Elsevier

Brain Research

Volume 1448, 11 April 2012, Pages 117-128
Brain Research

Research Report
Intraperitoneal injection of JNK-specific inhibitor SP600125 inhibits the expression of presenilin-1 and Notch signaling in mouse brain without induction of apoptosis

https://doi.org/10.1016/j.brainres.2012.01.066Get rights and content

Abstract

Presenilin-1 (PS1) is a multifunctional protein involved in many cellular functions including the processing of type 1 membrane proteins such as β-amyloid precursor protein (APP) and Notch 1 receptor. PS1 acts as the catalytic subunit of the γ-secretase complex, and participates in Notch 1 processing to release Notch intracellular domain (NICD) in the cytoplasm. NICD subsequently migrates to the nucleus and causes Notch signaling by increasing the expression of the Hes1 gene. We have previously shown that inhibition of basal activity of c-jun-NH2-terminal kinase (JNK) with JNK-specific inhibitor SP600125 represses the expression of PS1 and γ-secretase activity by increasing p53 level in SK-N-SH cell line in vitro (Lee and Das, 2008, 2010). However, it is largely unknown whether PS1 can be effectively suppressed in vivo in adult mouse brains. In this report we showed that intraperitoneal (i.p) injection of JNK-specific inhibitor SP600125 decreased p-JNK level, and reduced PS1 expression by increasing p53 level in adult mouse brains. We also showed that suppression of PS1 expression by SP600125 reduced γ-secretase activity which decreased Notch 1 processing to reduce NICD in mouse brains. Furthermore, inhibition of Notch 1 processing by SP600125 decreased Notch 1 signaling by reducing the expression of the NICD target Hes1 gene in mouse brains without induction of apoptosis. These results provide insights for further study on PS1-mediated reduction of Notch 1 and APP processing for the treatment of Alzheimer's disease.

Highlights

► JNK inhibitor SP600125 decreased p-JNK and PS1 in the mouse brain similar to what this laboratory has previously reported in cell culture system. ► Concurrently, i.p. injection of SP600125 increased the level of p53 in mouse brain. ► Suppression of PS1 expression by SP600125 reduced processing of Notch to NICD and expression of the NICD target, Hes1. ► The SP600125-induced changes observed in mouse brain did not correlate with induction of apoptosis.

Introduction

Presenilin-1 (PS1) is a multipass transmembrane protein (Dewji et al., 2004, Li et al., 2000, Wolfe et al., 1999) and PS1 mutations have been linked to early onset familial Alzheimer's disease (AD) (Sherrington et al., 1995, Tanzi et al., 1996). PS1 or PS2 is the catalytic subunit of γ-secretase: a multiprotein complex that has also been implicated in the development of AD (Chyung et al., 2005, De Strooper, 2003, Kimberly and Wolfe, 2003, Takasugi et al., 2003). PS1 and PS2 act as a catalyst or may be involved in the structure and metabolism of the complex itself. PS1 or PS2 containing γ-secretase has been implicated in the development of AD because of its role in the cleavage of the β-amyloid precursor protein (APP) and the production of Aβ peptide which is central to the pathogenesis of AD (De Strooper et al., 1998). Similarly the γ-secretase-mediated processing of the Notch receptor protein, which controls cell–cell communication, has implicated the role of PS1 and PS2 in embryonic development via Notch-mediated signaling pathway (Kopan and Goate, 2000, Shen et al., 1997). Notch 1 undergoes cleavage close to or within its transmembrane domain by PS1/γ-secretase to release Notch intracellular domain (NICD) to the cytoplasm (Ables et al., 2011, Artavanis-Tsakonas et al., 1999). NICD subsequently translocates to the nucleus and modifies transcription of target genes (Ables et al., 2011, Artavanis-Tsakonas et al., 1999). One of the Notch 1 downstream target genes is Hes1. NICD participates in the activation of Hes1 transcription (Ables et al., 2011, Artavanis-Tsakonas et al., 1999). Hes1 protein is translated in the cytoplasm and then localized in the nucleus to activate pro-neuronal genes (Ables et al., 2011). Regulation of downstream genes by NICD is called Notch signaling. It has been shown that the deletion of the PS1 gene is embryonic lethal and causes defects in brain development due to inhibition of Notch 1 signaling (Shen et al., 1997, Wong et al., 1997). PS1, PS2, and γ-secretase also cleave a variety of other type 1 transmembrane proteins which all release intracellular fragments (ICD) with the ability to interact with transcription co-activators (Koo and Kopan, 2004, Kopan and Goate, 2000). Hence PS1 and PS2 may affect the expression of many genes through intramembrane proteolysis (Thinakaran and Parent, 2004). Therefore, we have studied the transcriptional control of the PS1 gene.

We have identified DNA sequences required for the expression of the human PS1 gene. A promoter region has been mapped in SK-N-SH cells and includes sequences from − 118 to + 178 flanking the major initiation site (+ 1) (Pastorcic and Das, 1999, Pastorcic and Das, 2000). The − 10 Ets site controls 80% of transcription in SK-N-SH cells. We have previously shown that Ets transcription factors Ets1 and Ets2 bind specifically to the − 10Ets element and transactivate PS1 expression in SK-N-SH cells (Lee and Das, 2008, Pastorcic and Das, 2000). p53 has been shown to downregulate the expression of the endogenous PS1 gene (Roperch et al., 1998). We have reported previously that p53 inhibits PS1 transcription without binding to the PS1 promoter (Lee and Das, 2008, Pastorcic and Das, 2000). We also showed that c-jun-NH2-terminal kinase (JNK)-specific inhibitor SP600125 repressed PS1 expression and γ-secretase activity by augmenting p53 level in SK-N-SH cells in vitro (Lee and Das, 2008). While it is important to study PS1-mediated reduction of Notch 1 and APP processing for the treatment of Alzheimer's disease, we do not know whether SP600125 would repress PS1 expression and γ-secretase activity in vivo in adult mouse brains. In this report, we now show that i.p injection of JNK-specific inhibitor SP600125 also inhibits PS1 expression, γ-secretase mediated Notch 1 processing, and Notch signaling by augmenting total p53 level in mouse brains without induction of apoptosis.

Section snippets

Intraperitoneal (i.p) injection of JNK-specific inhibitor SP600125 reduces phosphorylation of JNK and PS1 protein expression in mouse brains

JNK-specific inhibitor SP600125 binds to JNK to inhibit the phosphorylation of JNK (p-JNK) and subsequently inactivates the function of JNK (Bennett et al., 2001, Bogoyevitch et al., 2010). It has been reported and confirmed that intravenous or intraperitoneal injection of JNK-specific inhibitor SP600125 drastically reduced JNK activity in brain extracts of C57BL/6 mice and had no off target effects of SP600125 (Chen et al., 2010, Gao et al., 2005, Wang et al., 2004). To determine whether basal

Mechanism of repression of PS1 and γ-secretase activity appears to be similar both in vitro in SK-N-SH cells and in vivo in mouse brains

PS1 is the catalytic subunit of the γ-secretase enzyme which participates in the proteolytic cleavage of several type I membrane proteins including APP and Notch 1. We have shown previously that regulation of PS1 transcription controls γ-secretase activity (Lee and Das, 2010). We have also ascertained the mechanism by which inhibition of PS1 transcription reduces γ-secretase activity in SK-N-SH cells (Lee and Das, 2008, Lee and Das, 2010). We have shown that p53 downregulates PS1 transcription,

Animals and experimental protocols

Three months old adult male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) weighing ~ 30 g were used. Mice were housed under standardized conditions with free access to a standard chow and water. Mice were divided into two groups with 4 animals in each group. Group 1 was vehicle control. Group 2 was treated with JNK inhibitor SP600125 (LC laboratories, Woburn, MA). Control animals in group-1 (n = 4) were given 250 μl of vehicle (45% w/v of 2-hydroxypropyl-β-cyclodextrin; Sigma Aldrich, St

Acknowledgment

This work was partially supported by NIA/NIH grant (R21AG031880) to Dr. Dong-Ming Su and research support from Graduate School of Biomedical Sciences of UNTHSC to Dr. Hriday K. Das.

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