Elsevier

Neurobiology of Disease

Volume 44, Issue 3, December 2011, Pages 317-326
Neurobiology of Disease

Cognitive impairment in humanized APP × PS1 mice is linked to Aβ1–42 and NOX activation

https://doi.org/10.1016/j.nbd.2011.07.012Get rights and content

Abstract

Cognitive impairment in Alzheimer's disease (AD) is strongly associated with both extensive deposition of amyloid β peptides and oxidative stress, but the exact role of these indices in the development of dementia is not clear. This study was designed to determine the relationship between cognitive impairment, activation of the free radical producing enzyme NADPH oxidase (NOX), and progressive changes in Aβ deposition and solubility in humanized APP × PS1 knock-in mice of increasing age. Data show that cognitive performance and expression of key synaptic proteins were progressively decreased in aging APP × PS1 mice. Likewise, NOX activity and expression of the specific NOX subunit NOX4 were significantly increased in APP × PS1 mice in an age-dependent manner, and NOX activity and cognitive impairment shared a significant linear relationship. Data further show that age-dependent increases in Aβ1–42 had a significant linear relationship with both NOX activity and cognitive performance in APP × PS1 knock-in mice. Collectively, these data show that NOX expression and activity are significantly upregulated with age in this humanized model of Aβ pathogenesis, and suggest that NOX-associated redox pathways are intimately linked to both the loss of cognitive function and the deposition of Aβ1–42.

Highlights

► Cognitive performance and expression of key synaptic proteins are progressively decreased with age in APP × PS1 mice. ► NOX activity and expression of the NOX4 are progressively increased with age in APP × PS1 mice. ► Cognitive function shows a significant linear correlation with NOX activity and Aβ1–42.

Introduction

Alzheimer's disease (AD) is characterized by a progressive and irreversible loss of cognitive function and is the most common dementing disorder of the elderly (Jalbert et al., 2008). As existing treatments unfortunately have only limited efficacy in slowing clinical decline, there has been concerted effort shared by basic, clinical, and epidemiological researchers to map the etiology of this disease. An important and primary pathological feature of AD is the progressive accumulation of amyloid β-protein (Aβ) into neuritic plaques (Jellinger, 2002, Selkoe, 2001, Vinters and Farag, 2003, Walsh and Selkoe, 2004), and the progression of cognitive decline is associated with accumulation of Aβ peptide, particularly Aβ1–42, and alterations in Aβ solubility (Johnson, 2000, Murphy et al., 2007). AD brains are also typified by ample pathological evidence of oxidative stress in affected regions, including the cerebral cortex and hippocampal formation (Aksenov et al., 2001, Markesbery and Lovell, 1998, Wang et al., 2006a). Markers of oxidative stress are elevated in mild cognitive impairment (MCI), which may be the earliest stage of clinical dementia (Butterfield et al., 2006, Keller et al., 2005, Mosconi et al., 2008, Williams et al., 2006). While collectively these data are consistent with the hypothesis that free radical production might drive the initiation of cognitive dysfunction, the nature of the relationship between free radical production and the pathogenesis of Aβ is still not fully understood.

Free radicals are produced in mammalian cells as secondary by-products by many different systems, including mitochondrial electron transport, xanthine oxidase, cyclooxygenases, and monoamine oxidases (reviewed in Droge, 2002, Reddy, 2006). However, the enzyme NADPH oxidase (NOX) is noteworthy as it is dedicated to the specific and deliberate production of free radicals. Although NOX was first described in phagocytic cells such as microglia, it is now well established that NOX subunits are also expressed in neurons and astrocytes (Kim et al., 2005, Noh and Koh, 2000). Indeed, experimental evidence points to a role for NOX in neuronal physiology, particularly in functions relating to hippocampal electrophysiology (Kishida et al., 2005, Tejada-Simon et al., 2005). These data suggest that NOX is very well situated to participate in perturbations to cognitive function, and indeed, many reports have proposed that NOX may be involved in AD pathogenesis (reviewed in Block, 2008, Wilkinson and Landreth, 2006). For example, early studies revealed the ability of Aβ to activate NOX and increase superoxide production (Bianca et al., 1999, Bruce et al., 1996, Jana and Pahan, 2004, Meda et al., 1995, Niikura et al., 2004, Thomas et al., 1996), suggesting that NOX may be involved in Aβ-induced neuronal injury. Furthermore, published reports show increased NOX activity in AD brains (Shimohama et al., 2000) and (Ansari and Scheff, 2011), and also in MCI brains (Bruce-Keller et al., 2010). Finally, genetic deletion of specific NOX subunits was recently shown to attenuate neurovascular dysfunction and cognitive decline in transgenic mice overexpressing the Swedish mutation of the human amyloid precursor protein (Park et al., 2008).

As there is ample support indicating that NOX could participate in AD, this study was undertaken to delineate the profile of NOX enzymatic activity and subunit expression in humanized APP × PS1 knock-in mice, which are a second generation, physiologically appropriate mouse model of Aβ pathogenesis. The use of knock-in technology, rather than a transgenic overexpression system, preserves the physiologically appropriate regulation of APP expression by endogenous promoters. Thus, data related to Aβ levels in these particular mice are not confounded by an artificial promoter system in unknown loci that drives supraphysiologic levels of APP expression. Indeed, previous reports from our group show that this model of Aβ pathogenesis is associated with progressive changes in Aβ solubility and deposition that mirror key changes in human AD (Flood et al., 2002, Murphy et al., 2007). Furthermore, this model recapitulates the development of both diffuse and neuritic plaques (Murphy et al., 2007), which is key to modeling AD pathology (Murphy et al., 2007). Finally, published reports document progressive and age-related increases in markers of oxidative stress in these mice (Abdul et al., 2008, Huang et al., 2010). Thus, these studies were designed to specifically evaluate the exact relationship between Aβ, NOX activation, and cognitive decline. To this end, APP × PS1 and wild type (WT) mice were examined for cognitive impairment, and cognitive function was analyzed in relation to NOX activation and to parameters of Aβ deposition and solubility. Studies were conducted in young (4–6 month-old) mice, in which neuritic plaques are not detected; and old (16–19 month-old) mice, in which neuritic plaques are present predominantly in the frontal and parietal cortex and (to a lesser degree) in the hippocampus (Murphy et al., 2007).

Section snippets

APP × PS1 knock-in mice

The Institutional Animal Care and Use Committee at the Pennington Center approved all experimental protocols, which were compliant with NIH guidelines on the use of experimental animals. All mice were housed in standard caging with 12:12 light:dark cycle, and had ad libitum access to food and water throughout the study. Young (age 4–6 months) and aged (16–19 months) mice were evaluated in this study, and data were compiled from 2 complete but separate cohorts of mice, for a total of 12–20 animals

Cognitive function in young and aged APP × PS1 and WT mice

Previous behavioral investigations of cognitive function in APP × PS1 knock-in mice employed the Morris water maze and the 6-arm water maze tasks of spatial memory (Chang et al., 2006). This study did not reveal a working memory deficit in APP × PS1 mice, although APP × PS1 mice did show impaired memory “flexibility”, i.e., the ability to learn the location of a new platform each day without perseverating on the previous location (Chang et al., 2006). However, it is possible that deficits in working

Discussion

This study uses a second generation mouse model of AD to evaluate the role of NOX in amyloid pathogenesis. Specifically, using APP × PS1 knock-in mice, experiments were designed to document changes in cognitive function and NOX activity, and to further evaluate these indices in relation to changes in Aβ. Data show that NOX activity is significantly increased in aged APP × PS1 mice, and furthermore shares a significant linear relationship with deficits in cognitive function. Data also show that

Acknowledgments

The authors are grateful to Dr. Barry Robert and Cynthia Kloster for exemplary veterinary assistance. Additional gratitude goes to Teresa Noel for expert training and leadership in animal husbandry and colony management. This work was supported by grants from the NIH (NS46267 and AG05119), and also used PBRC Core facilities (Animal Phenotyping) that are funded by the NIH (P20-RR021945 and P30-DK072476).

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