Tat-PRAS40 prevent hippocampal HT-22 cell death and oxidative stress induced animal brain ischemic insults.

Proline rich Akt substrate (PRAS40) is a component of mammalian target of rapamycin complex 1 (mTORC1) and is known to play an important role against reactive oxygen species-induced cell death. However, the precise function of PRAS40 in ischemia remains unclear. Thus, we investigated whether Tat-PRAS40, a cell-permeable fusion protein, has a protective function against oxidative stress-induced hippocampal neuronal (HT-22) cell death in an animal model of ischemia. We showed that Tat-PRAS40 transduced into HT-22 cells, and significantly protected against cell death by reducing the levels of H2O2 and derived reactive species, and DNA fragmentation as well as via the regulation of Bcl-2, Bax, and caspase 3 expression levels in H2O2 treated cells. Also, we showed that transduced Tat-PARS40 protein markedly increased phosphorylated RRAS40 expression levels and 14-3-3σ complex via the Akt signaling pathway. In an animal ischemia model, Tat-PRAS40 effectively transduced into the hippocampus in animal brain and significantly protected against neuronal cell death in the CA1 region. We showed that Tat-PRAS40 protein effectively transduced into hippocampal neuronal cells and markedly protected against neuronal cell damage. Therefore, we suggest that Tat-PRAS40 protein may be used as a therapeutic protein for ischemia and oxidative stress-induced brain disorders.

Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice.

Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E2 (PGE2) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases.

Down-regulation of MAPK/NF-κB signaling underlies anti-inflammatory response induced by transduced PEP-1-Prx2 proteins in LPS-induced Raw 264.7 and TPA-induced mouse ear edema model.

Excessive reactive oxygen species (ROS) production plays a crucial role in causing various diseases, including inflammatory disorders. The activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling is implicated in stimulating inflammatory response and cytokines. Peroxiredoxin 2 (Prx2) is a 2-cysteine (Cys) peroxiredoxin capable of removing endogenous hydrogen peroxide (H2O2). PEP-1 peptide, a protein transduction domain, consists of three domains which are used to transduce exogenous therapeutic proteins into cells. The correlation between effectively transduced PEP-1-Prx2 and ROS-mediated inflammatory response is not clear. In the present study, we investigated the protective effects of cell permeable PEP-1-Prx2 on oxidative stress-induced inflammatory activity in Raw 264.7 cells and in a mouse ear edema model after exposure to lipopolysaccharides (LPS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Transduced PEP-1-Prx2 suppressed intracellular ROS accumulation and inhibited the activity of MAPKs and NF-κB signaling that led to the suppression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and cytokines in LPS-induced Raw 264.7 cells and TPA-induced mouse ear edema model. Given these results, we propose that PEP-1-Prx2 has therapeutic potential in the prevention of inflammatory disorders.

Altered expression of adrenocorticotropic hormone in the epileptic gerbil hippocampus following spontaneous seizure.

We investigated the temporal alterations of adrenocorticotropic hormone (ACTH) immunoreactivity in the hippocampus after seizure onset. Expression of ACTH was observed within interneurons in the pre-seizure group of seizure sensitive gerbils, whereas its immunoreactivities were rarely detected in seizure resistant gerbil. Three hr after the seizure, ACTH immunoreactivity was significantly increased in interneurons within all hippocampal regions. On the basis of their localization and morphology through immunofluorescence staining, these cells were identified as GABAA α1-containing interneurons. At the 12 hr postictal period, ACTH expression in these regions was down-regulated, in a similar manner to the pre-seizure group of gerbils. These findings support the increase in ACTH synthesis that contributes to a reduction of corticotrophin-releasing factor via the negative feedback system which in turn provides an opportunity to enhance the excitability of GABAergic interneurons. Therefore, ACTH may play an important role in the reduction of excitotoxicity in all hippocampal regions.

Alterations in hyperpolarization-activated cyclic nucleotidegated cation channel (HCN) expression in the hippocampus following pilocarpine-induced status epilepticus.

To understand the effects of HCN as potential mediators in the pathogenesis of epilepsy that evoke long-term impaired excitability; the present study was designed to elucidate whether the alterations of HCN expression induced by status epilepticus (SE) is responsible for epileptogenesis. Although HCN1 immunoreactivity was observed in the hippocampus, its immunoreactivities were enhanced at 12 hrs following SE. Although, HCN1 immunoreactivities were reduced in all the hippocampi at 2 weeks, a re-increase in the expression at 2-3 months following SE was observed. In contrast to HCN1, HCN 4 expressions were un-changed, although HCN2 immunoreactive neurons exhibited some changes following SE. Taken together, our findings suggest that altered expressions of HCN1 following SE may be mainly involved in the imbalances of neurotransmissions to hippocampal circuits; thus, it is proposed that HCN1 may play an important role in the epileptogenic period as a compensatory response.

Altered PLCß-1 expression in the gerbil hippocampal complex following spontaneous seizure.

Although the phospholipase C (PLC)ß-1 isoform is associated with spontaneous seizure and distinctively expressed in the telencephalon, the distribution of PLCß-1 expression in the epileptic gerbil hippocampus remains controversial. Therefore, we determined whether PLCß-1 is associated with spontaneous seizure in an animal model of genetic epilepsy. In the present study, PLCß-1 immunoreactivity was down-regulated in seizure-sensitive (SS) gerbils more than in seizure-resistant (SR) gerbils. The expression of PLCß-1 within calretinin (CR)- positive neurons was rarely detected within the dentate hilar region of SS gerbils. PLCß-1 immunoreactivity in the hippocampus was significantly elevated as compared to that in pre-seizure SS gerbil 3 h post-ictal. These findings suggest that alterations in PLCß-1 immunoreactivity in the SS gerbil hippocampus may be closely related to the epileptic state of the gerbil brain and transiently elevated PLCß-1 protein levels following seizure episodes. Such alterations may be compensatory responses in the SS gerbil hippocampus.