Genetic deletion of fragile X mental retardation protein (FMRP) has been shown to enhance mGluR-dependent long-term depression (LTD). Herein, we demonstrate that mGluR-LTD induces a transient, translation-dependent increase in FMRP that is rapidly degraded by the ubiquitin-proteasome pathway. Moreover, proteasome inhibitors abolished mGluR-LTD, and LTD was absent in mice that overexpress human FMRP. Neither translation nor proteasome inhibitors blocked the augmentation of mGluR-LTD in FMRP-deficient mice. In addition, mGluR-LTD is associated with rapid increases in the protein levels of FMRP target mRNAs in wild-type mice. Interestingly, the basal levels of these proteins were elevated and their synthesis was improperly regulated during mGluR-LTD in FMRP-deficient mice. Our findings indicate that hippocampal mGluR-LTD requires the rapid synthesis and degradation of FMRP and that mGluR-LTD triggers the synthesis of FMRP binding mRNAs. These findings indicate that the translation, ubiquitination, and proteolysis of FMRP functions as a dynamic regulatory system for controlling synaptic plasticity.

The fully reduced hen egg white lysozyme (HEWL), which is a good model of random coil structure, has been converted to highly organized amyloid fibrils at low pH by adding ethanol. In the presence of 90% (v/v) ethanol, the fully reduced HEWL adopts ␤-sheet secondary structure at pH 4.5 and 5.0, and an ␣-to-␤ transition is observed at pH 4.0. A red shift of the Congo red absorption spectrum caused by the precipitation of the fully reduced HEWL in the presence of 90% (v/v) ethanol is typical of the presence of amyloid aggregation. EM reveals unbranched fibrils with a diameter of 2-5 nm and as long as 1-2 m. The pH dependence of the initial structure of the fully reduced HEWL in the presence of 90% (v/v) ethanol suggests that Asp and His residues may play an important role.Keywords: Amyloid fibril formation; hen lysozyme; disulfide reduction Protein amyloid aggregation has been recognized as a major cause of several important diseases, including Alzheimer's disease (the fourth most common cause of death in the Western world), Parkinson's disease, type II or noninsulindependent diabetes, and the transmissible spongiform encephalopathies. About 17 different proteins have been found to form amyloid in vivo. Amyloid fibrils formed from those proteins share some common morphological features, but these proteins do not have a conserved sequence or native structural motif (Kelly 1996;Lansbury 1999).Recent studies show that amyloid formation is not only possible with disease-associated proteins, but also with proteins that are not associated with any known amyloid diseases under certain conditions (Guijarro et al. 1998;Chiti et al. 1999;Dobson 1999;Fandrich et al. 2001;Kallberg et al. 2001). Hen egg white lysozyme (HEWL), an extensively studied small protein with four disulfide bonds, is a recent example (Goda et al. 2000;Krebs et al. 2000). Amyloid fibrils formed from all these proteins that are not associated with any known amyloid diseases not only share similar morphological features with amyloid fibrils from diseaseassociated proteins, but also can be inherently highly cytotoxic (Bucciantini et al. 2002). Based on these observations, Dobson proposed that amyloid aggregation was a generic property of all polypeptides (Guijarro et al. 1998;Dobson 1999).Study of the amyloid aggregation of nondisease-associated proteins cannot only help us understand the mechanism of amyloid fibrillogenesis, but also extend our understanding of the basic relationship between protein sequence and structure. A common strategy to convert a nondisease-associated protein to amyloid fibrils is destabilizing the protein either by mutation or by partial denaturation with heating or addition of alcohols (Chiti et al. 1999;Rochet and Lansbury 2000). Most of the above studies are based on partially denaturing the native structure of the target proteins; in other words, those target proteins still maintain a substantial amount of their native structures. One question remaining is whether or not unstructured polypeptides can form amyloid fibri...

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 gp91 phox and p47 phox , both of which are mouse models of human chronic granulomatous disease (CGD), also lack LTP. We also found that the gp91 phox and p47 phox mutant mice have mild impairments in hippocampus-dependent memory. The gp91 phox mutant mice exhibited a spatial memory deficit in the Morris water maze, and the p47 phox 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 (gp91 phox and p22 phox ) and three cytosolic subunits, which include p47 phox , p67 phox , 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 gp91 phox and p47 phox 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. gp91 phox (40) and p47 phox (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); ho...

Oxidative damage caused by reactive oxygen species (ROS) has been proposed to be critically involved in several pathological manifestations of aging, including cognitive dysfunction. ROS, including superoxide, are generally considered as neurotoxic molecules whose effects can be alleviated by antioxidant enzymes. However, ROS also are known to be necessary components of the signal transduction cascades underlying normal synaptic plasticity. Therefore, we reasoned that the role that ROS and antioxidant enzymes play in modulating neuronal processes varies over the lifespan of an animal. We examined hippocampal long-term potentiation (LTP) and memory-related behavioral performance in transgenic mice overexpressing extracellular superoxide dismutase (EC-SOD) and their wild-type littermates at different ages. We found that aged EC-SOD transgenic mice exhibited enhanced hippocampal LTP, better cerebellum-dependent motor learning, and better hippocampus-dependent spatial learning compared with their wild-type littermates. We also found that EC-SOD overexpression impaired contextual learning, but the impairment was decreased in the aged transgenic mice. At the molecular level, aged EC-SOD transgenic mice had lower superoxide levels, a decrease in protein carbonyl levels, and a decrease in p38 and extracellular signal-regulated kinase 2 phosphorylation compared with aged wild-type mice. Our findings suggest that elevated levels of superoxide contribute to aging-related impairments in hippocampal LTP and memory, and that these impairments can be alleviated by overexpression of EC-SOD. We conclude that there is an age-dependent alteration in the role of superoxide in modulating synaptic plasticity and learning and memory.

Superoxide has been shown to be critically involved in several pathological manifestations of aging animals. In contrast, superoxide can act as a signaling molecule to modulate signal transduction cascades required for hippocampal synaptic plasticity. Mitochondrial superoxide dismutase (SOD-2 or Mn-SOD) is a key antioxidant enzyme that scavenges superoxide. Thus, SOD-2 may not only prevent aging-related oxidative stress, but may also regulate redox signaling in young animals. We used transgenic mice overexpressing SOD-2 to study the role of mitochondrial superoxide in aging, synaptic plasticity and memory-associated behavior. We found that overexpression of SOD-2 had no obvious effect on synaptic plasticity and memory formation in young mice, and could not rescue the age-related impairments in either synaptic plasticity or memory in old mice. However, SOD-2 overexpression did decrease mitochondrial superoxide in hippocampal neurons, and extended the lifespan of the mice. These findings increase our knowledge of the role of mitochondrial superoxide in physiological and pathological processes in the brain.