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. 2014 Jan;35(1):179-90.
doi: 10.1016/j.neurobiolaging.2013.06.023. Epub 2013 Aug 15.

Dual-energy precursor and nuclear erythroid-related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species

Debolina Ghosh  1 , Kelsey R LeVault, Gregory J Brewer
Affiliations

Dual-energy precursor and nuclear erythroid-related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species

Debolina Ghosh et al. Neurobiol Aging. 2014 Jan.

Abstract

To determine whether glutathione (GSH) loss or increased reactive oxygen species (ROS) are more important to neuron loss, aging, and Alzheimer's disease (AD), we stressed or boosted GSH levels in neurons isolated from aging 3xTg-AD neurons compared with those from age-matched nontransgenic (non-Tg) neurons. Here, using titrating with buthionine sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase (GCL), we observed that GSH depletion increased neuronal death of 3xTg-AD cultured neurons at increasing rates across the age span, whereas non-Tg neurons were resistant to GSH depletion until old age. Remarkably, the rate of neuron loss with ROS did not increase in old age and was the same for both genotypes, which indicates that cognitive deficits in the AD model were not caused by ROS. Therefore, we targeted for neuroprotection activation of the redox sensitive transcription factor, nuclear erythroid-related factor 2 (Nrf2) by 18 alpha glycyrrhetinic acid to stimulate GSH synthesis through GCL. This balanced stimulation of a number of redox enzymes restored the lower levels of Nrf2 and GCL seen in 3xTg-AD neurons compared with those of non-Tg neurons and promoted translocation of Nrf2 to the nucleus. By combining the Nrf2 activator together with the NADH precursor, nicotinamide, we increased neuron survival against amyloid beta stress in an additive manner. These stress tests and neuroprotective treatments suggest that the redox environment is more important for neuron survival than ROS. The dual neuroprotective treatment with nicotinamide and an Nrf2 inducer indicates that these age-related and AD-related changes are reversible.

Keywords: 3xTg-AD; Aging; Glutathione; Neurodegeneration; Neuroprotection; Nrf2; ROS; Stress.

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Conflict of interest statement

Disclosure statement

The authors report no conflicts of interest.

Figures

Fig 1
Fig 1. Characterization of adult hippocampal neuron culture at 8 days in vitro
Bisbenzamide (blue nuclei) stained cells from 21 month old (A) non-Tg and (B) 3xTg-AD mice overlapping with either red MAP2 stain for differentiated adult neurons or green GFAP stain for astroglia shows about 90% of the cultured cell are neurons.
Fig. 2
Fig. 2. Glutathione depletion in neurons precedes elevated ROS levels
A) Dose dependent decline of glutathione and increase of ROS with increasing concentration of buthionine sulfoximine (BSO) in 2, 4, 8, 11 and 21 month non-Tg (ROS: gray dashed line, open circle, GSH gray solid line, open circle) and 3xTg- AD (ROS: black dashed line, filled circle, GSH black solid line, filled circle) neurons. * = p < 0.01. B) Relationship of ROS changes to GSH levels from the titrations in (A) in non-Tg (dashed line linear fit) and 3xTg-AD neurons (solid line linear fit) for each indicated age. C) GSH fluorescence per neuron at zero BSO indicates an early deficit in 3xTg-AD neurons while D) ROS fluorescence levels increase only after 4 months in both genotypes with ROS levels higher in 3xTg-AD than non-Tg neurons. E) Age-related rate of ROS generation as a function of GSH depletion is higher in 3xTg-AD neurons than the non-Tg neurons. N = 400–600 neurons from 3–4 animals per genotype per age.
Fig. 3
Fig. 3. HPLC from brain extracts of 21 month animals indicates that
A) GSH concentration from 3xTg-AD (black solid bar) is significantly lower than non-Tg (white bar) while B) GSSG concentrations (nmol/mg) are similar in both genotypes. N= 10–11 animals per genotype.
Fig. 4
Fig. 4. Genotype differences in neurodegeneration from glutathione depletion in neurons precedes elevated ROS levels, sustained into old age
A) Age and dose dependent increase in neuron death with decline of glutathione and B) increase in ROS with increasing concentration of buthionine sulfoximine (BSO) in 2, 4, 8, 11 and 21 month non-Tg (gray line, open circle, and 3xTg-AD (black solid line, filled circle) neurons. C) Neuron death with GSH loss expressed as a slope indicates greater susceptibility of neuron loss with GSH depletion across the age span in both non-Tg (black dashed line, open circle) and 3xTg-AD (black solid line, filled circle), with 3xTg-AD being more sensitive to GSH depletion. D) Neuron death with ROS elevation expressed as a slope indicates similar responses in both non-Tg and 3xTg-AD neurons, but neuron loss in oldest 21 month declined in both genotypes from 11 month with ROS elevation. N = 400–600 neurons from 3–4 animals per genotype per age.
Fig. 5
Fig. 5. 18α-glycyrrhetinic acid (18α-GA) stimulates Nrf2 mRNA and translocates Nrf2 protein to the nucleus
A) Age related Nrf2 mRNA levels from brain declines after 4 mo. in both non-Tg (gray dashed line, gray filled circle) and 3xTg-AD mice (black dashed line, black filled circle), N=6 animals/age/genotype. P values indicate genotype differences at specific ages. 18α-GA stimulates mRNAs for B) Nrf2 transcription factor and C) γ-glutamyl cysteine ligase, subunit c (Gclc) in neurons from 11 or 21 mo. brains. Vehicle (veh, DMSO) or 2 μg/ml 18α-GA in neurons from 11 (light grey bars) or 21 (black bars) month non-Tg and 3xTg-AD (11 month: white bars, 21 month: dark grey bars) brains. N=4–6 animals per genotype. D) Immunocytology for 21 mo untreated or 18α-GA treated non-Tg (upper panel) and 3xTg- AD (lower panel) neurons staining for Nrf2 (green) and bisbenzamide (blue) shows more green Nrf2 overlapping with blue bisbenzamide after 18α-GA treatment. E) Increased Nrf2 nuclear translocation with 18α-GA treatment in non-Tg (grey bars) and 3xTg-AD (black bars), n= 80–110 neurons from two 21 month animals/genotype.
Fig. 6
Fig. 6. The NAD(P)H precursor, nicotinamide and the redox enzyme inducer, 18α-GA, additively increase GSH and decrease ROS levels
A) Simultaneous measurement of glutathione with monochlorobimane (MCB) and ROS with dichlorofluorescein (DCF) and their merged effect on live 19 month cultured 3xTg-AD neurons with the indicated treatments. B) Additive increase in GSH levels in non-Tg (gray sold bars) and 3xTg-AD (black solid bars) and C) decrease in ROS levels with indicated treatments of 10 μM BSO, 2 mM nicotinamide (Nic), 2 μg/ml 18α-GA alone or in combination. N=200–300 neurons from 2 animals per genotype.
Fig. 7
Fig. 7. Nicotinamide and 18α-GA provide neuroprotection against GSH or beta-amyloid stress
11 month non-Tg (white bars) and 3xTg-AD neurons (grey bars) treated with nicotinamide or 18α-GA decreased neuron death against A) GSH depletion or B) Beta-amyloid stress. Note the additive neuroprotection provided by the combination of nicotinamide and 18α-GA in both models of stress toxicity in both genotypes.
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