P27 Protects Neurons from Ischemic Damage by Suppressing Oxidative Stress and Increasing Autophagy in the Hippocampus

Abstract

p27Kip1 (p27), a well-known cell regulator, is involved in the regulation of cell death and survival. In the present study, we observed the effects of p27 against oxidative stress induced by H2O2 in HT22 cells and transient ischemia in gerbils. Tat (trans-acting activator of transcription) peptide and p27 fusion proteins were prepared to facilitate delivery into cells and across the blood-brain barrier. The tat-p27 fusion protein, rather than its control protein Control-p27, was delivered intracellularly in a concentration and incubation time-dependent manner and showed its activity in HT22 cells. The localization of the delivered Tat-p27 protein was also confirmted in the HT22 cells and hippocampus in gerbils. In addition, the optimal concentration (5 μM) of Tat-p27 was determined to protect neurons from cell death induced by 1 mM H2O2. Treatment with 5 μM Tat-p27 significantly ameliorated H2O2-induced DNA fragmentation and the formation of reactive oxygen species (ROS) in HT22 cells. Tat-p27 significantly mitigated the increase in locomotor activity a day after ischemia and neuronal damage in the hippocampal CA1 region. It also reduced the ischemia-induced membrane phospholipids and ROS formation. In addition, Tat-p27 significantly increased microtubule-associated protein 1A/1B light chain 3A/3B expression and ameliorated the H2O2 or ischemia-induced increases of p62 and decreases of beclin-1 in the HT22 cells and hippocampus. These results suggest that Tat-p27 protects neurons from oxidative or ischemic damage by reducing ROS-induced damage and by facilitating the formation of autophagosomes in hippocampal cells.

Tat-Endophilin A1 Fusion Protein Protects Neurons from Ischemic Damage in the Gerbil Hippocampus: A Possible Mechanism of Lipid Peroxidation and Neuroinflammation Mitigation as Well as Synaptic Plasticity

“…In in vivo study, we observed the Tat-SH3GL2, not Control-SH3GL2, could cross the blood-brain barrier and be delivered to the cells in the hippocampal CA1 region. This result is consistent with previous studies that showed that Tat-cargo fusion proteins could cross the membrane and had the potential to be introduced into cells or the hippocampus [17,40,41,43]. In addition, we also observed the neuroprotective effects of Tat-SH3GL2 against 1 mM H 2 O 2 -induced oxidative damage in HT22 cells by WST-1 assay.…”

“…Mongolian gerbils (male, 3-month old) were purchased from Japan SLC Inc. (Shizuoka, Japan) and the experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Seoul National University, Seoul, Korea (SNU-190516-7). Transient forebrain ischemia in gerbils was induced by occlusion of both the common carotid arteries for 5 min under anesthesia with 2.5% isoflurane (Hana Pharm Co., Ltd., Hwaseong, South Korea), as described in previous studies [40,41]. Complete occlusion of both the arteries was confirmed by observing the central artery of the retina using an ophthalmoscope (HEINE K180 ® , Heine Optotechnik, Herrsching, Germany).…”

“…Five sections per animal that were obtained from regions separated from each other by at least 120 µm, were selected. Immunohistochemical staining for NeuN to detect mature neurons in the hippocampus was performed as described in previous studies [17,40,41,43]. Briefly, antibodies used in the present study were mouse anti-NeuN antibody (1:1000; EMD Millipore, Temecula, CA, USA) and biotinylated goat anti-mouse IgG (1:200; Vector,).…”

“…The number of NeuN-immunoreactive neurons was counted using OPTIMAS 6.5 software (CyberMetrics Corporation, Phoenix, AZ, USA) as described previously [41,43]. In addition, polyhistidine and endophilin A1 immunoreactivity was assessed based on its optical density and pixel numbers using ImageJ v. 1.80 software (National Institutes of Health, Bethesda, MD, USA), as described previously [40]. Data are presented as percent averages with standard deviation compared to the control group (which was set as 100%) and differences in averages were statistically analyzed by a one-way or two-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test using GraphPad Prism 5.01 software (GraphPad Software, Inc., La Jolla, CA, USA).…”

Neuroprotective Effects of Purpurin Against Ischemic Damage via Anti-inflammatory and MAPK Pathway

“…Murine hippocampal HT22 cells were obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modi ed Eagle's medium as described in previous studies [20,21]. Purpurin was dissolved in 200-mM dimethyl sulfoxide (DMSO) and various concentrations of purpurin (1-200 μM) were added to HT22 cells for 60 min.…”