Interaction of melatonin and Bmal1 in the regulation of PI3K/AKT pathway components and cellular survival

The circadian rhythm is driven by a master clock within the suprachiasmatic nucleus which regulates the rhythmic secretion of melatonin. Bmal1 coordinates the rhythmic expression of transcriptome and regulates biological activities, involved in cell metabolism and aging. However, the role of Bmal1 in cellular- survival, signaling, its interaction with intracellular proteins, and how melatonin regulates its expression is largely unclear. Here we observed that melatonin increases the expression of Bmal1 and both melatonin and Bmal1 increase cellular survival after oxygen glucose deprivation (OGD) while the inhibition of Bmal1 resulted in the decreased cellular survival without affecting neuroprotective effects of melatonin. By using a planar surface immunoassay for PI3K/AKT signaling pathway components, we revealed that both melatonin and Bmal1 increased phosphorylation of AKT, ERK-1/2, PDK1, mTOR, PTEN, GSK-3αβ, and p70S6K. In contrast, inhibition of Bmal1 resulted in decreased phosphorylation of these proteins, which the effect of melatonin on these signaling molecules was not affected by the absence of Bmal1. Besides, the inhibition of PI3K/AKT decreased Bmal1 expression and the effect of melatonin on Bmal1 after both OGD in vitro and focal cerebral ischemia in vivo. Our data demonstrate that melatonin controls the expression of Bmal1 via PI3K/AKT signaling, and Bmal1 plays critical roles in cellular survival via activation of survival kinases.


Resuls
Melatonin increases Bmal1 protein expression. N2A cells were transfected Bmal1 overexpression plasmid or short hairpin RNA (shRNA) plasmid targeting Bmal1 (shBmal1) or their control plasmids (pAcGFP1-N1 for Bmal1 or scrambled RNA (scRNA) for shBmal1) using Lipofectamine 3000 reagent. To determine the transfection efficiency, forty-eight hours after transfection, fluorescence-activated cell sorting (FACS) analysis was performed. Results demonstrated that transfection efficiency achieved approximately 75-80 percent ( Supplementary  Fig. 1). The expression of Bmal1 protein upon transfection of appropriate plasmids was assessed by Western blot under physiological conditions. Results demonstrated that Bmal1 protein level was increased 16-fold in the overexpression group and was reduced by 60 percent in the shRNA group when compared with their appropriate controls (Fig. 1a,b). Administration of 1 µM melatonin resulted in significantly enhanced expression of Bmal1 protein in Bmal1 overexpressing or knockdown N2A cells under normoxic conditions (Fig. 1a,b) and after OGD (Fig. 1c,d).

Both Bmal1 and melatonin increase cell survival after in vitro oxygen-glucose deprivation.
To mimic focal cerebral ischemia in vitro, oxygen-glucose deprivation (OGD) method was used. After 6 hours of OGD, 18-hour-reperfusion was performed, and cell survival was analyzed. Significantly increased cell survival was observed in both Bmal1 overexpression and 1 µM melatonin administration alone groups. However, no cumulative effect on cell survival was observed when melatonin was given in the presence of Bmal1 overexpression (Fig. 1e). When Bmal1 expression was reduced with shRNA, survival rate after OGD was significantly decreased (Fig. 1f). Therefore, our results indicate that increase in Bmal1 protein level could enhance cell survival after OGD.

Figure 3.
Regulation of PI3K/AKT signaling pathways. PI3K/AKT is an intracellular signal transduction pathway that is composed of a number of signaling molecules. AKT is activated via its upstream kinase PDK1, while PTEN is a major negative regulator of AKT signaling. When phosphorylated, inhibitory effect of PTEN on PI3K/AKT is decreased. Activated AKT stimulates mTOR phosphorylation, which is a central regulator of cell metabolism, growth, proliferation and survival. Both AKT, ERK-1/2 and mTOR are negative regulators of cell death (Kilic et al., 2017). Regulation of PI3K/AKT signaling pathways in the presence (a) and absence (b) of Bmal1, and contribution of melatonin in these conditions were also summarized (c,d). www.nature.com/scientificreports www.nature.com/scientificreports/ classified. Pathway analysis was performed according to the proteomic data (Fig. 5d). The top scored pathways were Huntington disease, Parkinson disease, ATP synthesis, glycolysis, apoptosis signaling, gastrin and cholecystokinin receptors mediated signaling map, cytoskeletal regulation by Rho GTPase and ubiquitin-proteasome pathway.

Melatonin modulates Bmal1 regulation through PI3K/AKT dependent pathway after in vitro OGD.
Phosphorylated AKT protein level was diminished 25-30 percent by 0.5 µM Wortmannin treatment. It was observed that inhibition of PI3K transduction pathway did not alter the Bmal1 expression, but significantly www.nature.com/scientificreports www.nature.com/scientificreports/ reduced cell survival after OGD (Fig. 6a). Conversely, administration of 1 µM melatonin after OGD increased p-AKT and Bmal1 protein expressions which led to an increase in cell survival. Correspondingly, AKT (Thr308) phosphorylation was significantly enhanced by melatonin and this increase in AKT phosphorylation was reversed by Wortmannin (Fig. 6b).

Melatonin leads to an increase in Bmal1 protein expression after focal cerebral ischemia. Laser
Doppler Flowmetry (LDF) was used to evaluate the changes in cerebral blood flow (CBF) during and after the onset of ischemia (Fig. 7a). An eighty five percent decrease in LDF values during the occlusion with a subsequent and rapid increase with the onset of reperfusion are routinely observed in our model of ischemic stroke 11,22 . Our data demonstrated that administration of 4 mg/kg melatonin increased neuronal survival and also reduced disseminate neuronal injury in the striatum after focal cerebral ischemia (Fig. 7c,d). It is well-known that especially 4 mg/kg melatonin increases phosphorylation of AKT (Thr308) after middle cerebral artery occlusion model in mice 12,23 . Similar to the results from in vitro OGD, melatonin increased p-AKT and Bmal1 protein expression and PI3K/AKT inhibitor Wortmannin diminished their levels (Fig. 8).

Discussion
Circadian rhythms dependent on daily environmental variations regulate almost all physiological and behavioral activities within the 24-hour light/dark cycle. Activities of the biological clock are controlled by the master pacemaker located in the SCN of the anterior hypothalamus and by circadian oscillators in most peripheral tissues. At the cellular-molecular level, circadian rhythms are regulated and generated by a complex network of feedback loops which involves core clock genes, such as Clock, Bmal1, Cry1, Cry2, Per1, Per2, and Per3 5,6 . Circadian rhythms-controlled daily physiological activities such as body temperature, hormone secretion, blood pressure or energy metabolism may be involved in pathophysiological processes 17,24 . In addition, it has been revealed through several clinical studies, the circadian system also contributes to cellular injury response mechanisms after neurodegenerative disorders including Alzheimer disease, Parkinson disease, and focal cerebral ischemia 17,21,25 . In a previous study, we demonstrated for the first time that differential response to ischemic stroke occurs according to the time of the injury 11 .
Transcription factor Bmal1 and its binding partner Clock are the core components of the circadian machinery. Bmal1/Clock coordinates and regulates various cellular signaling pathways, such as metabolic pathways or signal transduction pathways 26,27 . Due to its essential contribution to the molecular signaling mechanisms after pathophysiological processes, Bmal1 is the most prominent protein among the circadian rhythms proteins. It was demonstrated that Bmal1 is important for the regulation of oxidative stress and DNA damage responses 28,29 . Yang et al. in a perspectives paper in Science Translational Medicine demonstrated that knockdown of transcription factor Bmal1 leads to loss of circadian timing which results in acceleration of aging and shortened lifespan in mice 30 . In addition, our previous study suggests that Bmal1 could strongly influence disseminate neuronal injury through AKT signaling pathway after focal cerebral ischemia 11 .
The pineal hormone melatonin regulates synchronization of the master pacemaker in the SCN which is directing all behavioral and physiological processes in a dusk and dawn manner. Previous studies in humans and rats presumed that indolamine melatonin directly influences its receptors in the SCN to control circadian timing 31,32 . However, it is a matter of debate, how melatonin acts on core clock proteins. Recent studies suggested that indolamine melatonin could regulate circadian rhythms proteins through the ubiquitin-proteasome pathway which inhibits the destruction of transcription factors 33,34 . It is predicted that inhibition of ubiquitin-proteasome pathway regulates Cry, Period and Bmal1 transcription factors (reviewed by Vriend and Reiter, 2015). Whereas the effect of Bmal1 on circadian rhythms machinery is well-documented, its role in cellular injury mechanisms is still largely unknown.
The purpose of the current study is to characterize in detail the impact of the Bmal1 on survival mechanisms after in vitro OGD. In addition to this, the relationship between Bmal1 and neuro-hormone melatonin was investigated in both physiological and ischemic conditions. Therefore, LC-MS/MS and PSI based broad-scale protein analyses were performed to investigate the effect of Bmal1 alone or combination with melatonin on intracellular signaling pathways. In this context, we have also examined the relationship between Bmal1, melatonin and PI3K signaling pathway using in vivo and in vitro ischemia.
Bmal1 protein expression was increased or inhibited in a murine Neuro2A cell line (N2A) using plasmid-mediated overexpression or shRNA mediated knockdown. Forty-eight hours after inducing overexpression/knockdown of Bmal1, we performed Western blot experiments to analyze Bmal1 protein expression. Plasmid-mediated overexpression significantly increased Bmal1 protein expression and lentiviral shRNA mediated knockdown significantly reduced Bmal1 protein expression. Both in physiological and ischemic conditions, 1 µM melatonin enhanced Bmal1 protein expression. This effect of melatonin on Bmal1 was also seen in the Bmal1 overexpressed cells. These findings support the previous studies which speculated that melatonin increases circadian rhythms protein Bmal1 in rodents 35,36 . Although Bmal1 expression was inhibited by shRNA, melatonin administration significantly increased Bmal1 protein level in both physiological and ischemic conditions. To explore the effect of Bmal1 on cell survival, OGD/R was performed which is an in vitro model that is widely used to mimic pathological changes of cerebral ischemia 37,38 . The overexpression of Bmal1 predominantly increased cell survival while lentivirus-mediated shRNA targeting Bmal1 exacerbated cell damage after in vitro OGD. One µM melatonin significantly improved cell survival but no synergistic effect was observed when melatonin was given to Bmal1 overexpressing N2A cells after OGD. While knockdown of Bmal1 protein expression decreased cell survival, this effect was reversed by the addition of melatonin.
To www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 6. Inhibition of the PI3K signaling pathway prevents melatonin mediated Bmal1 expression after in vitro OGD. Blockage of PI3K signaling pathway by Wortmannin significantly reduced cell survival (the percentage of surviving cells was determined and normalized to the control group which was not subjected to OGD) and administration of 1 µm melatonin could not reverse its effect after OGD (a). p-AKT (b) and Bmal1 (c) levels were increased by melatonin administration and decreased by Wortmannin treatment. Representative images of Western blot bands were given above their corresponding graphs. β-actin was used as a loading control. Presented data were cropped from full immunoblots for (b,c) shown in Supplementary Figs. S7 and S8. Data are mean + S.D (three independent cell culture experiments were carried out (n = 3) and n = 3 blots/protein). **p < 0.01 compared with vehicle, ## p < 0.01/compared with melatonin group.  www.nature.com/scientificreports www.nature.com/scientificreports/ downstream proteins play an important role in cell proliferation, survival, apoptosis, protein synthesis, DNA repair, as well as other cellular functions. After OGD, Bmal1 increased cell survival through PI3K/AKT signaling pathway. Bmal1 regulates the activation of AKT phosphorylation at Thr308 which is activated via its upstream kinase PDK1. Up-regulation of AKT mediates multiple cellular processes such as cell proliferation, apoptosis, glucose metabolism, and neuronal injury mechanisms after pathophysiological conditions 39,40 . ERK-1/2 is a member of the mitogen-activated protein kinase (MAPK) which regulates a broad range of cellular functions such as proliferation, DNA fragmentation, and cell survival. In addition, it also controls cellular response to stress and influences several signal transduction pathways 41 . Bmal1 strongly increased phosphorylated Erk-1/2 protein level. The function of proapoptotic Bad protein is regulated by phosphorylation on either Ser112 or Ser136 residue 42 . It is important to note that melatonin slightly decreases phosphorylation of Bad, but Bmal1 significantly increases its phosphorylation. Bmal1 regulates phosphorylation of PTEN which increases GSK-3α/β activity by inhibiting PI3K/AKT signaling cascade. Importantly, Gsk3β specifically phosphorylates Bmal1, and primes it for ubiquitylation followed by proteasomal degradation 43 . Inhibition but not overactivation of Bmal1 attenuates www.nature.com/scientificreports www.nature.com/scientificreports/ phosphorylation of PRAS40 by AKT which relieves its inhibition of mTOR. Overactivation of mTOR which regulates several major cellular processes such as cell death, inflammation, and cell cycle, phosphorylates/activates rp-S6 and phosphorylates/inhibits the activity of 4EBP1. Taken together, our PSI results demonstrated that the modulation of Bmal1 protein regulates PI3K/AKT signaling cascade.
Our co-IP coupled LC-MS/MS analysis clearly demonstrated that, for the first time, 178 different proteins were in interaction with Bmal1. According to proteomic analysis, Bmal1 particularly interacted with nucleic acid binding proteins. Furthermore, it also interacted with hydrolyse, cytoskeletal, chaperone, enzyme modulator, ligase, oxidoreductase, transcription factor, transferase, and transporter proteins. Classification of identified proteins depending on molecular activity showed that Bmal1 interacted with binding, catalytic activity, structural molecular activity, translation regulator activity, and transporter activity related proteins. PANTHER classification based on biological process included 5 predominant groups; cellular process, metabolic process, cellular component organization or biogenesis, response to stimulus, and localization. In addition to this, 8 main Bmal1-related signaling pathways were identified as Huntington disease, Parkinson disease, ATP synthesis, apoptosis, CCKR signaling, cytoskeletal regulation, glycolysis, and ubiquitin-proteasome pathway.
Among the identified proteins, ten different proteins come into prominence. Class-III intermediate filament Vimentin (Vim) is an important protein which is responsible for filament dynamics. Furthermore, Zhang and colleagues suggest that continuous light causes post-translational modification of Vim and indolamine melatonin reverts the vimentin modification to the original form 44 . Histone H1.0 (H1f0) is a member of the H1 histone family of nuclear proteins which are a component of chromatin in eukaryotic cells 45 . Splicing factor, prolineand glutamine-rich (Sfpq) controls cell growth and regulates apoptosis-related genes 46 . Elongation factor 1-delta (Eef1d) is involved in eukaryotic protein synthesis and regulates cell cycle progression 47 . Spectraplakin family consists of two main members including dystonin (Dst) and microtubule-actin cross-linking factor 1 (Macf1). Dst is also known as Bullous pemphigoid antigen 1, and has a crucial role in anterior and retrograde protein transport 48 . Macf1 has various cellular functions such as cell proliferation, migration, signaling transduction and embryonic development 48,49 . Nucleolin (Ncl), contributes to cellular homeostasis mechanisms and has several cellular functions including ribosome biogenesis 48,50 . 39S ribosomal protein L1 (Mrpl1) is required for mitochondrial protein synthesis 51 . Nucleolar RNA helicase 2 (Ddx21), is an essential regulator for DNA polymerase I and II. Its activity is controlled by post-transcriptional regulatory mechanism which determines the activity of Ddx21, modulates genome dynamics, and safeguards genome integrity 52 . It was suggested that ATP-dependent RNA helicase DDX1 (Ddx1) plays an important role in NF-κB transcriptional activity 53 .
It is well-documented that free melatonin is a radical scavenger molecule that prevents disseminate cell injury after in vivo and in vitro ischemia through PI3K/AKT signaling pathway 12,54,55 . Our study has clearly demonstrated that blockage of PI3K/AKT pathway by Wortmannin significantly reduced cell survival after OGD in vitro (Fig. 6a) and after ischemia in vivo (Fig. 7c). As expected, cell survival was significantly increased with melatonin-mediated activation of PI3K/AKT signaling pathway. While administration of melatonin significantly increased the level of Bmal1 protein, inhibition of the PI3K/AKT signaling cascade with Wortmannin was shown to inhibit the protein expression of Bmal1 slightly. When the AKT signaling pathway is blocked by Wortmannin, melatonin could not display any effect on neither cell survival nor Bmal1 protein expression after ischemia.
Based on the findings of this study, Bmal1 increases cellular survival after oxygen-glucose deprivation in N2A cells, and this was associated with increased expression of AKT, ERK-1/2 and mTOR survival pathways. More profoundly, we demonstrated that neurohormone melatonin regulates Bmal1 protein expression in physiological conditions and after OGD via the activation of PI3K/AKT signaling pathways. In addition, Bmal1-interacting proteins, which were identified using co-IP coupled LC-MS/MS analysis, suggested the interplay of several other proteins; playing roles in neurodegenerative diseases, ATP synthesis, apoptosis and glycolysis. Also, we have not observed synergistic effects of melatonin and Bmal1 with the exception of mTOR activation, which needs to be investigated further in future studies. www.nature.com/scientificreports www.nature.com/scientificreports/ 5′-GACATGAAGTCGCTGATGG-3′, and 5′-CAAATTTCCCATCTATTGC-3′) and SMARTvector non-targeting mCMV-TurboRFP were purchased from Dharmacon.

Ethics statement for animal experiments. In vivo part of this study has been conducted in
Five ×10 5 cell/well were seeded on 6-well cell culture plates (3516, Corning). Next day, transfection was performed using Lipofectamine 3000 (L3000015, Thermo Fisher Scientific) as described by the manufacturer's protocol. Briefly, 2.5 µg DNA and 5 µl Lipofectamine TM 3000 reagent and 5 µl P3000 reagent were mixed in 250 µl Opti-MEM. After 10 min incubation at room temperature, the DNA-lipid complex was added to cells. Six hours after transfection, the medium was replaced with fresh DMEM incubated at 37 °C in a moist atmosphere containing 5 percent CO 2 . Forty-eight hours after transfection, OGD was performed.

Fluorescence-activated cell sorting (FACS).
To determine transfection efficiency cells were isolated from cell culture dish and rapidly sorted utilizing a high-speed cell sorter (BD Influx cell sorter, Becton Dickinson, New Jersey, USA). green fluorescent protein (GFP) positive cells for Bmal1 overexpression or red fluorescent protein (RFP) positive cells for shBmal1 were analyzed and collected.

Oxygen-Glucose deprivation (OGD).
To mimic in-vivo ischemia, OGD was performed 48 hours after transfection. For OGD, normal culture medium was replaced with equilibrated (exposed 5% CO 2 , 95%N 2 ) no-glucose Dulbecco's Modified Eagle Medium (DMEM; 11966, Gibco). Then, plates were transferred into a hypoxia incubator chamber (27310, Stemcell Technologies) supplemented with a gas mixture composed of 1 percent O 2 , 5 percent CO 2 , 94 percent N 2 . After six hours of incubation at 37 °C, OGD medium was replaced with fresh DMEM supplemented with 10 percent FBS and cells were incubated for 18 hours for re-oxygenation in an incubator maintained in a 5 percent CO 2 atmosphere at 37 °C. At the end of the reperfusion, cells were harvested for protein analysis experiments. cell survival analysis. Immediately before inducing OGD, number of cells were counted from nine different regions of interest (ROI). Then, six-hour OGD followed by eighteen-hour reperfusion was performed. At the beginning of the reoxygenation, plates were washed once with no-glucose DMEM to remove dead and unattached cells. At the end of the reperfusion, cells were counted from nine different ROIs again. Eventually, the percentage of surviving cells was determined and normalized to the control group which was not subjected to OGD. During in vitro experiments all cell counting was performed in a blinded manner.
Western blot. Cell lysates or brain tissue samples harvested from the ischemic striatum of mice exposed to 30 min middle cerebral artery occlusion (MCAO) were pooled, homogenized and treated with Protease/ Phosphatase inhibitor cocktail (5872, Cell Signaling). Protein concentration was determined via using Qubit 3.0 Fluorometer (Q33216, Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer's protocol. Twenty micrograms of protein were size-fractionated using 4-20% Mini-PROTEAN TGX (4561096, Bio-Rad, Life Sciences Research) gel electrophoresis and then transferred to a PVDF membrane using the Trans-Blot TurboTransfer System (1704155, Bio-Rad, Life Sciences Research). Thereafter, membranes were blocked in 5% nonfat milk in 50 mMol Tris-buffered saline (TBS) containing 0.1% Tween (TBS-T; blocking solution) for 1 h at room temperature, washed in 50 mMol TBS-T, and incubated overnight with monoclonal rabbit Bmal1 (14020; Cell Signaling) or polyclonal rabbit phospho-AKT (Thr308) (9275, Cell Signaling). Following day, blots were washed with 50 mM TBS-T and incubated with horseradish peroxidase-linked goat-anti-rabbit (sc-2004; Santa Cruz Biotechnology) antibody (diluted 1:2500) for 1 h at room temperature. All of the proteins to be examined were studied as triplicate. To control protein loading PVDF membranes were stripped and re-probed with polyclonal rabbit anti-β-actin antibody (4967; Cell Signaling Technology). PVDF membranes were developed via Clarity Western ECL Substrate kit (1705060, Bio-Rad; Life Sciences Research) and visualized using the ChemiDoc MP System (1708280, Bio-Rad; Life Sciences Research). Expression level of proteins were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), corrected with values determined on β-actin blots.

Sample preparation for liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis.
Sample preparation was performed as previously described 2,11 . Tryptic peptides were generated according to the Filter Aided Sample Preparation Protocol (FASP). The brain tissues were taken from ischemic striatum and were homogenized in 50 mM ammonium bicarbonate and lysed by heating at 95 °C in UPX buffer (Expedeon). After incubation at 4 °C for an hour, samples were centrifuged at 14,000 × g for 10 min and the supernatants were transferred to a clean 1.5 ml microcentrifuge tube. The total protein concentration was measured with the Qubit assay. Tryptic peptides were generated by FASP kit (Expedeon). Briefly, 50 µg protein was filtered with 6 M urea in a 30 kDa cut-off spin column, alkylated with 10 mM iodooacetamide in the dark for 20 min at room temperature and incubated with trypsin (1:100 trypsin to protein ratio, Pierce) overnight at 37 °C. The tryptic peptides were eluted from the columns and lyophilized. The peptides were dissolved in 0.1 percent formic acid (FA, Sigma-Fluka) and diluted to 100 ng/μl before injecting to the LC-MS/MS system.

LC-MS/MS analysis and data processing.
The LC-MS/MS analysis and the subsequent protein identifications were done according to a previously published protocol 2,11,56,57 . Briefly, the tryptic peptides were loaded onto the ACQUITY UPLC M-Class coupled to a SYNAPT G2-Si high definition mass spectrometer (Waters). The columns were equilibrated with 97% mobile phase A (0.1% FA in UHPLC grade water, Merck) and temperature was set to 55 °C. Peptides were separated by a 90 min gradient elution from the trap column (Symmetry C18, 5 μm, 180 μm i.d. × 20 mm, Waters) to the analytic column (CSH C18, 1.7 μm, 75 μm i.d. × 250 mm, Waters) at 0.400 μl/min flow rate with a gradient from 4% to 40% ACN containing 0.1% FA (v/v). Positive ion modes of MS and MS/MS scans with 0.7 sec cycle time were performed sequentially. 10 V was set as low collision energy and 30 V as high CE. The ions were separated by ion mobility separation (IMS). A wave velocity was ramped from 1000 m/s to 55 m/s over the full IMS cycle. The release time for mobility trapping was set as 500 μs, trap height was set to 15 V. IMS wave delay was 1000 μs for the mobility separation after trap release 58 . Without any precursor ion preselection, all the ions within 50-1900 m/z range were fragmented in resolution mode. Additionally, 100 fmol/μl Glu-1-fibrinopeptide B was infused as lockmass reference with a 60 s interval. To identify and quantify the peptides, Progenesis-QI for proteomics software (Waters) was used. All proteins were identified by at least 3 unique peptide sequences and then, expression ratio of proteins was calculated.

Inhibition of phosphatidylinositol 3-kinase/AKT signaling pathway in vitro and in vivo.
To inhibit PI3K/AKT signaling pathway, Wortmannin (Sigma Aldrich) was used for in vitro or in vivo ischemia. For cell culture experiments, vehicle (100 percent Dimethyl sulfoxide (DMSO) in 1 µl) or 0.5 µM Wortmannin (dissolved in 1 µl 100 percent DMSO) was performed 30 min before OGD.
For animal experiments, mice were anesthetized with 1% isoflurane (30% O 2 , reminder N 2 O) and placed into a stereotaxic device (World Precision Instruments). After a small midline scalp incision, vehicle (100 percent DMSO in 2 µl) or Wortmannin (0.1 mM in 2 µl of 100 percent DMSO) was carefully injected intrastriatally (2.5 mm lateral to the sagittal suture, 0.4 mm anterior to the bregma, and 3.5 mm deep) within 5 minutes. 30 min after the delivery of vehicle or Wortmannin, MCAO was performed 12 . Animal experiments. A total 28, 8-12 weeks male C57BL6/J mice (Jackson Laboratory, Sacramento, CA and Bar Harbor, ME) were randomly assigned to one of four groups and treated with intraperitoneal (i.p.) delivery of (i) vehicle (50 μl isotonic saline/5% ethanol; n = 7), (ii) melatonin (4 mg/kg, dissolved in 0.9% isotonic saline/5% ethanol; n = 7), (iii) Wortmannin (0.1 mM in 2 µl of 100 percent DMSO), (n = 7) and (ip) melatonin (4 mg/kg, dissolved in 0.9% isotonic saline/5% ethanol)/Wortmannin (0.1 mM in 2 µl of 100 percent DMSO (injected striatally)); (n = 7) immediately after reperfusion. Focal cerebral ischemia was induced using an intraluminal filament technique 11,22 . For the induction of focal cerebral ischemia, mice were anesthetized with 1% isoflurane (30% O 2 , reminder N 2 O), and rectal temperature was controlled between 36.5 and 37.0 °C using a feedback-controlled heating system. During the experiments, cerebral blood flow (CBF) was monitored via laser Doppler flowmetry (LDF) using a flexible 0.5 mm fiber optic probe (Perimed) which was attached with tissue adhesive to the intact skull overlying the MCA territory (2 mm posterior and 6 mm lateral from the bregma). After a midline neck incision, the left common and external carotid arteries were isolated and ligated. A microvascular clip (FE691; Aesculap) was temporarily placed on the internal carotid artery. A 7-0 silicon-coated nylon monofilament (701934PK5Re, Doccol) was inserted through a small incision into the common carotid artery and advanced 9 mm distal to the carotid bifurcation for MCAO. Reperfusion was initiated 30 min after the onset of ischemia by gentle monofilament removal. Thereafter, mice were placed back into their home cages. Seventy-two hours after ischemia, animals were deeply re-anesthetized and decapitated. All experimental procedures were performed in light period of the day to prevent mice from the diurnal variation. neuronal survival analysis. Neuronal survival was analyzed as previously described 11 . Brain sections from mid-striatum level were fixed with 4% paraformaldehyde (PFA)/0.1 M phosphate-buffered saline (PBS) and incubated for 1 h with blocking buffer (0.1 M PBS containing 0.3% Triton X-100 (PBS-T)/10% normal goat serum (NGS)) at RT. Next, brain sections were reacted overnight with Alexa Fluor 488-conjugated monoclonal mouse anti-NeuN (Mab377X; Chemicon) at 4 °C. Following day, sections were counterstained with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Stained sections were analyzed under a confocal Zeiss LSM 780 microscope (Carl Zeiss). In stained sections, NeuN and DAPI positive cells were counted from 9 different region of interest (ROI), each measuring 62,500 μm 2 , in both ischemic and non-ischemic (contralateral) striatum in a