Synaptic Plasticity Deficits and Mild Memory Impairments in Mouse Models of Chronic Granulomatous Disease

Abstract

Reactive oxygen species (ROS) are required in a number of critical cellular signaling events, including those underlying hippocampal synaptic plasticity and hippocampus-dependent memory; however, the source of ROS is unknown. We previously have shown that NADPH oxidase is required for N-methyl-d-aspartate (NMDA) receptor-dependent signal transduction in the hippocampus, suggesting that NADPH oxidase may be required for NMDA receptor-dependent long-term potentiation (LTP) and hippocampus-dependent memory. Herein we present the first evidence that NADPH oxidase is involved in hippocampal synaptic plasticity and memory. We have found that pharmacological inhibitors of NADPH oxidase block LTP. Moreover, mice that lack the NADPH oxidase proteins gp91phox and p47phox, both of which are mouse models of human chronic granulomatous disease (CGD), also lack LTP. We also found that the gp91phox and p47phox mutant mice have mild impairments in hippocampus-dependent memory. The gp91phox mutant mice exhibited a spatial memory deficit in the Morris water maze, and the p47phox mutant mice exhibited impaired context-dependent fear memory. Taken together, our results are consistent with NADPH oxidase being required for hippocampal synaptic plasticity and memory and are consistent with reports of cognitive dysfunction in patients with CGD. Chronic granulomatous disease (CGD) is caused by inherited mutations in genes encoding subunits of the NADPH oxidase complex (7, 14, 36, 43). NADPH oxidase is composed of two membrane-bound subunits (gp91phox and p22phox) and three cytosolic subunits, which include p47phox, p67phox, and Rac (49). The membrane-bound subunits form a heterodimer that stabilizes them within the membrane, whereas the cytosolic subunits are recruited to the membrane following stimulation. Complete complex assembly is necessary for full NADPH oxidase activity (2). Mutations in the gp91phox and p47phox genes are the most common mutations that cause CGD (58). These mutations disable the NADPH oxidase complex, thereby preventing the oxidation of NADPH and the subsequent production of superoxide (31, 45), which is required for pathogen destruction as well as most superoxide-dependent signal transduction in nonphagocytic cells (21, 24, 49). CGD patients suffer from frequent bacterial and fungal infections and inflammatory granulomas in the lungs, liver, skin, lymph nodes, and lining of the gastrointestinal and genitourinary tracts (34); loss of phagocytic oxidative burst activity due to mutations in genes encoding the NADPH oxidase subunits is the cause of these symptoms. gp91phox (40) and p47phox (22) mutant mice have been generated and used as models for the most common mutations that cause CGD. These mutant mouse lines, which lack their respective gene products due to targeted homologous recombinant gene disruption, have been utilized to understand the molecular mechanisms underlying CGD (22, 40). NADPH oxidase has been studied primarily for its role in the phagocyte oxidative burst in the immune system (43); however, its regulation and expression pattern suggest that it may be an important source of superoxide in the brain (9, 46, 54). Interestingly, some CGD patients also suffer from cognitive dysfunction (37), indicating that the lack of a fully functional NADPH oxidase impairs higher-order brain function. NADPH oxidase function in the nervous system has been well studied in microglia (9), and a role for NADPH oxidase in astrocyte function has been described (1). Moreover, it has been demonstrated that NADPH oxidase is expressed in neurons (51, 54) and localized at synapses (51). These findings indicate that NADPH oxidase may be a source of superoxide that is required for normal brain function. Superoxide is known to be required for hippocampal synaptic plasticity, specifically long-term potentiation (LTP) (25-27, 52, 53), as well as hippocampus-dependent memory (27, 52, 53). However, the source of superoxide required for LTP and memory formation has yet to be determined. We previously have shown that superoxide is required for N-methyl-d-aspartate (NMDA) receptor-dependent activation of extracellular signal-regulated kinase (ERK) in the hippocampus (24) and that a likely source of superoxide required for ERK activation is NADPH oxidase (24). Thus, we hypothesized that NADPH oxidase could be a source of superoxide that is required for hippocampal LTP and memory. In order to test this hypothesis directly, we examined LTP in hippocampal slices treated with two structurally and functionally different NADPH oxidase inhibitors. We also examined LTP in hippocampal slices prepared from either gp91phox or p47phox mutant mice. In addition, we tested the ability of the NADPH oxidase mutant mice to perform hippocampus-dependent learning and memory tasks. Our data indicate that the loss of a functional NADPH oxidase results in deficient LTP and mild hippocampus-dependent memory impairments. Our data are the first to show a direct role of NADPH oxidase involvement in hippocampal synaptic plasticity and memory formation. Overall, these findings are consistent with the idea that superoxide produced by NADPH oxidase is required for normal hippocampal synaptic plasticity and memory and may explain why some CGD patients exhibit cognitive dysfunction.

Publication
Molecular and Cellular Biology

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