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. 2014 Jun;35(6):1459-68.
doi: 10.1016/j.neurobiolaging.2013.12.002. Epub 2013 Dec 9.

Role of antioxidant enzymes in redox regulation of N-methyl-D-aspartate receptor function and memory in middle-aged rats

Wei-Hua Lee  1 , Ashok Kumar  2 , Asha Rani  2 , Thomas C Foster  3
Affiliations

Role of antioxidant enzymes in redox regulation of N-methyl-D-aspartate receptor function and memory in middle-aged rats

Wei-Hua Lee et al. Neurobiol Aging. 2014 Jun.

Abstract

Overexpression of superoxide dismutase 1 (SOD1) in the hippocampus results in age-dependent impaired cognition and altered synaptic plasticity suggesting a possible model for examining the role of oxidative stress in senescent neurophysiology. However, it is unclear if SOD1 overexpression involves an altered redox environment and a decrease in N-methyl-D-aspartate receptor (NMDAR) synaptic function reported for aging animals. Viral vectors were used to express SOD1 and green fluorescent protein (SOD1 + GFP), SOD1 and catalase (SOD1 + CAT), or GFP alone in the hippocampus of middle-aged (17 months) male Fischer 344 rats. We confirm that SOD1 + GFP and SOD1 + CAT reduced lipid peroxidation indicating superoxide metabolites were primarily responsible for lipid peroxidation. SOD1 + GFP impaired learning, decreased glutathione peroxidase activity, decreased glutathione levels, decreased NMDAR-mediated synaptic responses, and impaired long-term potentiation. Co-expression of SOD1 + CAT rescued the effects of SOD1 expression on learning, redox measures, and synaptic function suggesting the effects were mediated by excess hydrogen peroxide. Application of the reducing agent dithiolthreitol to hippocampal slices increased the NMDAR-mediated component of the synaptic response in SOD1 + GFP animals relative to animals that overexpress SOD1 + CAT indicating that the effect of antioxidant enzyme expression on NMDAR function was because of a shift in the redox environment. The results suggest that overexpression of neuronal SOD1 and CAT in middle age may provide a model for examining the role of oxidative stress in senescent physiology and the progression of age-related neurodegenerative diseases.

Keywords: Aging; Catalase; Glutathione peroxidase; Learning and memory; NMDAR; Superoxide dismutase; Synaptic plasticity.

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Figures

Fig. 1
Fig. 1
Effect of treatment on acquisition of cue and spatial discrimination on the water maze. (A) Mean distance to reach the escape platform for 5 blocks of cue and 6 blocks of spatial discrimination training. GFP expressing rats exhibited longer distance to reach the visible escape platform during block 2 and 3 of cue discrimination training. However, all groups (GFP black circles, n=14; SOD1+GFP rats: gray circles, n=11; SOD1+CAT rats: open circles, n=12) learned to reach the visible platform, as indicated by a significant overall decrease in distance across blocks. Spatial discrimination training was initiated 3 days later. The initial learning phase consisted of 6 blocks with an acquisition probe trial inserted between blocks 5 and 6 (arrow). Probe trial measures indicated impaired acquisition of a spatial search strategy in SOD1+GFP animals. (B) Mean+SEM discrimination index scores for GFP (black bars), SOD1+GFP (gray bars), and SOD1+CAT (open bars). Pound signs indicate a significant (p < 0.05) difference from chance (a score = 0) for SOD1+CAT and GFP animals. (C) Mean+SEM number of platform crossings and (D) latency for first platform crossing during the probe trial. Asterisk indicates a significant (p < 0.05) difference relative to SOD1+GFP.
Fig. 2
Fig. 2
Co-localization of viral mediated overexpression of antioxidant enzymes in the hippocampus. (A) Merged images for cells in the hippocampus from a rat co-transduced with AAV-SOD1 (myc staining shown in red) and AAV-GFP (green) and (B) co-transduced with AAV-SOD1 (myc staining shown in red) and AAV-CAT (CAT staining shown in green). The merged figures indicate co-expression in neurons observed as yellow or orange. Calibration bars in (A) and (B) represent 100 μm.
Fig. 3
Fig. 3
Quantification of viral mediated overexpression of antioxidant enzymes in the hippocampus. (A) Western blots of hippocampal lysates from rats injected with viral vectors to express GFP, SOD1+GFP or SOD1+CAT. For SOD1, two bands were observed representing endogenous rat SOD1 (rSOD1 at 19 kDa) and the human myc tagged SOD1 (hSOD1 at 23 kDa). Antibodies against myc revealed a band at 23 kDa, only in animals injected with AAV-SOD1. GFP was only detected in rats injected with virus to express GFP or SOD1+GFP. Lipid peroxidation was visualized using an anti-4-hydroxy-2-nonenal (HNE) antibody. GAPDH was used as a loading control. (B) Quantification of western blot data of hippocampal lysates from rats injected with viral vectors to express GFP, SOD1+GFP, or SOD1+CAT. All densitometry was normalized by GAPDH and each bar represents the mean+SEM (n=3-6). Asterisk indicates a significant (p < 0.05) difference relative to GFP animals.
Fig. 4
Fig. 4
Overexpression of SOD1 results in a loss of GSH, which is rescued by co-expression of SOD1+CAT. Bars represent the means+SEM. Asterisk indicates an increase relative to SOD1+GFP group.
Fig. 5
Fig. 5
Overexpression of SOD1 results in a decrease in GPx, which is rescued by co-expression of SOD1+CAT. Bars represent the means+SEM for (A) GPx and (B) GR activity. Asterisk indicates an increase relative to SOD1+GFP group.
Fig. 6
Fig. 6
SOD1 overexpression has little effect on the total EPSP and decreases NMDAR synaptic responses. Input-output curves for (A) Presynaptic fiber volley (PFV) amplitude for the total synaptic response, (B) the total EPSP slope, (C) the total EPSP slope/PFV, (D) PFV amplitude for the NMDAR synaptic response, (E) the NMDAR EPSP slope, (F) the NMDAR EPSP slope/PFV for rats injected with GFP (filled circle), SOD1+GFP (gray circle), and SOD1+CAT (open circle). The numbers in the brackets indicate the number of slices/rats. The inserts in B and E illustrate total and NMDAR-mediated synaptic responses and the arrow indicate the PFV (open) and slope (filled) of the synaptic response.
Fig. 7
Fig. 7
SOD1 expression in the hippocampus of middle-age rats depresses LTP. (A) Time course of changes in the field EPSP obtained from hippocampal slices 10 min before and 60 min after LTP-inducing stimulation (arrow) to induce LTP for the rats expressing GFP (filled circle, n = 11/8, slices/animals), SOD1+GFP (gray circle, n = 9/7, slices/animals), SOD1+CAT (open circle, n = 7/5, slices/animals), and non-tetanized control path (line, n = 27/20, slices/animals). (B) Bar diagram showing the average magnitude of LTP during the last 5 min of recording [dotted area in (A)]. Asterisk indicates a significant (p < 0.05) difference from GFP. Pound signs indicate a significant difference from base line for GFP and SOD1+CAT animals.
Fig. 8
Fig. 8
Antioxidant enzymes contribute to redox regulation of NMDAR function. Time course of changes in the slope of NMDAR–fEPSP obtained from hippocampal slices 10 min before and 60 min after bath application of the reducing agent DTT (0.5 mM, solid line) for slices from SOD1+GFP (gray circle, n = 13/7, slices/animals) and SOD1+CAT (open circle, n = 13/8, slices/animals) animals.
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