Heat shock protein 22 modulates NRF1/TFAM-dependent mitochondrial biogenesis and DRP1-sparked mitochondrial apoptosis through AMPK-PGC1α signaling pathway to alleviate the early brain injury of subarachnoid hemorrhage in rats

Abstract

Mitochondrial dysfunction has been widely accepted as a detrimental factor in subarachnoid hemorrhage (SAH)-induced early brain injury (EBI), which is eminently related to poor neurologic function outcome. Previous studies have revealed that enhancement of heat shock protein 22 (hsp22) under conditions of stress is a friendly mediator of mitochondrial homeostasis, oxidative stress and apoptosis, thus accelerating neurological recovery. However, no study has confirmed whether hsp22 attenuates mitochondrial stress and apoptosis in the setting of SAH-induced EBI. Our results indicated that endogenous hsp22, p-AMPK/AMPK, PGC1α, TFAM, Nrf1 and Drp1 were significantly upregulated in cortical neurons in response to SAH, accompanied by neurologic impairment, brain edema, neuronal degeneration, lower level of mtDNA and ATP, mitochondria-cytosol translocation of cytochrome c, oxidative injury and caspase 3-involved mitochondrial apoptosis. However, exogenous hsp22 maintained neurological function, reduced brain edema, improved oxidative stress and mitochondrial apoptosis, these effects were highly dependent on PGC1α-related mitochondrial biogenesis/fission, as evidenced by co-application of PGC1α siRNA. Furthermore, we demonstrated that blockade of AMPK with dorsomorphin also compromised the neuroprotective actions of hsp22, along with the alterations of PGC1α and its associated pathway molecules. These data revealed that hsp22 exerted neuroprotective effects by salvaging mitochondrial function in an AMPK-PGC1α dependent manner, which modulates TFAM/Nrf1-induced mitochondrial biogenesis with positive feedback and DRP1-triggered mitochondrial apoptosis with negative feedback, further reducing oxidative stress and brain injury. Boosting the biogenesis and repressing excessive fission of mitochondria by hsp22 may be an efficient treatment to relieve SAH-elicited EBI.

Keywords: AMPK-PGC1α; Heat shock protein 22; Mitochondria; Subarachnoid hemorrhage.

Graphical abstract

Fig. 1

Typical images of subarachnoid hemorrhage (SAH) model and expression alterations of Hsp22 (heat shock protein 22), p-AMPK/AMPK(adenosine 5'monophosphate-activated protein kinase) and PGC1α (peroxisome proliferative activated receptor γ (PPARγ) coactivator 1α) after SAH. (A) Representative photographs of the bottom of rat brains and HE staining from sham and 24 h after SAH. (B) Representative microphotographs of immunofluorescence staining for Hsp22 (green) co-localization on neurons (NeuN, red) after SAH. n = 2 per group. Scale bar = 50 μm. (C) Western blot assay and quantitative analysis of the temporal profiles of Hsp22, p-AMPK/AMPK and PGC1α expressions from the injured cortex at 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h post-SAH. n = 6 per group. Bars represent mean ± SD. *P < 0.05, **P < 0.01 vs Sham group. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Hsp22 is bound up with the severity of EBI post-SAH and mediates the alterations of p-AMPK/AMPK, PGC1α, DRP1, and NRF1. (A) Beam balance scores, Modified Garcia scores, and Brain water content at diverse groups. n = 6 per group. (B) FJC staining and quantitative analyses. n = 4 per group. Scale bar = 100 μm. (C) Representative Western blot images and quantitative analyses of p-AMPK/AMPK, PGC1α, NRF1, and DRP1. n = 6 per group. (D) Double immunofluorescence staining revealed the variations of PGC1α, NRF1, and DRP1(green) in the neuron (NeuN, red). n = 4 in each group. Scale bar = 50 μm. Bars represent mean ± SD. *P < 0.05, **P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. SAH + Vehicle group. ^&P < 0.05, ^&&P < 0.01 vs. SAH + hsp22+scramble siRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Effects of PGC1α siRNA on hsp22-induced improvements in neurologic function and brain water content as well as apoptotic cascades after SAHBeam balance scores, Modified Garcia scores and Brain water content in different groups. n = 6 per group. (B) Typical photomicrographs of TUNEL staining and quantitative analyses in the indicated groups. n = 4. Scale bar = 100 μm. (C) Representative Western blot images and quantitative analyses of cleaved Caspase-3/Caspase-3, Bax and Bcl-2. n = 6 per group. Bars represent mean ± SD. *P < 0.05, **P < 0.01 vs. Sham group. ^#P < 0.05, ^##P < 0.01 vs. SAH + Vehicle group. ^&P < 0.05, ^&&P < 0.01 vs. SAH + hsp22+scramble siRNA.

Fig. 4

Effects of PGC1α siRNA on hsp22-mediated protection against redox imbalance after SAH DHE staining in various groups. n = 4 per group. Scale bar = 100 μm (B) Immunohistochemical staining of 8-OHdG in the indicated groups. n = 4 per group. Scale bar = 100 μm (C) Elisa assay and quantitive analysis of different markers of oxidative damage (8-OHdG, MDA and PCO) and antioxidative indices (GSH-Px and SOD). n = 6 per group. (D) Representative Western blot images and quantitative analysis of UCP2. n = 6 per group. Bars represent mean ± SD. *P < 0.05, **P < 0.01 vs. Sham group. ^#P < 0.05, ^##P < 0.01 vs. SAH + Vehicle group. ^&P < 0.05, ^&&P < 0.01 vs. SAH + hsp22+scramble siRNA.

Fig. 5

Effects of PGC1α siRNA on hsp22-mediated protection of mitochondrial structure and function following SAH Representative Western blot images and quantitative analyses of TFAM, Nrf1 and Drp1 of different groups. n = 6 per group. (B) Double immunofluorescence staining for Nrf1 and Drp1 (green) in the neuron (NeuN, red) in diverse groups. n = 4 per group. Scale bar = 50 μm. (C) mtDNA copy number and ATP content in the indicated groups. n = 6 per group. (D) Representative Western blot images and quantitative analyses of mitochondrial cytochrome c and cytoplasmic cytochrome c from five groups. n = 6 per group. (E) Transmission electron microscope (TEM) of cortical neurons. n = 4 per group. The red star stands for mitochondria. Bars represent mean ± SD. *P < 0.05, **P < 0.01 vs. Sham group. ^#P < 0.05, ^##P < 0.01 vs. SAH + Vehicle group. ^&P < 0.05, ^&&P < 0.01 vs. SAH + hsp22+scramble siRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Hsp22 regulates PGC1α via AMPK signaling pathway in rats after SAH Beam balance scores, Modified Garcia scores and Brainwater content in various groups. n = 6 per group. (B) Representative photomicrographs of TUNEL staining and quantitative analyses in the indicated groups. n = 4 per group. Scale bar = 100 μm. (C) Typical photomicrographs showing double immunofluorescence staining of PGC1α (green) and NeuN (red) in diverse experimental groups. n = 4 per group. Scale bar = 50 μm. (D) Western blot images and quantitative analyses of p-AMPK/AMPK, PGC1α, Drp1, Nrf1, TFAM, UCP2, Cleaved caspase-3/Caspase-3, Bcl2, Bax, Cytosolic and mitochondrial cytochrome c. n = 6 per group. Bars represent mean ± SD. **P < 0.01, *P < 0.05 vs. Sham group. ^##P < 0.01, ^#P < 0.05 vs. SAH + Vehicle group. ^&&P < 0.01, ^&P < 0.05 vs. SAH + hsp22+scramble siRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

figs1

figs2

figs3

figs4