documented sex differences in the Aβ deposition in the APP SW /PS1 dE9 strain 13 , short-term and the long-term 2-PMAP treatment experiments were conducted only in female mice that were heterozygous for the APP SW /PS1 dE9 transgene.…”

“…Four hours after the last dose of 2-PMAP or vehicle animals were killed by ip injection of sodium pentobarbital (150 mg/kg) and transcardially perfused. The brains were extracted from the skulls and the cortical mantle was dissected out and ultrasonically homogenized (1:10 w/v at 4°C) using Misonix XL-2000 ultrasonic homogenizer (Qsonica, LLC; Newtown, CT) in the brain homogenization buffer consisting of 20mM Tris-HCL (pH 7.4), 250mM sucrose, 1mM EDTA, 1mM PMSF, and 10μg/ml of a Complete Protease Inhibitor Cocktail (Roche Applied Science; Indianapolis, IN) supplemented with leupeptin, antipain, pepstatin (5 μg/ml each) 21,22 . The total protein concentration in the brain homogenate was determined using BCA method as described above.…”

“…At the conclusion of 2-PMAP treatment the load of Aβ plaques (% of cross-sectional area occupied by Aβ plaques) was determined in the cerebral cortex of the right hemisphere in serial sections stained with Thioflavin-S for fibrillar Aβ deposits, or immunostained with anti-Aβ mAbs specific for C-terminal configuration of Aβ x-40 and Aβ x-42 peptides following our published protocols 11,21 . To detect Aβ x-40 a mouse mAb (clone 11A50-B10; Covance Inc., Princeton, NJ) was used in 1:250 dilution followed by Texas Red conjugated goat anti-mouse IgG secondary antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) diluted 1:200.…”

“…The brain cortex including the hippocampus was dissected out, homogenized, and subjected to formic acid (FA) extraction 21,22 . The levels of FA-extractable Aβ x-40 and Aβ x-42 were quantified by C-terminal specific sandwich ELISAs as described above.…”

“…The levels of FA-extractable Aβ x-40 and Aβ x-42 were quantified by C-terminal specific sandwich ELISAs as described above. In addition, the level of oligomeric Aβ in the brain homogenate was measured using ELISA specifically detecting aggregates of multiple Aβ copies 21 . Results of all ELISAs were presented as μg of Aβ per gram of wet brain accounting for all dilutions made during brain homogenization and subsequent processing of the homogenate.…”