Nrf2 Deficiency Attenuates Testosterone Efficiency in Ameliorating Mitochondrial Function of the Substantia Nigra in Aged Male Mice

Abstract

Reduced testosterone level is a common feature of aging in men. Aging, as a risk factor for several neurodegenerative disorders, shows declined mitochondrial function and downregulated mitochondrial biogenesis and mitochondrial dynamics. Mitochondrial biogenesis and mitochondrial dynamics are crucial in maintaining proper mitochondrial function. Supplementation with testosterone is conducive to improving mitochondrial function of males during aging. Nuclear factor erythroid 2-related factor 2 (Nrf2), a regulator of redox homeostasis, is involved in the ameliorative effects of testosterone supplementation upon aging. To explore Nrf2 role in the effects of testosterone supplementation on mitochondrial function during aging, we studied the efficiency of testosterone supplementation in improving mitochondrial function of Nrf2 knockout- (KO-) aged male mice by analyzing the changes of mitochondrial biogenesis and mitochondrial dynamics. It was found that wild-type- (WT-) aged male mice showed low mitochondrial function and expression levels of PGC-1α, NRF-1\NRF-2, and TFAM regulating mitochondrial biogenesis, as well as Drp1, Mfn1, and OPA1 controlling mitochondrial dynamics in the substantia nigra (SN). Nrf2 KO aggravated the defects above in SN of aged male mice. Testosterone supplementation to WT-aged male mice significantly ameliorated mitochondrial function and upregulated mitochondrial biogenesis and mitochondrial dynamics, which were not shown in Nrf2 KO-aged male mice due to Nrf2 deficiency. Testosterone deficiency by gonadectomy (GDX) decreased mitochondrial function, downregulated mitochondrial biogenesis, and altered mitochondrial dynamics balance in young male mice. Supplementation with testosterone to Nrf2 KO-GDX mice only ameliorated the alterations above but did not reverse them to sham level. Nrf2 deficiency attenuated testosterone efficiency in ameliorating mitochondrial function in the SN of aged male mice through mitochondrial biogenesis and mitochondrial dynamics to some extent. Activation of Nrf2 might contribute to testosterone-upregulating mitochondrial biogenesis and mitochondrial dynamics in the SN during aging to produce efficient mitochondria for ATP production.

Figure 1

Effects of TP supplementation on open-field activity and walking gait of Nrf2 KO-aged male mice: (a) total path length, (b) walking, (c) climbing, (d) rearing, (e) sniffing, (f) number of grooming, (g) duration of grooming, (h) latency of grooming, (i) forelimb stride length, (j) hindlimb stride length, (k) overlap of left footprints, and (l) overlap of right footprints. Data were presented as mean ± SD; n = 10 for open-field test; n = 5 for footprint test. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 2

Effects of TP supplementation on dopaminergic activity in the caudate putamen of Nrf2 KO-aged male mice. (a, b) TH and (a, c) DAT were detected by immunoblotting. (d) DA, (e) DOPAC, and (f) HVA were measured by LC-MS/MS assay. Data were presented as mean ± SD; n = 5 for TH and DAT; n = 10 for DA, DOPAC, and HVA. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 3

Effects of TP supplementation on oxidative balance in the substantia nigra of Nrf2 KO-aged male mice. (a) GSH/GSSG, (b) MDA, and (c) mitochondrial H₂O₂ were assessed by spectrophotometry. (d, e) Mitochondrial 3-NT was measured by immunoblotting; (f–h) 3-NT in the SN was detected by immunohistochemistry. Data were presented as mean ± SD; n = 5 for GSH/GSSG, MDA, mitochondrial H₂O₂, and mitochondrial 3-NT; n = 3 for 3-NT immunohistochemistry. Scale bars = 50 μm (lower panel); scale bars = 200 μm (upper panel). ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 4

Effects of TP supplementation on mitochondrial function in the substantia nigra of Nrf2 KO-aged male mice. (a) Mitochondrial membrane potential was revealed using the Rh123 fluorescence method. (b) Mitochondrial ATP level, (c) mitochondrial complex I activity, (d) mitochondrial complex II activity, (e) mitochondrial complex III activity, (f) mitochondrial complex IV activity, and (g) mitochondrial complex V activity were revealed by spectrophotometry. Data were presented as mean ± SD; n = 5. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 5

Effects of TP supplementation on mitochondrial biogenesis in the substantia nigra of Nrf2 KO-aged male mice. (a) PGC-1α, (b) NRF-1, (c) NRF-2, and (d) TFAM mRNAs were revealed by qPCR. (e, i) PGC-1α, (f, j) NRF-1, (g, k) NRF-2, and (h, l) TFAM proteins were detected by immunoblotting. Data were presented as mean ± SD; n = 5. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 6

Effects of TP supplementation on mitochondrial content in the substantia nigra of Nrf2 KO-aged male mice. (a) CS activity was assessed by spectrophotometry. (b) mtDNA/nDNA was detected by qPCR. (c) Mitochondrial number was counted using Image-Pro Plus 6.0 by an electron microscope. (d) Mitochondrial ultrastructure images were taken by an electron microscope. Data were presented as mean ± SD; n = 5 for CS activity and mtDNA/nDNA; n = 2 for an electron microscope (mitochondrial number from an analysis of 5 images per sample from each group). Scale bar = 500 nm. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 7

Effects of TP supplementation on mitochondrial dynamics in the substantia nigra of Nrf2 KO-aged male mice. (a) Drp1, (b) Mfn1, and (c) OPA1 mRNA were revealed by qPCR. (d, e) Drp1, (d, f) pDrp1-S616, (d, g) Mfn1, (d, h) L-OPA1, and (d, i) S-OPA1 proteins were detected by immunoblotting. Data were presented as mean ± SD; n = 5. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 8

Effects of TP supplementation on Nrf2 in the substantia nigra of aged male mice. (a, d) Nrf2 in the SN, (b, e) Nrf2 in nucleus fraction, and (c, f) HO-1 protein in the SN were detected by immunoblotting. Data were presented as mean ± SD; n = 5. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 9

Effects of TP supplementation on open-field activity and walking gait of Nrf2 KO GDX male mice: (a) total path length, (b) walking, (c) climbing, (d) rearing, (e) sniffing, (f) number of grooming, (g) duration of grooming, (h) latency of grooming, (i) forelimb stride length, (j) hindlimb stride length, (k) overlap of left footprints, and (l) overlap of right footprints. Data were presented as mean ± SD; n = 8 for open-field test; n = 5 for footprint test. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 10

Effects of TP supplementation on dopaminergic activity in the caudate putamen of Nrf2 KO GDX male mice. (a, b) TH and (a, c) DAT were detected by immunoblotting. (d) DA, (e) DOPAC, and (f) HVA were measured by LC-MS/MS assay. Data were presented as mean ± SD; n = 5 for TH and DAT; n = 8 for DA, DOPAC, and HVA. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 11

Effects of TP supplementation on oxidative balance in the substantia nigra of Nrf2 KO GDX male mice. (a) GSH/GSSG, (b) MDA, and (c) mitochondrial H₂O₂ were assessed by spectrophotometry. (d, e) Mitochondrial 3-NT was measured by immunoblotting; (f–h) 3-NT in the SN was detected by immunohistochemistry. Data were presented as mean ± SD; n = 8 for GSH/GSSG; n = 7 for MDA; n = 5 for mitochondrial 3-NT; n = 3 for 3-NT immunohistochemistry. Scale bars = 50 μm (lower panel); scale bars = 200 μm (upper panel). ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 12

Effects of TP supplementation on mitochondrial function in the substantia nigra of Nrf2 KO GDX male mice. (a) Mitochondrial membrane potential was revealed using the Rh123 fluorescence method. (b) Mitochondrial ATP levels, (c) mitochondrial complex I activity, (d) mitochondrial complex II activity, (e) mitochondrial complex III activity, (f) mitochondrial complex IV activity, and (g) mitochondrial complex V activity were revealed by spectrophotometry. Data were presented as mean ± SD; n = 8 for MMP and mitochondrial ATP levels; n = 5 for mitochondrial complexes I-V. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 13

Effects of TP supplementation on mitochondrial biogenesis in the substantia nigra of Nrf2 KO GDX male mice. (a) PGC-1α, (b) NRF-1, (c) NRF-2, and (d) TFAM mRNA were revealed by qPCR. (e, i) PGC-1α, (f, j) NRF-1, (g, k) NRF-2, and (h, l) TFAM proteins were detected by immunoblotting. Data were presented as mean ± SD; n = 8 for qPCR; n = 5 for western blot. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 14

Effects of TP supplementation on mitochondrial content in the substantia nigra of Nrf2 KO GDX male mice. (a) CS activity was assessed by spectrophotometry. (b) mtDNA/nDNA was detected by qPCR. Data were presented as mean ± SD; n = 5. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 15

Effects of TP supplementation on mitochondrial dynamics in the substantia nigra of Nrf2 KO GDX male mice. (a) Drp1, (b) Mfn1, and (c) OPA1 mRNA were revealed by qPCR. (d, e) Drp1, (d, f) pDrp1-S616, (d, g) Mfn1, (d, h) L-OPA1, and (d, i) S-OPA1 proteins were detected by immunoblotting. Data were presented as mean ± SD; n = 8 for qPCR; n = 5 for immunoblotting. ^#P < 0.05 main effect of genotype by two-way ANOVA; ^&P < 0.05 main effect of treatment by two-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01.