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. 2024 Apr;30(4):e14703.
doi: 10.1111/cns.14703.

SIRT3 alleviates painful diabetic neuropathy by mediating the FoxO3a-PINK1-Parkin signaling pathway to activate mitophagy

Jing Yang  1 , Zhuoying Yu  1 , Ye Jiang  1 , Zixian Zhang  2   3   4 , Yue Tian  2   3   4 , Jie Cai  2   3   4 , Min Wei  1 , Yanhan Lyu  1 , Dongsheng Yang  1 , Shixiong Shen  1 , Guo-Gang Xing  2   3   4 , Min Li  1
Affiliations

SIRT3 alleviates painful diabetic neuropathy by mediating the FoxO3a-PINK1-Parkin signaling pathway to activate mitophagy

Jing Yang et al. CNS Neurosci Ther. 2024 Apr.

Abstract

Introduction: Painful diabetic neuropathy (PDN) is a common complication of diabetes. Previous studies have implicated that mitochondrial dysfunction plays a role in the development of PDN, but its pathogenesis and mechanism have not been fully investigated.

Methods: In this study, we used high-fat diet/low-dose streptozotocin-induced rats as a model of type 2 diabetes mellitus. Behavioral testing, whole-cell patch-clamp recordings of dorsal root ganglion (DRG) neurons, and complex sensory nerve conduction velocity studies were used to assess peripheral neuropathy. Mitochondrial membrane potential (MMP), ATP, tissue reactive oxygen species, and transmission electron microscopy were used to evaluate the function and morphology of mitochondria in DRG. Real-time PCR, western blot, and immunofluorescence were performed to investigate the mechanism.

Results: We found that damaged mitochondria were accumulated and mitophagy was inhibited in PDN rats. The expression of sirtuin 3 (SIRT3), which is an NAD+-dependent deacetylase in mitochondria, was inhibited. Overexpression of SIRT3 in DRG neurons by intrathecally administered LV-SIRT3 lentivirus ameliorated neurological and mitochondrial dysfunctions. This was evidenced by the reversal of allodynia and nociceptor hyperexcitability, as well as the restoration of MMP and ATP levels. Overexpression of SIRT3 restored the inhibited mitophagy by activating the FoxO3a-PINK1-Parkin signaling pathway. The effects of SIRT3 overexpression, including the reversal of allodynia and nociceptor hyperexcitability, the improvement of impaired mitochondria and mitophagy, and the restoration of PINK1 and Parkin expression, were counteracted when FoxO3a siRNA was intrathecally injected.

Conclusion: These results showed that SIRT3 overexpression ameliorates PDN via activation of FoxO3a-PINK1-Parkin-mediated mitophagy, suggesting that SIRT3 may become an encouraging therapeutic strategy for PDN.

Keywords: SIRT3; dorsal root ganglion; mitochondria; mitophagy; painful diabetic neuropathy.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effects of mitochondria and mitophagy in PDN rats. Effects of MMP, ATP, ROS, mitochondria morphology, the area of mitochondria, the perimeter of mitochondria, and the expression of P62, Beclin‐1, and LC3B in PDN rats. (A) MMP levels of mitochondria in the DRG of rats (***p < 0.001, n = 8). (B) ATP levels of mitochondria in the DRG of rats (*p < 0.05, n = 8). (C) ROS levels of mitochondria in the DRG of rats (***p < 0.001, n = 8). (D–I) TEM examination of mitochondria and mitophagosomes in DRG (*p < 0.05, ***p < 0.001, n = 3). (D) Representative electron micrographs of mitochondria structures (red arrows indicate abnormal mitochondria; m: mitochondria; scale bar: 500 nm). (E) The ratio of abnormal mitochondria counts to mitochondrial counts. (F) The area of mitochondria. (G) The perimeter of mitochondria. (H) Representative electron micrographs of mitophagosome structures; scale bar: 100 nm. (I) The number of mitophagosome structures. (J–M) Western blot analysis of P62, Beclin‐1, and LC3B expression in mitochondrial extracts (*p < 0.05, n = 3). (J) Representative immunoblots. (K) Quantification for the ratio of P62 to TOM20. (L) Quantification for the ratio of Beclin‐1 to TOM20. (M) Quantification for the ratio of LC3II to LC3I. (N) Co‐localization analysis of confocal laser scanning microscopy images of COX IV (red) and LC3B (green) staining (Scale bar = 5 μm). (O) The degree of co‐localization between COX IV and LC3B (***p < 0.001, n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test for (A–C), (E–G), (I), (K–M), and (O).
FIGURE 2
FIGURE 2
The localization and expression of SIRT3 in PDN rats. (A–C) Distribution of SIRT3 in the DRG on days 21 after STZ injection. (A) SIRT3 was colocalized mostly with neurons (NeuN), and a minority with astrocytes (GFAP) in the DRG (n = 3, Scale bar = 25 μm). (B) Representative images of immunofluorescence staining with SIRT3 and large‐diameter NF‐200+ neurons, peptide‐rich, medium‐diameter CGRP+ neurons, peptide‐poor, small‐diameter isolectin B4 (IB4)‐positive in DRG neurons in PDN rats (n = 3, Scale bar = 100 μm). (C) The percentage of NF‐200+, CGRP+, and IB4+ (green) neurons relative to SIRT3 (red) positive cells (**p < 0.01, NF‐200 vs. CGRP; ## p < 0.01, NF‐200 vs. IB4). (D) mRNA expression levels of SIRT3 detected by qRT‐PCR in DRG. The BL indicates the baseline, and the numbers below the x‐axis indicate the time after STZ injection (**p < 0.01, n = 6–8). (E) The time‐course of SIRT3 protein in the DRG of PDN rats on days 21 after STZ injection (*p < 0.05, n = 4). (F) Representative images of immunofluorescence staining with SIRT3 and NeuN in DRG neurons in Control and PDN rats (scale bar = 100 μm). (G) The mean fluorescence intensity of SIRT3 immunostaining in DRG neurons (***p < 0.001, n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ## p < 0.01, one‐way ANOVA with Tukey's post hoc test for (C); unpaired t test for (D), (E), and (G).
FIGURE 3
FIGURE 3
Effects of SIRT3 overexpression on pain hypersensitivity, and DRG neuron hyperexcitability in PDN rats. (A–H) Validation of overexpression viruses on primary cultured DRG neurons (*p < 0.05, ***p < 0.001, n = 3–7) and DRG tissues (**p < 0.01, ***p < 0.001, n = 3–6). (A and E) qRT‐PCR of SIRT3 overexpression in Sirt3 mRNA abundance in vitro and in vivo. (B and F) Protein blot of SIRT3 overexpression in vitro and in vivo. (C and G) Immunostaining of SIRT3 with GFP in SIRT3 overexpressing DRG neurons in vitro (scale bar = 10 μm) and in vivo (scale bar = 100 μm). (D and H) Statistical analysis of SIRT3 mean fluorescence intensity in vitro and in vivo. (I and J) PWT and PWL in the hind paws were measured before STZ injection (BL), before LV‐SIRT3 or LV‐GFP injection (day‐13), and at days 7, 14, 21, 23, 25, 27, and 29 after STZ injection (*p < 0.05, **p < 0.01, ***p < 0.001, n = 11). (K–O) Electrophysiological analysis of excitability of DRG neurons after LV‐SIRT3 or LV‐GFP injection (*p < 0.05, ***p < 0.001, n = 14–17 cells for each group of five rats). (K) Representative traces of neuronal action potentials. (L–O) Statistical analysis of the AP number, ISI, AHP 80% duration, AP amplitude, RMP, and TP in DRG neurons of PDN rats in the LV‐SIRT3 group compared with rats in the LV‐GFP group. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test for (A), (B), (D–F), (H), and (M–O); two‐way ANOVA with Sidak's post hoc test for (I), (J), and (L).
FIGURE 4
FIGURE 4
Effects of SIRT3 overexpression on mitochondria and mitophagy in PDN rats. Effects of SIRT3 overexpression on MMP, ATP, ROS, mitochondria morphology, the area of mitochondria, the perimeter of mitochondria, and the expression of P62, Beclin‐1, and LC3B in PDN rats. (A) MMP levels of mitochondria in the DRG of PDN rats treated with LV‐GFP or LV‐SIRT3 (**p < 0.01, n = 8). (B) ATP levels of mitochondria in the DRG of rats (*p < 0.05, n = 8). (C) ROS levels of mitochondria in the DRG of rats (*p < 0.05, n = 8). (D–I) TEM examination of mitochondria and mitophagosomes in PDN rats after LV‐GFP or LV‐SIRT3 injection (*p < 0.05, n = 3). (D) Representative electron micrographs of mitochondria structures (red arrows indicate abnormal mitochondria; m: mitochondria; scale bar: 500 nm). (E) The ratio of abnormal mitochondria counts to mitochondrial counts. (F) The area of mitochondria. (G) The perimeter of mitochondria. (H) Representative electron micrographs of mitophagosome structures; scale bar: 100 nm. (I) The number of mitophagosome structures. (J–M) Western blot analysis of P62, Beclin‐1, and LC3B expression in mitochondrial extracts (*p < 0.05, **p < 0.01, ***p < 0.001, n = 4–6). (J) Representative immunoblots. (K) Quantification for the ratio of P62 to TOM20. (L) Quantification for the ratio of Beclin‐1 to TOM20. (M) Quantification for the ratio of LC3II to LC3I. (N) Co‐localization analysis of confocal laser scanning microscopy images of COX IV (red) and LC3B (green) staining. (O) the degree of co‐localization between COX IV and LC3B (***p < 0.001, n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test for (A–C), (E–G), (I), (K–M), and (O).
FIGURE 5
FIGURE 5
The localization and expression of FoxO3a, PINK1, and Parkin. The localization and expression of FoxO3a, PINK1, and Parkin and impacts of SIRT3 overexpression on the expression FoxO3a, PINK1, and Parkin in PDN rats. (A) Representative images of immunofluorescence staining with FoxO3a and NeuN in DRG neurons in PDN rats. (B) Representative images of immunofluorescence staining with PINK1 and NeuN in DRG neurons in PDN rats. (C) Representative images of immunofluorescence staining with Parkin and NeuN in DRG neurons in PDN rats. (D–K) Protein blot analysis of FoxO3a, PINK1, and Parkin in whole‐cell extracts was performed in the DRG of rats. In H‐K, PDN rats were injected with LV‐SIRT3 or LV‐GFP (*p < 0.05, n = 3–6). (D) The representative blots graph. (E–G) Statistical analysis showing the relative intensities of FoxO3a, PINK1, and Parkin. (H) The representative blots graph. (I–K) Statistical analysis of the relative intensities of FoxO3a, PINK1, and Parkin. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test for (E–G) and (I–K).
FIGURE 6
FIGURE 6
FoxO3a knockdown counteracted the effect of SIRT3 overexpression. (A and B) PWT and PWL in the hind paws were measured before STZ injection (BL), before LV‐SIRT3+scramble or LV‐SIRT3+siFoxO3a injection (day−13), and at days 7, 14, 21, 23, 25, 27, and 29 after STZ injection (**p < 0.01, ***p < 0.001, n = 11). (C–G) Electrophysiological analysis of DRG neurons after LV‐SIRT3+scramble or LV‐SIRT3+siFoxO3a injection (*p < 0.05, **p < 0.001, ***p < 0.001, n = 16–17 cells for each group of five rats). (C) Representative traces of neuronal AP. (D–G) Statistical analysis of the AP number, ISI, AHP 80% duration, AP amplitude, RMP, and TP in DRG neurons of PDN rats. (H) MMP levels of mitochondria in the DRG of rats (**p < 0.01, n = 8). (I) ATP levels of mitochondria in the DRG of rats (*p < 0.05, n = 8). (J) ROS levels of mitochondria in the DRG of rats (*p < 0.05, n = 8). (K) Representative electron micrographs of mitochondria structures (red arrows indicate abnormal mitochondria; m: mitochondria; scale bar: 500 nm). (L–O) Western blot analysis of P62, Beclin‐1, and LC3B expression in mitochondrial extracts (*p < 0.05, n = 3–6). (L) Representative immunoblots. (M) Quantification for the ratio of P62 to TOM20. (N) Quantification for the ratio of Beclin‐1 to TOM20. (O) Quantification for the ratio of LC3II to LC3I. (P) Representative blots of FoxO3a, PINK1, and Parkin in whole‐cell extracts after LV‐SIRT3+scramble or LV‐SIRT3+siFoxO3a injection. (Q–S) Statistical analysis of the relative intensities of FoxO3a, PINK1, and Parkin (*p < 0.05, **p < 0.01, ***p < 0.001, n = 5–6). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two‐way ANOVA with Sidak's post hoc test for (A), (B), and (D); unpaired t test for (E–J), (M–O), and (Q–S).
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