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Intraperitoneal injection of JNK-specific inhibitor SP600125inhibits the expression of presenilin-1 and Notch signaling inmouse brain without induction of apoptosis
Moshiur Rahmana, Zhijie Zhanga, Avani A. Modyb, Dong-Ming Sua, Hriday K. Dasb, c, d,⁎aDepartment of Molecular Biology & Immunology, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard,Fort Worth, TX 76123, USAbDepartment of Pharmacology & Neuroscience, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard,Fort Worth, TX 76123, USAcInstitute of Cancer Research, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth,TX 76123, USAdInstitute of Aging & Alzheimer's Disease Research, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard,Fort Worth, TX 76123, USA
A R T I C L E I N F O
⁎ Corresponding author at: Department of PWorth, 3500 Camp Bowie Boulevard, Fort Wo
E-mail address: [email protected] (Abbreviations: AD, Alzheimer's disease; AP
Article history:Accepted 27 January 2012Available online 3 February 2012
Presenilin-1 (PS1) is a multifunctional protein involved in many cellular functions includingthe processing of type 1 membrane proteins such as β-amyloid precursor protein (APP) andNotch 1 receptor. PS1 acts as the catalytic subunit of the γ-secretase complex, and partici-pates 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 theexpression of the Hes1 gene. We have previously shown that inhibition of basal activity ofc-jun-NH2-terminal kinase (JNK) with JNK-specific inhibitor SP600125 represses the expres-sion of PS1 and γ-secretase activity by increasing p53 level in SK-N-SH cell line in vitro (Leeand Das, 2008, 2010). However, it is largely unknown whether PS1 can be effectively sup-pressed 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 expres-sion by increasing p53 level in adult mouse brains. We also showed that suppression of PS1expression by SP600125 reduced γ-secretase activity which decreased Notch 1 processing toreduce NICD in mouse brains. Furthermore, inhibition of Notch 1 processing by SP600125decreased Notch 1 signaling by reducing the expression of the NICD target Hes1 gene inmouse brains without induction of apoptosis. These results provide insights for further
Presenilin-1 (PS1) is a multipass transmembrane protein(Dewji et al., 2004; Li et al., 2000; Wolfe et al., 1999) and PS1mutations have been linked to early onset familial Alzheimer'sdisease (AD) (Sherrington et al., 1995; Tanzi et al., 1996). PS1 orPS2 is the catalytic subunit of γ-secretase: a multiprotein com-plex 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 ormay be involved in the structure and metabolism of the com-plex itself. PS1 or PS2 containing γ-secretase has been implicat-ed in the development of AD because of its role in the cleavageof the β-amyloid precursor protein (APP) and the production ofAβ peptide which is central to the pathogenesis of AD (DeStrooper et al., 1998). Similarly the γ-secretase-mediated pro-cessing of the Notch receptor protein, which controls cell–cellcommunication, has implicated the role of PS1 and PS2 in em-bryonic development via Notch-mediated signaling pathway(Kopan and Goate, 2000; Shen et al., 1997). Notch 1 undergoescleavage close to or within its transmembrane domain by PS1/γ-secretase to release Notch intracellular domain (NICD) to thecytoplasm (Ables et al., 2011; Artavanis-Tsakonas et al., 1999).NICD subsequently translocates to the nucleus and modifiestranscription of target genes (Ables et al., 2011; Artavanis-Tsakonas et al., 1999). One of the Notch 1 downstream targetgenes is Hes1. NICD participates in the activation of Hes1 tran-scription (Ables et al., 2011; Artavanis-Tsakonas et al., 1999).Hes1 protein is translated in the cytoplasm and then localizedin the nucleus to activate pro-neuronal genes (Ables et al.,2011). Regulation of downstream genes by NICD is calledNotch signaling. It has been shown that the deletion of the PS1gene is embryonic lethal and causes defects in brain develop-ment due to inhibition of Notch 1 signaling (Shen et al., 1997;Wong et al., 1997). PS1, PS2, and γ-secretase also cleave a varietyof other type 1 transmembrane proteins which all release intra-cellular fragments (ICD) with the ability to interact with tran-scription co-activators (Koo and Kopan, 2004; Kopan andGoate, 2000). Hence PS1 and PS2 may affect the expression ofmany genes through intramembrane proteolysis (Thinakaranand Parent, 2004). Therefore, we have studied the transcription-al control of the PS1 gene.
We have identified DNA sequences required for the ex-pression of the human PS1 gene. A promoter region hasbeen mapped in SK-N-SH cells and includes sequences from−118 to +178 flanking the major initiation site (+1) (Pastorcicand Das, 1999, 2000). The −10 Ets site controls 80% of tran-scription in SK-N-SH cells. We have previously shown thatEts transcription factors Ets1 and Ets2 bind specifically to the−10Ets element and transactivate PS1 expression in SK-N-SHcells (Lee and Das, 2008; Pastorcic and Das, 2000). p53 hasbeen shown to downregulate the expression of the endoge-nous PS1 gene (Roperch et al., 1998). We have reported previ-ously 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 cellsin vitro (Lee and Das, 2008). While it is important to studyPS1-mediated reduction of Notch 1 and APP processing for thetreatment of Alzheimer's disease, we do not know whetherSP600125would repress PS1 expression and γ-secretase activityin vivo in adult mouse brains. In this report, we now show thati.p injection of JNK-specific inhibitor SP600125 also inhibits PS1expression, γ-secretase mediated Notch 1 processing, and Notchsignaling by augmenting total p53 level in mouse brains withoutinduction of apoptosis.
2. Results
2.1. Intraperitoneal (i.p) injection of JNK-specific inhibitorSP600125 reduces phosphorylation of JNK and PS1 proteinexpression in mouse brains
JNK-specific inhibitor SP600125 binds to JNK to inhibit thephosphorylation of JNK (p-JNK) and subsequently inactivatesthe function of JNK (Bennett et al., 2001; Bogoyevitch et al.,2010). It has been reported and confirmed that intravenousor intraperitoneal injection of JNK-specific inhibitor SP600125drastically reduced JNK activity in brain extracts of C57BL/6mice and had no off target effects of SP600125 (Chen et al.,2010; Gao et al., 2005; Wang et al., 2004). To determine wheth-er basal JNK activity controls PS1 protein expression in vivo,mice were treated i.p once a day with 250 μl of vehicle (45%w/v of 2-hydroxypropyl-β-cyclodextrin in water) control and250 μl of SP600125 solution (16 mg/kg/day) respectively, forcontinuous 14 days. The maximum solubility of SP600125 inthe vehicle was determined by us to be 1.92 mg/ml. We alsodetermined that maximum 250 μl of vehicle or SP600125 solu-tion can be injected to mice without harmful effect. Conse-quently, we chose to administer maximum amount ofSP600125 (16 mg/kg/day) to each mouse. Control and treatedmice appeared to have no health problems after 14 days of ex-periments with the particular dose of SP600125 (16 mg/kg/day). Brains were removed from the animals at day 15 for per-forming immunofluorescent staining (IFS) and biochemicalanalysis.
We first examined the levels of p-JNK and PS1 in hemi-brain slices. We performed immunofluorescent staining (IFS)with p-JNK antibody and PS1 antibody on cryosections. Asshown in Fig. 1, both p-JNK (Fig. 1A) and PS1 protein (Fig. 1B)levels were reduced significantly in the brains of mice treatedwith SP600125 compared to controls. Co-immunofluorescentstaining of p-JNK and PS1 also suggested that PS1 protein ex-pression was decreased in the area of the brain accompanyingwith the reduction of p-JNK (Fig. 1C). Because IFS could notdistinguish different brain regions in detail, we generally
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Fig. 1 – Intraperitoneal administration of JNK-inhibitor (SP600125) reduced expression of p-JNK and PS-1 proteins in mousebrains. (A) Immunofluorescent staining results show a significant decrease of p-JNK in SP600125-treated mouse brain. Leftpanels show representative images of p-JNK (red) and nucleus (blue) on cryosections from mouse brain tissues; right panelshows a summary of ratios of p-JNK/DAPI positive percentage area analyzed by Image-J software from 4 mice in each group.Each symbol represents one animal. (B) Immunofluorescent staining results show a significant decrease of PS1 in SP600125-treatedmouse brain. Left panels show representative images of PS1 (red) and nucleus (blue) on cryosections from mouse brain tissues;right panel shows a summary of ratios of PS-1/DAPI positive percentage area analyzed by Image-J software from 4 mice ineach group. Each symbol represents one animal. (C) A representative co-staining images of PS1 (red) and p-JNK (green) againstcounterstaining of DAPI (blue) provides the same information as A and B single staining. Scale bar=50μm.
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looked all the regions of the brain. We could not find obviousdifference among different brain regions. To confirm our IFSdata, we carried out immunoblot analysis with protein extractsfrom vehicle-treated control and SP600125-treated mouse cor-tex because PS1-mRNA, PS1 protein, and PS1/γ-secretase activi-ty are significantly increased in the frontal cortex of late-onsetsporadic AD patients relative to controls (Borghi et al., 2010).As shown in Fig. 2, i.p injection of SP600125 reduced the levelsof p-JNK and PS1 significantly in mouse cortex but the totalamount of JNK remained unchanged.
2.2. Administration of JNK-specific inhibitor SP600125 invivo increases p53 level in mouse brains
We tested if administration of SP600125 in vivo can increasep53 protein levels in mouse brains. The results from IFSwith p53 antibody and p-JNK antibody in cryosections areshown in Fig. 3A. p53 protein level was increased morethan 2 fold in SP600125-treated mouse brains relative tovehicle-treated controls. On the contrary, p-JNK was reducedsubstantially in SP600125 treated mouse brain relative to
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Fig. 2 – Immunoblot analysis to show that intraperitoneal injection of JNK-specific inhibitor SP600125 decreases p-JNK and PS1expression in mouse cortex. (A) Control extracts (40 μg) (C1, C2, C3) from the cortex of vehicle treated 3 mice and 40 μg extracts(S1, S2, S3) from the cortex of SP600125 treated mice were subjected to immunoblot analysis with anti-p-JNK, anti-JNK, andanti-PS1. Same protein samples (C1, C2, C3, S1, S2, S3) were run on 4 separate gels (n=3). Four blots were developed bychemiluminescence and protein gel bands in four blots were quantified using Labworks Image Analysis Software and forstatistical analyses. Arrows mark the positions of p54 subunit of JNK1/3 (total JNK), as well as p54 and p46 subunits of p-JNK.Arrows also mark the position of 45 kDa full length (PS1 FL) and 20 kDa carboxyterminal fragment (PS1CTF) of mouse PS1.(B) Data were analyzed by prism software (Graphpad software Inc.) and presented as means U±USEM. Comparison was madebetween two groups by unpaired Student's t-test. A probability was considered to be significant with less than 0.05. p-JNK(sum total of p54 and p46) , and PS1CTFwere normalized with total JNK (p54). Bar graphs represent percent changes of p-JNK inSP600125-treated mice compared to vehicle-treated controls. Statistical significance was denoted by asterisks. ***P<0.002.(C) Statistical analysis is same as (B). Bar graphs represent percent changes of PS1CTF (20 kDa) in SP600125 treated micecompared to vehicle treated controls. Statistical significance was denoted by asterisks. **P<0.01.
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control (Fig. 3B). Both p-JNK and p53 proteins were localizedin the cytosol (Fig. 3B). These in vivo data are in agreementwith our published in vitro data in SK-N-SH cells (Lee andDas, 2008).
2.3. Accumulation of p53 and reduction of PS1 by SP600125do not cause apoptosis in mouse brains due to constant amountof phosphorylated-p53
JNK-specific inhibitor SP600125 was shown to accumulatenon-phosphorylated p53 (Miyamoto-Yamasaki et al., 2007).As increase of p53 and its downstream target proteins areusually involved in increase of apoptosis (Miyamoto-Yamasaki et al., 2007), we want to know whetherSP600125-induced decrease of p-JNK and PS1 is related toincrease of apoptosis in the SP600125-treated brain. Fur-thermore, PS1 is an anti-apoptotic molecule and deletionof the PS1 gene causes defects in brain development dueto neuronal apoptosis in fetus (Shen et al., 1997). In orderto test if p53 accumulation and reduction of PS1 bySP600125 are associated with apoptosis, we assessed thenumber of apoptotic cells in the brains of mice treatedwith vehicle or SP600125 by TUNEL assay. As shown inFig. 4, a similar number of apoptotic cells were detected in
the brains of mice treated with vehicle or SP600125. Activa-tion and phosphorylation of p53 is often induced by DNAdamage and apoptosis (Levine, 1997). DNA damage-induced phosphorylation of p53 occurs at multiple sites invivo, including phosphorylation at serine 15 (Ser15) and ser-ine 20 (Ser20), which lead to a reduced interaction betweenp53 and its negative regulator, the oncoprotein Mdm2(Shieh et al., 1997). p53 phosphorylation at threonine 18(Thr18) is also causally associated with p53-mediated apo-ptosis (Nakamizo et al., 2008). Therefore, we performed IFSwith phospho-p53 (p-p53) antibody (anti-Ser15) in braincryosections to check whether expression of apoptosis-related p-p53 is increased after treatment of SP600125.As shown in Fig. 5, p-p53 (Ser15) protein levels wereunchanged in the brains of mice treated with SP600125 orvehicles, and p-p53 was localized in the nucleus. On thecontrary, p53 levels were significantly increased in thebrains of mice treated with SP600125 compared to the con-trols, and p53 was localized in the cytosol (Fig. 3A). There-fore, treatment of mice with SP600125 did not increaseapoptosis because both TUNEL positive cells and p-p53were not increased in the SP60012-treated mouse brain tis-sues. This data also suggests that SP600125 reduces PS1protein expression by increasing the amount of non-
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Fig. 3 – Intraperitoneal administration of JNK-specific inhibitor (SP600125) elevated expression of total p53 protein in mousebrains. (A) Left panels: representative immunofluorescent staining results show a significant increase of total p53 (red) inSP600125-treated mouse brain tissues (low panels). Right panel: a summary of ratios of p53 (red)/DAPI (blue) positive percentagearea analyzed by Image-J software from 4mice in each group. Each symbol represents one animal. (B) A representative co-stainingimages of p53 (red) and p-JNK (green) shows increased total p53 and decreased p-JNK in the same SP600125-treated mouse braintissue (bottom panels) compared to vehicle-treated controls (top panels). Scale bar=50 μm.
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phophorylated p53 (p53) and without induction of apoptosisin mouse brains.
2.4. Administration of JNK-specific inhibitor SP600125 invivo inhibits Notch 1 processing and signaling in mouse brains
We want to determine whether inhibition of PS1 proteinexpression by SP600125 also inhibits Notch 1 processingand Notch 1 signaling in adult mouse brains without dele-terious consequences. We examined the levels of NICDand Hes1 in brain slices. We performed IFS with NICD anti-body and Hes1 antibody on cryosections of mouse braintissues. As shown in Fig. 6, both NICD and Hes1 proteinlevels were reduced drastically in the brains of mice trea-ted with SP600125. Immunoblot analysis (Fig. 7) showed
that i.p injection of SP600125 reduced the levels of NICDand Hes1 proteins in mouse cortex compared to controls.Our data also suggest that inhibition of PS1 by SP600125reduces PS1/γ-secretase activity and Notch 1 signaling inadult mouse brains without lethal effect or induction ofapoptosis.
2.5. Administration of JNK-specific inhibitor SP600125 invivo inhibits Hes1-mRNA expression in mouse brains
We performed RT-PCR to show that i.p injection of JNK-specific inhibitor SP600125 reduced the levels of Hes1-mRNAin mouse cortex compared to controls (Fig. 8). This result sug-gests that SP600125 inhibits Notch 1 signaling by decreasingthe transcription of the Hes 1 gene.
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Fig. 4 – TUNEL assay did not show significant difference of apoptotic cells between SP600125 treated and control mousebrains. Top panels: images show a representative merged fluorescent staining result of TUNEL positive cells (green) and DAPIcounterstaining (blue) on cryosections from mouse brain tissues of control and treated groups.Bottom panels: a summary ofratios of TUNEL (green)/DAPI (blue) positive percentage area analyzed by Image-J software from 3 mice in each group. Scalebar=50 μm.
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Fig. 5 – p-p53 expression did not show significant difference between SP600125 treated and control mouse brains. Top panels:images show a representative immunofluorescent staining of p-p53 (red) and DAPI (blue) on cryosections from mouse braintissues. The left panels in each sample show p-p53 (red) single staining, while the right panels in each sample show p-p53 andDAPI merger. Bottom panels: a summary of ratios p-p53 (red)/DAPI (blue) positive percentage area analyzed by Image-J softwarefrom 3 mice in each group. Scale bar=50μm.
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Fig. 6 – Inhibition of NICD and Hes1 in the brain of mouse treated with JNK-specific inhibitor SP600125. (A) Immunofluorescentstaining results show a significant decrease of NICD in SP600125-treated mouse brain tissues. Top panel shows images of arepresentative merged immunofluorescent staining of NICD (red) and DAPI (blue) on cryosections of mouse brain tissues.Bottom panel shows summarized ratios of percentage of NICD positive area versus percentage of DAPI regions analyzed by NIHsoftware image-J. Each symbol represents one animal. (B) Immunofluorescent staining results show a significant decrease ofNotch target Hes1 gene in SP600125-treated mouse brain tissues. Top panel shows images of a representative mergedimmunofluorescent staining of Hes1 (red) and DAPI (blue) on cryosections of mouse brain tissues. Bottom panel showssummarized ratios of percentage of Hes1 positive area versus percentage of DAPI regions analyzed by NIH softwareimage-J. Each symbol represents one animal. Scale bar=50 μm.
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3. Discussion
3.1. Mechanism of repression of PS1 and γ-secretase activ-ity appears to be similar both in vitro in SK-N-SH cells and invivo in mouse brains
PS1 is the catalytic subunit of the γ-secretase enzyme whichparticipates in the proteolytic cleavage of several type I mem-brane proteins including APP and Notch 1. We have shownpreviously that regulation of PS1 transcription controls γ-secretase activity (Lee and Das, 2010). We have also ascer-tained the mechanism by which inhibition of PS1 transcrip-tion reduces γ-secretase activity in SK-N-SH cells (Lee andDas, 2008, 2010). We have shown that p53 downregulates PS1transcription, PS1 protein expression, and PS1-mediated γ-secretase activity in vitro in SK-N-SH cells (Lee and Das, 2008,2010). p53 does not bind to the PS1 promoter but inhibits PS1transcription by protein–protein interaction with Ets1/Ets2transcription factors resulting in the dissociation of Ets1/Ets2from the PS1 promoter and repression of PS1 expression (Leeand Das, 2008; Pastorcic and Das, 2000). We have also shown
that inhibition of basal activity of c-jun-NH2-terminal kinase(JNK) by JNK-specific inhibitor SP600125 or JNK1-specific siRNArepresses PS1 expression and PS1-mediated γ-secretase activityby increasing the amount non-phosphorylated p53 proteinwithout increasing p53-mRNA levels and without induction ofapoptosis in vitro in SK-N-SH cells. We have shown thatSP600125-mediated inhibition of PS1 expression is very specificfor JNK pathway (Lee and Das, 2008). On the contrary, PI3K-specific inhibitor LY294002 and ERK-specific inhibitor PD98059donot inhibit PS1 expression inSK-N-SH cells ruling out the pos-sible off-target effects of SP600125 (LeeandDas, 2008). In our cur-rent study, we show that i.p injection of JNK-specific SP600125also inhibits PS1 expression and γ-secretase-mediated Notch 1processing in vivo inmouse brainswithout induction ofneuronalapoptosis and deleterious effects. Administration of SP600125augments the amount of non-phosphorylated forms of p53 pro-tein, and also reduces PS1 expression and γ-secretase activity inmouse brains. Given the correspondence between these resultsand those previously obtained with SK-N-SH cells in whichmore mechanistic experiments were possible (Lee and Das,2008) we conclude that these changes are obtained in a p53-dependent manner. Phosphorylation of p53 at serine 15 (Ser15),
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Fig. 7 – Immunoblot analysis to show that intraperitoneal injection of JNK-specific inhibitor SP600125 decreases NICD and Hes1expression in mouse cortex.(A) Immunoblot analysis shows a significant reduction of NICD and Hes1 expression in the cortexof mice treated with SP600125. Control extracts (40 μg) (C1, C2, C3) from the cortex of vehicle-treated 3 mice and 40 μg extracts(S1, S2, S3) from the cortex of SP600125-treated mice were subjected to immunoblot analysis with anti-NICD, anti-Hes1, andanti-βActin. Same protein samples (C1, C2, C3, S1, S2, S3) were run on 4 separate gels (n=3). Four blots were developed bychemiluminescence and protein gel bands in four blots were quantified using Labworks Image Analysis Software and forstatistical analyses. Positions of 80 kDa NICD, 16 kDa Hes1, and 42 kDa β-Actin are marked. (B) Data were analyzed by prismsoftware (Graphpad software Inc.) and presented as means U±U SEM. Comparisonwasmade between two groups by unpairedStudent's t-test. A probability was considered to be significant with less than 0.05. NICD was normalized with β-Actin. Bargraphs represent percent changes of NICD in SP600125 treated mice compared to vehicle treated controls. Statistical significancewas denoted by asterisks. ***P<0.003. (C) Statistical analysis is same as (C). Bar graphs represent percent changes of Hes1 inSP600125 treated mice compared to vehicle treated controls. Statistical significance was denoted by asterisks. ***P<0.002.
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threonine 18 (Thr18), and serine 20 (Ser20) is causally associatedwith p53-mediated apoptosis (Nakamizo et al., 2008; Shieh et al.,1997). Moreover, we could not detect the induction of apoptosisin mouse brains because the amount of p-p53 was unaffectedby administration of SP600125.
3.2. Inhibition of JNK by SP600125 stabilizes p53 withoutinduction of apoptosis in mouse brains
The steady state level of p53 is regulated byMdm2–ubiquitinin–proteosome degradation pathway (Morrison and Kinoshita,2000). Mdm2 is an ubiquitin ligase which binds to p53 to formMdm2–p53 complex andaddsubiquitin to p53molecule for deg-radation (Morrison and Kinoshita, 2000). Certain proteins bindto p53 and increase the stability of p53 by preventing p53from undergoing ubiquitination via interaction with Mdm2(Morrison and Kinoshita, 2000).
JNK activity determines p53 protein level (Wang andFriedman, 2000). It has been reported that JNK-specific inhibitorSP600125 can upregulate cellular p53 levels (Miyamoto-Yamasaki et al., 2007). SP600125 is an anthrapyrazolone inhibi-tor which binds to JNK to inhibit the phosphorylation and sub-sequently blocks the functional activation of JNK (Bennett etal., 2001). Activated JNK (p-JNK) catalyzes the phosphorylationat the NH2-terminus of c-jun. Phosphorylated c-jun forms
heterodimers with phosphorylated c-fos to form activatedAP-1 transcription factor which regulates the transcription ofgenes containing AP-1 binding sites in their promoters. There-fore, by binding to JNK, SP600125 inactivates the function ofJNK. Anti-sense JNK1 treatment also increased the level of p53in human fibroblast (Tafolla et al., 2005). JNK1-siRNA increasedp53 protein level in human neuroblastoma SK-N-SH cells with-out increasing p53 transcription (Lee and Das, 2008). Moreover,sustained activation of JNK1 downregulated p53 during apopto-sis (Tafolla et al., 2005). It has been reported that JNK directlybinds to p53 to form JNK–p53 complex (Fuchs et al., 1998a). Bydirect binding, JNK also targets p53 for ubiquitin-mediated deg-radation involving Mdm2–p53 degradation pathway (Fuchs etal., 1998b) Therefore, inactivation of JNK by anti-sense JNK1 orSP600125 would decrease the amount of JNK–p53 and/orMdm2–p53 complex to increase the steady state level of p53 bypreventing p53-degradation in non-stressed cells. On the otherhand, JNK also phosphorylates p53 (p-p53) resulting in p-p53 ac-cumulation in non-stressed cells (Fuchs et al., 1998b; Milne etal., 1995). The accumulated p-53 acts as an activator of genescontaining p53-response elements. On the contrary, adminis-tration of JNK-specific inhibitor SP600125 increased the totalamount of p53 (Fig. 3A) but did not alter p-p53 level in the brainsof treated mice relative to controls (Fig. 5). These data suggestthat JNK-specific inhibitor SP600125 may have increased the
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Fig. 8 – RT-PCR analysis to show that intraperitoneal injection of JNK-specific inhibitor SP600125 decreases Hes1-mRNA expressionin mouse cortex.(A) Hes1-mRNA level was measured in the cortex of mice treated with vehicle (C1, C2, C3) or SP600125 (S1, S2, S3)by RT-PCR. RT-PCR products were run on 5% plyacylamide gel and visualized by ethidium bromide staining. DNA gel bands werequantified using Labworks Image Analysis Software. RT-PCR products (C1, C2, C3, S1, S2, S3) from three different RT-PCR withsame first strand cDNA were run on 3 separate gels (n=3). DNA bands in three gels (n=3) were quantified using Labworks ImageAnalysis Software and for statistical analyses. Positions of Hes1 (309 bp), and GAPDH (599 bp) are marked. M represents DNA sizemarker. (B) Data were analyzed by prism software (Graphpad software Inc.) and presented as means U±U SEM. Comparison wasmade between two groups by unpaired Student's t-test. A probability was considered to be significant with less than 0.05. Hes1was normalized with GAPDH. Bar graphs represent percent changes of Hes1-mRNA in SP600125-treated mice compared tovehicle-treated controls. Statistical significance was denoted by asterisks. ***P<0.0012.
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steady state level of p53 by inhibiting the formation of JNK–p53and/or Mdm2–p53 complex. Therefore, accumulation of non-phophorylated p53 may be responsible for compensating theapoptotic cell deaths that would have been otherwise causedby p53-mediated inhibition of PS1 expression and Notch 1 sig-naling in the brains of mice treated with SP600125.
3.3. Inhibition of Notch signaling and APP processing withSP600125 may have beneficial effect in Alzheimer's diseasetherapy
The Notch signaling pathway is mostly regarded as a develop-mental pathway (Ables et al., 2011; Artavanis-Tsakonas et al.,1999). Notch is also a key regulator of adult neural stem cells(Ables et al., 2011). Decrease in Notch activity leads to neuro-nal stem cell (NSC) proliferation and an increased net numberof adult-born neurons because the cell exits the cell cycle anddifferentiates into neuron (Ables et al., 2011). In addition,Notch signaling plays a crucial role in regulation of migration,morphology, synaptic plasticity, and survival of mature neu-rons (Ables et al., 2011). Notch activation leads to activationof Hes genes which inhibit NGN3 expression and neurite out-growth (Ables et al., 2011). Therefore, inhibition of Notch sig-naling in adult brain leads to increase neurite outgrowth,survival of mature and immature neurons, and restore synap-tic plasticity (Ables et al., 2011). PS1/γ-secretase cleavage iscommon to both Notch signaling and APP processing. Proces-sing of Notch 1 by γ-secretase generates NICD (Ables et al.,
2011) whereas processing of APP by γ-secretase generatesAβ40 and Aβ42 peptides (Borchelt et al., 1996). Aβ42 aggregatesfaster than Aβ40 and produces amyloid plaques in the brainsof AD patients resulting in neurodegeneration and cognitivedeficits. The amount of Aβ40 in C57BL/6 wild-type mousebrain is very low. So we could not accurately determine theamount of Aβ40 in wild-type mouse brain using ELISA. SinceAβ42 level is very high in the brain of APPTg mouse (Sisodiaet al., 1990), JNK-specific inhibitor SP600125 will be tested inAPPTg mouse model of AD to determine if it reduces Aβ42 asan alternative remedy for Alzheimer's disease.
Processing of Notch was increased in brains of patientswith Alzheimer's disease compared to controls (Berezovskaet al., 1998). Thus increased Notch 1 cleavage and Notch 1 sig-naling exacerbate the pathology of Alzheimer's disease (Ableset al., 2011). Therefore, reducing γ-secretase activity by γ-secretase inhibitors was expected to control Alzheimer's dis-ease. Unfortunately, thus far, γ-secretase inhibitors have notbeen very successful as potential treatment for Alzheimer'sdisease. It has been reported that JNK is upregulated in thedegenerating neurons of Alzheimer's disease patients com-pared to controls (Zhu et al., 2001). Therefore, JNK-specificinhibitor SP600125 may potentially reduce JNK activity to pre-vent neuronal degeneration. Our current study indicates thatNotch processing and Notch signaling can be inhibited simul-taneously in adult mouse brains by peripheral administrationof JNK-specific inhibitor SP600125. SP600125 likely reduces γ-secretase activity and Notch 1 signaling in mouse brains by
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repressing PS1 transcription via increasing the accumulationof p53. Reduced PS1 expression and Notch 1 signaling byJNK-specific inhibitor should potentially result in apoptosisin mouse brains. It is possible that apoptotic cell deathscaused by p53-mediated reduction of PS1 and Notch signalingmay have been compensated by the anti-apoptotic effect ofaccumulated p53 in the brains of mice treated with SP600125.
4. Experimental procedures
4.1. Animals and experimental protocols
Three months old adult male C57BL/6 mice (Jackson Laborato-ry, Bar Harbor, Maine) weighing ~30 g were used. Mice werehoused under standardized conditions with free access to astandard chow and water. Mice were divided into two groupswith 4 animals in each group. Group 1 was vehicle control.Group 2was treatedwith JNK inhibitor SP600125 (LC laboratories,Woburn, MA). Control animals in group-1 (n=4) were given250 μl of vehicle (45% w/v of 2-hydroxypropyl-β-cyclodextrin;Sigma Aldrich, St Louis, MO) by i.p injection once a day for con-tinuous 14 days. Treated animals in group-2 (n=4) were given250 μl of SP600125 (16 mg/kg/day of SP600125 in vehicle) by i.p in-jection once a day for continuous 14 days. Mice were sacrificedon day 15. One hemi-brain from each mouse was frozen forimmunofluorecent staining (IFS). The other hemi-brain wasused for biochemical studies. For IFS brain tissueswere snap fro-zen with OCT (optimal cutting temperature) compound (Tissue-Tek-Cat. No. 4583; ThermoFisher Scientific, Atlanta, GA) at−70 °C. The frozen brain tissue was cut on sagittal plane for sec-tions by cryostat (MICROM HM 525, Thermo scientific, Atlanta,GA). All animal experiments were in compliance with the proto-cols approved by the Institutional Animal Care andUse Commit-tee of theUniversity ofNorthTexasHealth ScienceCenter at FortWorth, in accordance with guidelines of the NIH.
4.2. Immunoblot analysis
Cortex from mouse hemi-brain (n=3) was homogenized for30 s using a mechanical homogenizer with homogenizationbuffer (10 mM Tris 7.2, 150 mM NaCl, 5 mM EDTA, 1% TritonX100, 1% sodium deoxycholate, 5 mM sodium orthovanadate,50 mM NaF,) containing proteinase inhibitors. The homoge-nate was incubated for 2–3 h with shaking at 4 °C, sonicatedfor 10 s, and centrifuged at 12,000×g for 30 min. The superna-tant (protein extract) was used for determination of proteinconcentration using Biorad reagent. 40 μg of Protein extractwas mixed with equal volume 2× SDS-PAGE loading dye solu-tion containing β-mercaptoethanol and heated for 10 min at90 °C. Proteins were separated by 16% SDS-PAGE and trans-ferred to PVDF membrane at 200 mA for 3 h. The membraneswere blocked with 2% BSA in TBST (10 mM Tris 7.5, 150 mMNaCl and 0.05% Tween 20) for 2 h at room temperature fol-lowed by overnight incubation with primary antibodies at4 °C. Following antibodies were used: Anti-PS1 (Cat. No.MAB5232; Millipore, Billercia, MA), anti-phospho-SAPK/JNK(Cat. No. 9255; Cell signaling Tech, Boston, MA), anti-JNK(Cat. No. sc-474; Santa Cruz Biotech, Santa Cruz, CA), anti-activated Notch1 (Cat. No. ab8925; Abcam, Cambridge, MA),
anti-Hes1 (Cat. No. ab71559: Abcam, Cambridge, MA), andanti-βActin (Cat. No A5441; Sigma Aldrich Inc., St Louis, MO).
The blots were developed by ECL system (Pierce, IL).
Homogenates from mouse cortex (n=3) were centrifuged andcell pellets were used to prepare total RNA using trizol reagent(Invitrogen, CA) according to manufacturer protocol. Firststrand cDNA was synthesized using oligodT primers andreverse transcriptase (Invitrogen, CA). 34 cycles of PCR wereperformed with the primers for mouse Hes1 and GAPDH.250 ng of cDNA was used in PCR for Hes1 and 62.5 ng of thesame cDNA was used in PCR for GAPDH. PCR was carried outat 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s. PCR productswere run on a 5% poly-acylamide gel, stained with ehidiumbromide, and visualized under UV light. The sequences formouse Hes1 and GAPDH primers are
For immunofluorescent staining (IFS), each 10 μm-thick cryosec-tion was fixed in cold acetone, blocked with 10% donkey serumin TBST, and stained with optimum dilution of primary anti-bodies, then optimum dilution of fluorochrome-conjugated sec-ondary antibodies. Primary antibodies were anti-presenilin-1(Cat. No. MAB5232; Millipore, Billercia, MA), phospho-SAPK/JNK(Cat. No. 9255; Cell Signaling Tech, Boston, MA), anti-p53 (Cat.No. sc-98; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53 (Cat. No. 9284L; Cell Signaling Tech, Boston, MA),activated Notch1 (Cat. No. ab8925; Abcam, Cambridge, MA), andHes1 (Cat. No. ab71559: Abcam, Cambridge, MA). Fluorochrome-conjugated secondary antibodies were Cy3-conjugated donkeyanti-mouse IgG (Cat.No. 715-166-151; Jackson ImmunoResearch,Mill Valley, CA), Cy3-conjugated donkey anti-rabbit IgG (Cat. No.711-166-152; Jackson Immuno Research, Mill Valley, CA), andAlexa-Fluor-488-conjugated chicken anti-goat IgG (Cat. No.A21467; Invitrogen, Carlsbad, CA). Antibody-stained immu-nofluorescent samples were mounted by anti-fading aqueousmounting medium containing 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI) and covered by cover slips. The magnifi-cation indicated in each figure shows that of the objective lensin Nikon Eclipse Ti-U fluorescentmicroscope. The ratio of % pos-itive staining areas versus % DAPI regions was analyzed by NIHsoftware image-J.
4.5. TUNEL assay
For TUNEL assay, each 10 μm-thick cryosection was fixed in4% paraformaldehyde, permeabilized with 0.1% TritonX-100and pH 7.2. Terminal transferase reactions (containing TdTand fluorescein-dUTP) were then performed with the in situCell Death Detection Kit (Cat. No. 11684795910; Roche, SanFrancisco, CA) for the TUNEL assay. Labeled samples weremounted by anti-fading aqueous mounting medium
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containing DAPI and covered by cover slips. The magnificationin the figures shows that of the objective lens in Nikon EclipseTi-U fluorescent microscope.
4.6. Statistical analysis for IFS and TUNEL assays
For IFS and TUNEL assays, the statistical significance betweenany two groups was analyzed by unpaired Student's t-test. If theF-test comparisonof variancewas less than0.05 (i.e.nonparamet-ric distribution), the unpaired t-test with Welch's correction wasused. Differences were considered statistically significant atvalues of p<0.05. Allmeasures of variance are presented as SEMs.
Acknowledgment
This work was partially supported by NIA/NIH grant(R21AG031880) to Dr. Dong-Ming Su and research supportfrom Graduate School of Biomedical Sciences of UNTHSC toDr. Hriday K. Das.
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