External cys/cySS redox state modification controls the intracellular redox state and neurodegeneration via Akt in aging and Alzheimer's disease mouse model neurons

Abstract

The extracellular redox environment of cells is mainly set by the redox couple cysteine/cystine (cys/cySS) while intracellular redox is buffered by reduced/oxidized glutathione (GSH/GSSG), but controlled by NAD(P)H/NAD(P). With aging, the extracellular redox environment shifts in the oxidized direction beyond middle-age. Since aging is the primary risk factor in Alzheimer's disease (AD), here our aim was to determine if a reduced extracellular cys/cySS redox potential of cultured primary mouse neurons changes the intracellular redox environment, affects pAkt levels, and protects against neuron loss. A reductive shift in cys/cySS in the extracellular medium of neuron cultures from young (4 month) and old (21 month) neurons from non-transgenic) and triple transgenic AD-like mice (3xTg-AD) caused an increase in intracellular NAD(P)H and GSH levels along with lower reactive oxygen species levels. Importantly, the imposed reductive shift decreased neuron death markedly in the 21 month neurons of both genotypes. Moreover, a reduced cys/cySS redox state increased the pAkt/Akt ratio in 21 month aging and AD-like neurons that positively correlated with a decreased neuron loss. Our findings demonstrate that manipulating the extracellular redox environment toward a more reduced redox potential is neuroprotective in both aging and AD-like neurons and may be a powerful and pragmatic therapeutic tool in aging and age-related diseases like AD.

Keywords: Aging; Akt; Alzheimer's disease; NAD(P)H; cys/cySS; glutathione; neurodegeneration.

Figure 1. A sufficiently reducing external cys/cySS redox potential maximizes internal NAD(P)H determined by intrinsic fluorescence (A, B) and internal redox state as NAD(P)H/FAD (C, D)

External reducing levels of cys/cySS increased NAD(P)H concentrations in neurons from A) 4 month mice (ANOVA genotype F(1,193)=79, p < 0.001; redox potential F(4,193)= 37, p < 0.001) and B) 21 month mice (genotype F(1,178)=140, p < 0.001; redox potential F(4,178)= 27, p < 0.001). The genotype difference was significant at the most oxidized levels studied (p=0.02). The NAD(P)H levels in 3xTg-AD neurons (black solid circles, solid line) resisted a shift as large as those of non-Tg neurons (gray open circles, gray dashed line). Similarly, the internal redox ratio was increased by external cys/cySS in C) 4 month neurons (ANOVA genotype F(1,193)=79, p < 0.001, redox potential F(4,193)=38, p < 0.001) and D) 21 month neurons F(1,178)=140, p < 0.001; redox potential F(4,178)= 28, p < 0.001). Note that 3xTg-AD neurons were unable to attain the maximized NAD(P)H concentration in non-Tg neurons. The normal cys/cySS redox state for the culture medium is ~−50 mV (indicated by arrow in figures) at the juncture of a redox inflection point. N= neurons from 4 animals per genotype per redox potential. The bars and p values indicate post-hoc significance at each of the indicated external cys/cySS levels.

Figure 2. A reductive shift in external cyS/cySS caused large increases in intracellular GSH and small decreases in ROS

Simultaneous GSH and ROS fluorescence at indicated redox cys/cySS potentials in live non-Tg (gray dashed line, open circle) and 3xTg-AD (black solid line, solid circle) neurons caused increased GSH levels in A) 4 month neurons (ANOVA genotype F(1,632)= 65, p < 0.001; redox potential F(4, 632)= 57, p < 0.005)) and B) 21 month neurons (ANOVA genotype F(1,713)= 77, p < 0.001; redox potential, F(4, 713)= 35, p < 0.001). A reduced cys/cySS also decreased ROS in C) 4 month neurons (ANOVA genotype F(1,632)= 38, p = 0.058; redox potential F(4, 532)= 95, p < 0.004) and B) 21 month neurons (ANOVA genotype F(1,713)= 56 p < 0.001; redox potential F(4, 713)= 71, p < 0.001). Arrow indicates redox state of control culture medium at ~−50 mV. N = neurons from 4 animals per genotype per redox potential.

Figure 3. A reductive extracellular cys/cySS shift increases neuron survival in non-Tg and AD-neurons in old age

Altered external redox potentials of A) 4 month neurons fails to change the percentage of dead non-Tg neurons (gray dashed line, open circle) and slightly improves survival of 3xTg-AD neurons (black solid line, solid circle) (ANOVA genotype F(1,125) =1, p =0.26; redox potential F(4, 125)=2, p =0.065). For B) 21 month neurons, a reducing external redox shift increases neuron survival for both genotypes (ANOVA genotype F(1,72) =17, p < 0.001; redox potential F(4, 72)=40, p < 0.001). N = > 4 fields from 2–3 animals per genotype per redox potential.

Figure 4. Aging and AD-genotype alterations in pAkt and Akt levels in response to shifts in an extracellular cys/cySS redox clamp

A) Immunocytological analysis of 21 month 3xTg-AD neurons at oxidized shift to 0 mV reveals less green pAkt and higher red Akt stain than B) when shifted to reducing (−150 mV) external redox clamp with higher green (pAkt) and lower red (Akt) staining. These types of images were subjected to quantitative image analysis. In C), pAkt levels in 4 month non-Tg (gray dashed line, open circle) and 3xTg-AD (black solid line, solid circle) neurons at indicated cys/cySS redox potential (ANOVA genotype F(1, 793)= 21, p = 0.064; redox potential F(3, 793) = 66, p < 0.001 and D) 21 month neurons (ANOVA genotype F(1, 838)= 0.07, p = 0.8; redox potential F(3, 838) = 30, p < 0.001). Akt levels in E) 4 month neurons (ANOVA genotype F(1, 793)= 144, p < 0.001; redox potential F(3, 793) = 1, p = 0.4) and F) 21 month neurons (ANOVA genotype F(1, 838)= 30, p < 0.001; redox potential F(3, 838) = 35, p < 0.001). pAkt/Akt ratios in G) 4 month neurons (ANOVA genotype F(1, 793)= 137, p < 0.001; redox potential F(3, 793) = 10, p < 0.001) and H) 21 month (ANOVA genotype F(1, 838)= 9, p = 0.003; redox potential F(3, 838) = 26, p < 0.001). N > 350 neurons per genotype per redox potential from 3 mice.

Figure 5. An increase in pAkt or pAkt /Akt levels decreases neuron loss in neurons from both genotypes and ages

A) As pAkt levels increased, death decreased in non-Tg (grey line, open black circle), slope = −0.11 R² = 0.87 and in 3xTg-AD (black line, black filled circle), slope = −0.18; R² = 0.79 neurons. Extrapolation to the threshold of 150 for pAkt indicated a maximum 48% death in non-Tg neurons and a maximum 62% death in 3xTg-AD neurons. B) In both non-Tg (black open circle) and 3xTg-AD (black solid circle) neuron death decreases with a decrease in the pAkt/Akt ratio. R² = 0.98 (with two most oxidized (lowest pAkt/Akt) non-Tg points redacted).