FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

Abstract

Mitochondrial dysfunction and oxidative stress are important mechanisms for secondary injury after traumatic brain injury (TBI), which result in progressive pathophysiological exacerbation. Although the Fibronectin type III domain-containing 5 (FNDC5) was reported to repress oxidative stress by retaining mitochondrial biogenesis and dynamics, its possible role in the secondary injury after TBI remain obscure. In present study, we observed that the level of serum irisin (the cleavage product of FNDC5) significantly correlated with the neurological outcomes of TBI patients. Knockout of FNDC5 increased the lesion volume and exacerbated apoptosis and neurological deficits after TBI in mice, while FNDC5 overexpression yielded a neuroprotective effect. Moreover, FNDC5 deficiency disrupted mitochondrial dynamics and function. Activation of Sirtuin 3 (SIRT3) alleviated FNDC5 deficiency-induced disruption of mitochondrial dynamics and bioenergetics. In neuron-specific SIRT3 knockout mice, FNDC5 failed to attenuate TBI-induced mitochondrial damage and brain injuries. Mechanically, FNDC5 deficiency led to reduced SIRT3 expression via enhanced ubiquitin degradation of transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), which contributed to the hyperacetylation and inactivation of key regulatory proteins of mitochondrial dynamics and function, including OPA1 and SOD2. Finally, engineered RVG29-conjugated nanoparticles were generated to selectively and efficiently deliver irisin to the brain of mice, which yielded a satisfactory curative effect against TBI. In conclusion, FNDC5/irisin exerts a protective role against acute brain injury by promoting SIRT3-dependent mitochondrial quality control and thus represents a potential target for neuroprotection after TBI.

Fig. 1. Association of serum irisin levels with outcomes in TBI patients.

a Serum irisin levels were decreased in patients with poor outcomes compared to those with good outcomes (P < 0.001). b ROC analysis of the discriminative ability of serum irisin levels for risk of poor outcome in patients with TBI. c Linear correlation between serum irisin levels and short-term neurological outcome assessed using GOS scores of patients with TBI. Significance was determined by Student’s t test (a) and one-way ANOVA (c). Values are presented as the mean ± SD.

Fig. 2. Knockout of FNDC5 increases lesion volume, worsens brain edema, and aggravates neurological deficits after TBI.

a Schematic of MRI test and behavioral studies. b MRI scan of the brain in WT + TBI group and FNDC5-KO + TBI group; n = 6 for each group. c Quantification of lesion volume of MRI. d measurement of brain water content. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT + TBI group. e–g Several behavioral tests at different time points after TBI as measured by the grid-walking test (e), cylinder test (f) and adhesive removal test (g) (n = 9 for each group). Significance was determined by Student’s t test (c, d) and two-way repeated ANOVA (e–g) with Bonferroni post hoc tests. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT+Sham group, ^#P < 0.05 and ^##P < 0.01 vs. WT + TBI group. Values are presented as the mean ± SEM.

Fig. 3. FNDC5 suppresses oxidative stress and reduces neural apoptosis after TBI.

a, b Representative DHE staining images and the quantitative results (n = 6 for each group). Scale bar: 200 μm. c The effects of FNDC5 on MDA levels. d The effects of FNDC5 on mitochondrial MnSOD activity. e, f Representative images and statistical analysis of TUNEL staining of the perilesional cortex after TBI. Scale bar: 200 μm. g, h Representative Western blots and statistical analysis of the levels of Bax and Bcl-2 (n = 6 for each group). Significance was determined by one-way ANOVA (b–d, f, h) with Bonferroni post hoc tests. *P < 0.05 and **P < 0.01 vs. sham group, ^#P < 0.05 and ^##P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Fig. 4. FNDC5 deficiency disrupts mitochondrial dynamics and bioenergetics, and activation of SIRT3 alleviates FNDC5 deficiency-induced damage to mitochondrial dynamics and bioenergetics.

a, b Western blot and statistical analysis of FNDC5 expression. c Representative MitoTracker fluorescence images illustrating mitochondrial morphology. Scale bar: 10 μm. d, e Mitochondrial morphological characteristics were quantified by ImageJ software. f ATP content of HT22 cells. g NAD⁺/NADH of HT22 cells. h, i Representative fluorescence intensity of JC-1 staining illustrating the MMP. Scale bar: 20 μm. j, k Representative MitoSOX fluorescence images of mitochondria-derived ROS. Scale bar: 50 μm (n = 6 for each group). l, m Western blot and statistical analysis of SIRT3 expression. n Relative mRNA level of SIRT3. o Total acetylation level of mitochondrial proteins. p Relative acetylation level of mitochondrial proteins. Significance was determined by Student’s t test (b, m, n, p) and one-way (d–g, i, k) ANOVA with Bonferroni post hoc tests. *P < 0.05 and **P < 0.01 vs. Control group, ^#P < 0.05 and ^##P < 0.01 vs. FNDC5-KO + vel group. Values are presented as the mean ± SEM.

Fig. 5. Neuronal SIRT3 deficiency reduces FNDC5-mediated protection of mitochondria in mice.

a Experimental flowchart of neuronal-specific SIRT3 homozygous knockout mice (SIRT3 cKO). b Identification of neuronal-specific SIRT3 homozygous knockout mice (SIRT3 cKO). c Representative ultrastructure of neurons in each group and the magnified images of the ultrastructure of neurons shown in (a). Scale bar: 0.3 μm and 0.05 μm (n = 6 for each group). d Percentage of damaged mitochondria. e ATP content in brain tissue. f NAD⁺/NADH ratio in brain tissue. g Relative level of ROS in brain tissue, n = 6 for each group. Significance was determined by two-way repeated ANOVA (d–g) with Bonferroni post hoc tests. *P < 0.05 and **P < 0.01 vs. sham group, ^#P < 0.05 and ^##P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Fig. 6. Neuronal SIRT3 knockout counters FNDC5-mediated effects on oxidative stress and neuronal apoptosis after TBI in mice.

a Representative DHE staining images in SIRT3^CKO mice and SIRT3^f/f mice. Scale bar: 200 μm. b The quantitative results of DHE staining images. c The effects of FNDC5 on MDA levels in SIRT3^CKO mice and in SIRT3^f/f mice. d The effects of FNDC5 on mitochondrial MnSOD activity in SIRT3^CKO mice and in SIRT3^f/f mice. e Representative images of TUNEL staining of the perilesional cortex 24 h in SIRT3^CKO mice and SIRT3^f/f mice. Scale bar: 200 μm. f, g Representative Western blot and statistical analysis of the levels of Bax and Bcl-2 in SIRT3^CKO mice and in SIRT3^f/f mice. (n = 6 for each group). Significance was determined by two-way repeated ANOVA (b, c, d, g) with Bonferroni post hoc tests. *P < 0.05 and **P < 0.01 vs. sham group, ^#P < 0.05 and ^##P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Fig. 7. FNDC5 upregulates the level of SIRT3, and mechanically, through modulating the protein stability of NRF2.

a, b Western blot and statistical analysis of the levels of NRF2 expression in normal HT22 cell line and FNDC5 KO HT22 cell line. c Relative mRNA level of SIRT3 after treating with an NRF2 inhibitor (ML385). d, e Western blot and statistical analysis of the levels of SIRT3 expression. f FNDC5 deficiency does not affect the mRNA of NRF2. g The protein level of NRF2 was degraded faster over time in FNDC5 knockout cells after incubating with the mRNA synthesis inhibitor CHX. h statistical analysis of the levels of NRF2. i The protein level of NRF2 expression after incubating with the proteasome inhibitor MG132. j Statistical analysis of the levels of NRF2. k The protein level of NRF2 expression after incubating with the lysosomal inhibitor NH4Cl. l statistical analysis of the levels of NRF2. m Ubiquitination level of NRF2. Significance was determined by Student’s t test (b, f) and one-way ANOVA (c, e) or two-way repeated ANOVA (h, j, l) with Bonferroni post hoc tests. *P < 0.05 and **P < 0.01 vs. Control group, ^#P < 0.05 and ^##P < 0.01 vs. TBI + vector group, & P < 0.05 and ^&&P < 0.01 vs. TBI + FNDC5 + veh group. Values are presented as the mean ± SEM.

Fig. 8. Nanoparticle-mediated irisin delivery to the brain significantly promotes recovery after TBI in mice.

a TEM observation of nap-irisin and RVG-nap-irisin. b Particle size analysis of nap-irisin and RVG-nap-irisin. c The drug release kinetics of nap-irisin and RVG-nap-irisin. d–f Real-time fluorescence imaging, and corresponding fluorescence analysis of mice after intravenous injection of Cy5.5-labeled nap-irisin and RVG-Nap-irisin. g Immunofluorescence of RVG-nap-irisin. h–m several behavioral tests with treatment with irisin or RVG-nap-irisin after TBI (n = 9 for each group). Significance was determined by one-way ANOVA (j, m) or two-way repeated ANOVA (f, h, i, l) with Bonferroni post hoc tests. *P < 0.05 and **P < 0.01 vs. sham group, ^#P < 0.05 and ^##P < 0.01 vs. TBI + irisin group. Values are presented as the mean ± SEM.