Tat-Cannabinoid Receptor Interacting Protein Reduces Ischemia-Induced Neuronal Damage and Its Possible Relationship with 14-3-3η

Abstract

Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the C-terminal domain of cannabinoid 1 receptor (CB1R) and regulates CB1R activities. In this study, we made Tat-CRIP1a fusion proteins to enhance CRIP1a penetration into neurons and brain and to evaluate the function of CRIP1a in neuroprotection following oxidative stress in HT22 hippocampal cells and transient forebrain ischemia in gerbils. Purified exogenous Tat-CRIP1a was penetrated into HT22 cells in a time and concentration-dependent manner and prevented H2O2-induced reactive oxygen species formation, DNA fragmentation, and cell damage. Tat-CRIP1a fusion protein also ameliorated the reduction of 14-3-3η expression by H2O2 treatment in HT22 cells. Ischemia–reperfusion damage caused motor hyperactivity in the open field test of gerbils; however, the treatment of Tat-CRIP1a significantly reduced hyperactivity 1 day after ischemia. Four days after ischemia, the administration of Tat-CRIP1a restored the loss of pyramidal neurons and decreased reactive astrocytosis and microgliosis induced by ischemic damage in the hippocampal cornu Ammonis (CA)1 region. Ischemic damage decreased 14-3-3η expression in all hippocampal sub-regions 4 days after ischemia; however, the treatment of Tat-CRIP1 ameliorated the reduction of 14-3-3η expression. These results suggest that Tat-CRIP1a attenuates neuronal damage and hyperactivity induced by ischemic damage, and it restores normal expression levels of 14-3-3η protein in the hippocampus.

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

“…Tat ( trans -acting activator of transcription) was discovered in human immunodeficiency virus-1, and it is widely used to deliver proteins, DNA phages, and liposomes intracellularly [ 22 , 23 , 24 ]. In addition, Tat-cargo fusion proteins are successfully delivered into the hippocampus [ 25 ], and they show neuroprotective effects against ischemic damage [ 26 , 27 ]. Several studies have demonstrated that the Tat-p27 fusion protein is efficiently delivered to cardiomyocytes, and treatment with Tat-p27 protects cardiomyocytes from myocardial infarction [ 17 , 18 ].…”

Tat-malate dehydrogenase fusion protein protects neurons from oxidative and ischemic damage by reduction of reactive oxygen species and modulation of glutathione redox system

“…Transformed proteins were harvested after disruption with 5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9) containing 6 M urea. Proteins were purified using Ni 2+ -nitrilotriacetic acid Sepharose affinity column (Qiagen, Valencia, CA, USA) and PD-10 column chromatography, and the purified protein was identified by western blotting for His-tag, as previously described 24,25 .…”

“…Thereafter, the membranes were incubated with mouse anti-His-tag (1:1,000; Abcam, Cambridge, UK) and mouse anti-β-actin (1:5,000; Cell Signaling, Danvers, MA, USA). After washing the membranes, the protein bands were visualized using chemiluminescent reagents as previously described 25,26 .…”

“…To confirm the in vivo delivery of proteins, animals (n = 5 per group) were intraperitoneally injected with 1 mg/kg Tat-MDH1 or MDH1 and anesthetized with 5% isoflurane (Baxtor, Deerfield, IL, USA). Transcardiac perfusion was performed for morphological study 6 h after ischemia/reperfusion, and both hippocampi were quickly removed for immunohistochemical staining for His-tag as previously described 25,26 . Animals were anesthetized with 5% isoflurane and transcardiac perfusion was performed via the left ventricular injection of 0.1 M phosphate-buffered saline (PBS) and 4% paraformaldehyde in 0.1 M PBS.…”

“…Oxidative stress was induced by incubation with 200 μM hydrogen peroxide (H 2 O 2 ) for 1 h and Tat-MDH1 was simultaneously treated with H 2 O 2 in HT22 cells to assess the protective effects and optimal concentration of proteins using a water-soluble tetrazolium salt (WST-1) assay kit. In addition, surviving cells were visualized by reaction with 1 μM 5-carboxyfluorescein diacetate acetoxymethyl ester (5-CFDA AM) (Invitrogen, Carlsbad, CA, USA) with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI; Roche Applied Science, Mannheim, Germany) for 15 min at 37 °C after exposure to the optimal concentration of proteins, as previously described 25,26 .…”