Possible divergence of serum- and glucocorticoid-inducible kinase function in ischemic brain injury

Possible divergence of serum- and glucocorticoid-inducible kinase function in ischemic brain injury

As recent medical progress decreases the incidence of certain diseases, ischemic brain injury remains one of the major diseases that threaten human lives, especially in western countries. Ischemic brain injury occurs as a result of lack of oxygen and nutrients due to obstruction of blood flow in the brain, and often leads to neurological disorders such as cerebral palsy, depression, and ultimately, death. Around 800,000 Americans suffer a new or recurrent stroke, and more than 130,000 people die annually in the United States (Goldstein et al., 2011). Despite much effort in searching for an effective treatment, at most a few reagents are approved for therapeutic medication in many countries. One of them, tissue plasminogen activator, is able to positively recover blood flow but unfortunately has a limited therapeutic time window and the potential side effect of intracranial hemorrhage. Therefore, identifying new targets for this disorder (and subsequent development of therapeutic reagents) may shed light on future solutions for treating ischemic brain injury. Incidentally, the incidence rate is higher among patients with hypertension and/ or diabetes mellitus (diabetes) (Goldstein et al., 2011), with both conditions involved in metabolic diseases. Moreover, these two conditions and ischemic stroke together represent the main causes of morbidity and mortality in western countries after cancer. Notably, given that the prevalence of hypertension and diabetes is rising as a result of the modern lifestyle, the incidence rate of ischemic brain injury may further increase interactively. To resolve this complication, it would be preferable if anti-hypertensive and/ or anti-diabetic reagents also work against ischemic brain injury. In this regards, one suitable candidate is serum- and glucocorticoid-inducible kinase (SGK) inhibitors, which we have recently reported the effect of in rodent models (Inoue et al., 2016).

Mechanism underlying the significance of SGKs: SGK1 encodes a serine/threonine kinase that regulates the function of many proteins including ion channels and transporters (Lang et al., 2006). One of the prominent roles of SGK1 is in blood pressure control, which is mediated through regulation of epithelial Na+channels (ENaCs) located at apical membranes in renal convoluted kidney tubules (Lang et al., 2006). ENaCs contain proline-rich PY motifs that bind to an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated gene 4-like (Nedd4-2) and this interaction facilitates ENaC ubiquitination followed by its degradation via the 26S proteasome. Nedd4-2 is a substrate of SGK1, and does not interact with ENaCs when phosphorylated by SGK1. As a result, these channels remain on the surface membrane, with elevated SGK1 activity facilitating Na+accumulation, and subsequently, higher blood pressure. Similarly, the SGK1/Nedd4-2 pathway can also be linked to diabetes and obesity through a soluble glucose transporter sodium glucose transporter 1 (Lang et al., 2009). Further, a genetic approach using single nucleotide polymorphism analysis reiterates the association between SGK1 and both hypertension and diabetes (Dahlberg et al., 2011). Apart from the Nedd4-2 system, SGKs also phosphorylate intracellular signaling molecules such as IκB kinases and glycogen synthase kinase 3β, and alter their downstream action (Lang et al., 2006).

SGKs in brain function: SGK1 is also expressed in the brain, and recent studies have revealed a contribution of SGK1 to physiological functions such as fear retention and learning and memory. Interestingly, neuronal SGK1 may be protective against ischemic brain injury while endothelial SGK1 in the brain microvasculature appears to exacerbate outcome after stroke (Zhang et al., 2014, 2015). Thus, as SGK1 activity appears to work in both a beneficial and detrimental manner against ischemic brain injury, and at least partly in a distribution-dependent manner, whether SGK1 should be used as a therapeutic target for specific drugs is uncertain. Furthermore, SGK1 is part of the SGK family that also contains SGK2 and SGK3, with all three family members reported to be expressed in the brain, although their detailed tissue distribution is not fully known (Lang et al., 2006). In addition, many splicing variants are recognized in all the SGK subunits, with these variants potentially having distinct substrates (Lang et al., 2006; Arteaga et al., 2008). Consequently, targeting a single subunit of the SGK family may present too many problems in developing a therapeutic strategy. Because currently available reagents for SGK1 also work on the other SGK isoforms, this convoluted situation allows us to consider investigating the effect of SGK inhibitors on ischemic brain injury. There are two commercially available SGK inhibitors, gsk650394 and EMD638683, which have been used for in vivo animal studies without any remarkable side effects (Sherk et al., 2008; Ackermann et al., 2011). Interestingly, EMD638683 even shows an effect on blood pressure by oral intake (Ackermann et al., 2011). These inhibitors may be attractive candidates for human therapeutic intervention if they are shown to work beneficially on stroke outcome in animal models.

Effect of SGK inhibitors in an ischemic brain injury model: After intracerebroventricular injection of SGK inhibitors, middle cerebral artery occlusion (MCAO) for 1 hour was performed in mice. Brain lesion volumes were examined 24 hours after reperfusion using triphenyltetrazolium chloride staining, a common procedure to evaluate brain injury. We found that both SGK inhibitors significantly reduced infarct volumes. To determine the underlying mechanism, we examined the effect of SGK inhibition on N-methyl-D-aspartate (NMDA) toxicity in cultured mouse cortical neurons. Excessive increase in intracellular Ca2+via NMDA receptors (NMDA-Rs) is a main cause of neuronal injury under oxygen and glucose-deprivation conditions, and SGKs appear to facilitate the response of NMDA-Rs via the Nedd4 system (Gautam et al., 2013). We found that SGK inhibition ameliorates NMDA-induced neurotoxicity and NMDA-R-mediated intracellular Ca2+elevation by lactate dehydrogenase-releasing toxicity assay and fluorescence Ca2+imaging using a fluorescent indicator Fluo-4, respectively. These findings suggest that the beneficial effect of SGK inhibitors on ischemic brain injury is, at least in part, mediated by inhibition of NMDA-Rs. Further, we subsequently determined using electrophysiological recordings that SGK inhibitors significantly decrease NMDA current amplitude. Additionally, SGK inhibition decreased activity of voltage-gated Na+channels, with Nedd4-2 phosphorylation diminished at the same time. This suggests that attenuation of NMDA and voltage-gated Na+currents is owing to the facilitation of their interaction with dephosphorylated Nedd4-2, which results in accelerated turnover. Of note, we also explored the effect of SGK inhibition in an MCAO model that employs a diabetic condition, and detected similar observations. This suggests that our results are also suitable for pathophysiological situations.

Overall, our studies show that SGK inhibition relieves outcome after ischemic brain injury. As mentioned, Zhang et al. (2014) antecedently reported that neuronal SGK1 protects cells in a stroke model. Assuming that SGK1 acts predominantly among SGKs, their study has a shared point of view to ours in that the role of SGKs was examined in a rodent model of transient brain ischemia. However, these two findings are likely to be confronted. We believe that variation in the detail needs to be considered, for example, species and ischemic duration. We may also have to overcome the disparity in the genetic approach and use of compounds. In addition,their study suggested that SGK1 overexpression activates Akt/protein kinase B (PKB) signaling, which may be a considerable factor regarding anti-apoptotic outcome, as this signaling pathway is regarded to be pro-survival, although at this point it is not clear how SGK1 increases Akt/PKB phosphorylation. Also, Zhang et al. (2014) focused on SGK1, while all SGK subunits were treated in our study. If both findings are taken together, while ignoring the disagreement between conditions (such as species and MCAO duration), there may be two considerable possibilities for the underlying mechanism (Figure 1). First, SGK1 is protective, while other SGKs such as SGK2 and SGK3 may be apoptotic in neurons. Indeed, activity of GluR1-containing glutamate receptor is enhanced by SGK2 and SGK3 and not by SGK1 (Lang et al., 2006), which may lead to an SGK2/3-dependent cytotoxic effect. In line with the contribution of SGKs other than SGK1, a dominant presence of SGK1.1 is also detected in the brain (Arteaga et al., 2008). The impact of SGK1.1 in the study by Zhang et al. is not clear because downstream targets between SGK1 and SGK1.1 are distinct (Zhang et al., 2014; Arteaga et al., 2008). Another possibility is that SGKs expressed in non-neuronal cells play more important roles during and/or after ischemic brain injury than those of neuronal cells. In this case, assuming that SGK activity in certain non-neuronal cells impairs brain function and overwhelms neuronal SGKs (including SGK1), SGK inhibitors would behave as shown in our study. In fact, although all SGK subunits are expressed in the brain (Lang et al., 2006), their detailed distribution and function have only been minimally examined. There are reports that designate the apparent existence of SGK1 in neurons, astrocytes, and oligodendrocytes (Lang et al., 2006). In contrast, W?rntges et al. (2002) showed the existence of SGK1 in a minor proportion of brain microglia. Indeed, in support of this, Zhang et al. (2015) identified a possible role of endothelial SGK1 in high salt-dependent exacerbation of brain ischemia. Thus, to obtain a comprehensive understanding of SGK operation in ischemic brain injury, certain areas still need to be elucidated. This resolution may contribute to the development of new therapeutic drugs not only for ischemic brain injury but also for other neurological disorders. Development of specific inhibitors for each SGK isoform might also help more profound explication, and these inhibitors may become greater candidates for future therapeutic strategies.

Figure 1 Scheme of potential interpretation of serum- and glucocorticoid-inducible kinase (SGK) action in ischemic brain injury.

Summary: Our new findings suggest that SGK activity exacerbates ischemic brain injury, and that SGK inhibitors may be useful candidates for a stroke therapeutic strategy. Although we are not clear at this point how critical the discussed experimental disparities are, they may need to be overcome to determine the effect of SGKs on ischemic brain injury. Although SGK inhibitors are still far from clinical application because of unknown and unsolved issues, such as their ability to cross the blood-brain barrier and development of non-invasive administration, it is worth investigating the further significances of these targets for future application.

Koichi Inoue*

Department of Integrative Anatomy, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

*Correspondence to: Koichi Inoue, M.D., Ph.D., ino-k@umin.ac.jp.

Accepted: 2016-08-30

orcid: 0000-0002-5481-569X (Koichi Inoue)

How to cite this article: Inoue K (2016) Possible divergence of serumand glucocorticoid-inducible kinase function in ischemic brain injury. Neural Regen Res 11(9):1396-1397.

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10.4103/1673-5374.191202