Maitea Guridi

Blocking the Interaction between Apolipoprotein E and Aβ Reduces Intraneuronal Accumulation of Aβ and Inhibits Synaptic Degeneration

Accumulation of β-amyloid (Aβ) in the brain is a key event in Alzheimer disease pathogenesis. Apolipoprotein (Apo) E is a lipid carrier protein secreted by astrocytes, which shows inherent affinity for Aβ and has been implicated in the receptor-mediated Aβ uptake by neurons. To characterize ApoE involvement in the intraneuronal Aβ accumulation and to investigate whether blocking the ApoE/Aβ interaction could reduce intraneuronal Aβ buildup, we used a noncontact neuronal-astrocytic co-culture system, where synthetic Aβ peptides were added into the media without or with cotreatment with Aβ12-28P, which is a nontoxic peptide antagonist of ApoE/Aβ binding. Compared with neurons cultured alone, intraneuronal Aβ content was significantly increased in neurons co-cultured with wild-type but not with ApoE knockout (KO) astrocytes. Neurons co-cultured with astrocytes also showed impaired intraneuronal degradation of Aβ, increased level of intraneuronal Aβ oligomers, and marked down-regulation of several synaptic proteins. Aβ12-28P treatment significantly reduced intraneuronal Aβ accumulation, including Aβ oligomer level, and inhibited loss of synaptic proteins. Furthermore, we showed significantly reduced intraneuronal Aβ accumulation in APPSW/PS1dE9/ApoE KO mice compared with APPSW/PS1dE9/ApoE targeted replacement mice that expressed various human ApoE isoforms. Data from our co-culture and in vivo experiments indicate an essential role of ApoE in the mechanism of intraneuronal Aβ accumulation and provide evidence that ApoE/Aβ binding antagonists can effectively prevent this process.

Blocking the apoE/Aβ interaction ameliorates Aβ-related pathology in APOE ε2 and ε4 targeted replacement Alzheimer model mice

Accumulation of β-amyloid (Aβ) in the brain is essential to Alzheimer’s disease (AD) pathogenesis. Carriers of the apolipoprotein E (APOE) ε4 allele demonstrate greatly increased AD risk and enhanced brain Aβ deposition. In contrast, APOE ε2 allele carries show reduced AD risk, later age of disease onset, and lesser Aβ accumulation. However, it remains elusive whether the apoE2 isoform exerts truly protective effect against Aβ pathology or apoE2 plays deleterious role albeit less pronounced than the apoE4 isoform. Here, we characterized APPSW/PS1dE9/APOE ε2-TR (APP/E2) and APPSW/PS1dE9/APOE ε4-TR (APP/E4) mice, with targeted replacement (TR) of the murine Apoe for human ε2 or ε4 alleles, and used these models to investigate effects of pharmacological inhibition of the apoE/Aβ interaction on Aβ deposition and neuritic degeneration. APP/E2 and APP/E4 mice replicate differential effect of human apoE isoforms on Aβ pathology with APP/E4 mice showing a several-fold greater load of Aβ plaques, insoluble brain Aβ levels, Aβ oligomers, and density of neuritic plaques than APP/E2 mice. Furthermore, APP/E4 mice, but not APP/E2 mice, exhibit memory impairment on object recognition and radial arm maze tests. Between the age of 6 and 10 months APP/E2 and APP/E4 mice received treatment with Aβ12-28P, a non-toxic, synthetic peptide homologous to the apoE binding motif within the Aβ sequence, which competitively blocks the apoE/Aβ interaction. In both lines, the treatment significantly reduced brain Aβ accumulation, co-accumulation of apoE within Aβ plaques, and neuritic degeneration, and prevented memory deficit in APP/E4 mice. These results indicate that both apoE2 and apoE4 isoforms contribute to Aβ deposition and future therapies targeting the apoE/Aβ interaction could produce favorable outcome in APOE ε2 and ε4 allele carriers.

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

Proteolytic cleavage of the amyloid precursor protein (APP) generates β‐amyloid (Aβ) peptides. Prolonged accumulation of Aβ in the brain underlies the pathogenesis of Alzheimer disease (AD) and is regarded as a principal target for development of disease‐modifying therapeutics.

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

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimers disease (AD) including memory deficit, presence of β-amyloid (Aβ) 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 Aβ 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 4 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 4 and 15 months old APP(SW) and 4 months old APP(SW)/PS1(L166P) 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. Aβ oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5±3.6ng/g in 4 months old APP(SW) mice to 60.8±15.9ng/g in 4 months old APP(SW)/PS1(L166P) mice. Four months old APP(SW) mice had no Aβ amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient Aβ 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 Aβ oligomers but predating onset of Aβ amyloidosis.

Modulation of amyloid precursor protein expression reduces β-amyloid deposition in a mouse model: Targeting of APP Expression in AD Mice

Proteolytic cleavage of the amyloid precursor protein (APP) generates β‐amyloid (Aβ) peptides. Prolonged accumulation of Aβ in the brain underlies the pathogenesis of Alzheimer disease (AD) and is regarded as a principal target for development of disease‐modifying therapeutics.

Modulation of APP Expression Reduces Aβ Deposition in a Mouse Model

Proteolytic cleavage of the amyloid precursor protein (APP) generates β‐amyloid (Aβ) peptides. Prolonged accumulation of Aβ in the brain underlies the pathogenesis of Alzheimer disease (AD) and is regarded as a principal target for development of disease‐modifying therapeutics.

Blocking the apoE/Aβ interaction ameliorates Aβ-related pathology in APOE ε 2a ndε4 targeted replacement Alzheimer model mice

Accumulation of β-amyloid (Aβ) in the brain is essential to Alzheimer’s disease (AD) pathogenesis. Carriers of the apolipoprotein E (APOE) e4 allele demonstrate greatly increased AD risk and enhanced brain Aβ deposition. In contrast, APOE e2 allele carries show reduced AD risk, later age of disease onset, and lesser Aβ accumulation. However, it remains elusive whether the apoE2 isoform exerts truly protective effect against Aβ pathology or apoE2 plays deleterious role albeit less pronounced than the apoE4 isoform. Here, we characterized APPSW/PS1dE9/ APOE e2-TR (APP/E2) and APPSW/PS1dE9/APOE e4-TR (APP/E4) mice, with targeted replacement (TR) of the murine Apoe for human e 2o re4 alleles, and used these models to investigate effects of pharmacological inhibition of the apoE/Aβ interaction on Aβ deposition and neuritic degeneration. APP/E2 and APP/E4 mice replicate differential effect of human apoE isoforms on Aβ pathology with APP/E4 mice showing a several-fold greater load of Aβ plaques, insoluble brain Aβ levels, Aβ oligomers, and density of neuritic plaques than APP/E2 mice. Furthermore, APP/E4 mice, but not APP/E2 mice, exhibit memory impairment on object recognition and radial arm maze tests. Between the age of 6 and 10 months APP/E2 and APP/E4 mice received treatment with Aβ12-28P, a non-toxic, synthetic peptide homologous to the apoE binding motif within the Aβ sequence, which competitively blocks the apoE/Aβ interaction. In both lines, the treatment significantly reduced brain Aβ accumulation, co-accumulation of apoE within Aβ plaques, and neuritic degeneration, and prevented memory deficit in APP/E4 mice. These results indicate that both apoE2 and apoE4 isoforms contribute to Aβ deposition and future therapies targeting the apoE/Aβ interaction could produce favorable outcome in APOE e 2a nde4 allele carriers.

Impairment of NMDA receptor signaling in the CA1 sector of APPSW/PS1dE9 AD Tg mice precedes Aß deposition

Background: Alzheimer’s disease (AD) is associated with synaptic loss and memory impairment. AD transgenic mice (Tg) models, over-expressing mutated forms of the amyloid precursor protein demonstrate memory deficit and inhibition of the long-term potentiation prior to the appearance of Aß plaques, which implicates toxicity of soluble Aß oligomeric species in the early pathogenesis of memory dysfunction. Experiments in primary cultures of hippocampal neurons showed preferential binding of Aß oligomers to Nmethyl-D-aspartic acid (NMDA) receptors resulting over time in their down regulation. NMDA receptor signaling involves Ca for intracellular signal transduction and is pivotal for the synaptic plasticity in the hippocampus, which underlies memory formation. The goal of our experiments was to characterize impairment of NMDA receptor signaling AD Tg mice, which has not been done before. Methods: NMDA receptor signaling in the CA1 sector of the hippocampus was investigated using microdialysis in awake and behaving 3.5-4 month old APPSW/PS1dE9 AD Tg mice. Under physiological conditions stimulation of NMDA receptor results in a massive increase in the intracellular Ca concentration, which via Ca/ calmodulin / nitric oxide synthase / nitric oxide / soluble guanyl cyclase pathway stimulates production of cGMP. Thus cGMP released to the interstitial fluid can serve as a measure of NMDA receptor function. Results: Stimulation of the NMDA receptor with 500mM NMDA (a selective NMDA agonist) administered through reverse microdialysis resulted in 32% increase in the baseline cGMP level in four month old wild type mice (p < 0.05, paired ttest). In contrast, only 2%, non-significant change in 3.5-4 month old APPSW/PS1dE9 Tg mice was noticed (n 1⁄4 8). Examination of APPSW/ PS1dE9 Tg mice brains revealed minimal Aß plaque load in the CA1 hippocampal sector. These mice had high tissue concentration of Aß species, with predominance of oligomerization prone Aß42. Conclusions: Our experiments provide direct proof of the NMDA receptor signaling impairment in AD Tg mice, which is associated with presence of Aß oligomers but not Aß deposits. Biochemical analysis of NMDA receptor and other post-synaptic proteins is close to explaining whether impairment of the NMDA receptor signaling is associated with down regulation of its expression.