Elevating sestrin2 attenuates endoplasmic reticulum stress and improves functional recovery through autophagy activation after spinal cord injury
Spinal cord injury (SCI) is a devastating central neurological trauma that causes a loss of motor and sensory function. Sestrin2, also known as hypoxia inducible gene 95 (Hi95), is emerging as a critical determinant of cell homeostasis in response to cellular stress. However, the role of sestrin2 in neuron response to endoplasmic reticulum (ER) stress and potential mechanism remains undefined. In this study, we investigated the effects of sestrin2 on ER stress and delineated a underlying
... a underlying molecular mechanism after SCI. Methods The induction of sestrin2 after traumatic injury and ER stress insult were investigated in vitro and in vivo. West blot and immunofluorescence were used to analyze the potential mechanisms of sestrin2 on autophagy and ER stress after SCI. Behavior assessment were used to evaluated the effect of sestrin2 on function recovery in vivo. Results Elevated sestrin2 is a protective process in neurons against chemical ER stress induced by tunicamycin (TM) or traumatic invasion. While treatment with PERK inhibitor or knockdown of ATF4 reduces sestrin2 expression upon ER stress. In addition, overexpression of sestrin2 limits ER stress, promotes the survival of neuron and improves functional recovery after SCI, which is associated with activation of autophagy and restoration of autophagic flux mediated by sestrin2. Meanwhile, sestrin2 activates autophagy dependent on AMPK-mTOR signaling pathway. Consistently, inhibition of AMPK abrogates the effect of sestrin2 on activation of autophagy, and blockage of autophagic flux abolishes the effect of sestrin2 on limiting ER stress and neural death. Conclusion Upregulation of sestrin2 as an important resistance mechanism of neuron to ER stress, and its potential role as a therapeutic target for SCI. Background Traumatic spinal cord injury (SCI) is a devastating central neurological disorder that leads to sensory deficits and physical disabilities and affects thousands of individuals worldwide . The physiological progression of SCI is characterized by a series of secondary molecular events including inflammatory response, mitochondrial dysfunction and oxidative stress, axonal demyelination, which cause neuron death and injury beyond the initial mechanical damage  . Despite there is yet no completely satisfactory therapy for the treatment of SCI in clinical trials, separately or combined targeting these 4 secondary reactions have been regarded as proper therapeutic strategies and promoted functional recovery in almost animal cases [3-6]. As a stress inducible protein induced upon various stresses, sestrin2 is essential for maintaining cell metabolism and homeostasis by regulating a series of kinases and pathways, and loss of sestrin2 can result in metabolic disturbance and mitochondrial dysfunction . Sestrin2 has been shown to be capable against oxidative stress and apoptosis by regulating mammalian target of rapamycin complex (mTOR) by activating AMP-activated protein kinase (AMPK). Importantly, sestrin2 is also known as hypoxia inducible gene 95 (Hi95) and originally discovered in hypoxia condition, and the responsive induction of sestrin2 is associated with HIF-1α stabilization [7, 10]. Extensive evidences confirmed that upregulated sestrin2 protect heart against ischemic heart injury, alleviates insulin resistance, and delays the process of neurodegenerative diseases . In addition, numerous evidences implicated that sestrin2 provides beneficial effects on acute central nervous system injury. As previous study reported, sestrin2 was upregulated in cortical region in an acute stroke model in rats  . Further studies revealed that silence of sestrin2 aggravates cerebral infarct but overexpression of sestrin2 enhanced angiogenesis and attenuates focal cerebral ischemic injury under ischemic condition, the potential mechanisms are related to the phosphorylation of AMPK and activation of nuclear factor erythroid 2 related factor 2 (Nrf2) [15, 16] . Previous studies have indicated that neuron is vulnerable to the various stresses stimulation induced by the fierce damage of microcirculation and mechanical destruction, maintaining neuron homeostasis is a feasible method for functional recovery [17, 18]. As the versatile function of sestrin2 exhibited above, we hypothesized that sestrin2 is highly advantageous to neuron homeostasis in spinal cord. However, the role of sestrin2 in neuron response to traumatic SCI is not well-elucidated. The endoplasmic reticulum (ER) serves the major site for protein synthesis and controls the quality of newly synthesized . The perturbation of ER environment homeostasis lead to overabundance of misfolded protein and ER stress [20, 21]. Though unfolded protein response (UPR) provides effect on attenuating misfolded proteins, durative ER stress without treatment ultimately exceeds the compensation and turns UPR into pro-death [22, 23]. Accordingly, maintaining ER homeostasis is 5 available for neuron regeneration and function [24, 25]. In particular, a study reported by park et al  showed that ER stress suppressed by sestrin2 is important for hepatic rehabilitation. Therefore, we reasoned that sestrin2 plays a critical role in regulating neural ER stress. Numerous evidences indicated that a link between UPR and autophagy, in which the initiated of ER stress often stimulates the autophagic activity and autophagy acts as a compensatory protection for eliminating aggregated misfolded proteins . Meanwhile, it has been found that sestrin2 can activate autophagy under various pathological stresses and deficient of sestrin2 results in impairment of autophagy [9, 30]. As described previously, ER stress limited by sestrin2 may related to the activation of autophagy. However, the relationship of sestrin2, autophagy and ER stress in neural injury remains elusive. Given that sestrin2 performs such critical and versatile physiological functions and prevents the progression of diverse pathologies, we choose to determine the role of sestrin2 in ER stress and autophagy in neuron and therapeutic effect on SCI. In the current study, we hypothesized that ER stress is involved in sestrin2 regulation, the induction of sestrin2 would activate autophagy, enhance ER homeostasis and reduce neurons apoptosis in a mouse model of acute SCI for decreasing damage size, enhancing neural regeneration and function recovery.