Therapeutic Options of Crystallin Mu and Protein Disulfide Isomerase A3 for Cuprizone-Induced Demyelination in Mouse Hippocampus.

This study investigates the changes in hippocampal proteomic profiles during demyelination and remyelination using the cuprizone model. Employing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry for protein profiling, we observed significant alterations in the expression of ketimine reductase mu-crystallin (CRYM) and protein disulfide isomerase A3 precursor (PDIA3) following exposure to and subsequent withdrawal from cuprizone. Immunohistochemical staining validated these protein expression patterns in the hippocampus, revealing that both PDIA3 and CRYM were downregulated in the hippocampal CA1 region during demyelination and upregulated during remyelination. Additionally, we explored the potential protective effects of CRYM and PDIA3 against cuprizone-induced demyelination by synthesizing cell-permeable Tat peptide-fusion proteins (Tat-CRYM and Tat-PDIA3) to facilitate their crossing through the blood-brain barrier. Our results indicated that administering Tat-CRYM and Tat-PDIA3 mitigated the reduction in proliferating cell and differentiated neuroblast counts compared to the group receiving cuprizone alone. Notably, Tat-PDIA3 demonstrated significant effects in enhancing myelin basic protein expression alongside phosphorylation of CREB in the hippocampus, suggesting its potential therapeutic role in the prevention or treatment of demyelination, and by extension, in conditions such as multiple sclerosis.

Cu,Zn-Superoxide Dismutase has Minimal Effects Against Cuprizone-Induced Demyelination, Microglial Activation, and Neurogenesis Defects in the C57BL/6 Mouse Hippocampus.

Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.

Comparison of the Effects of Cuprizone on Demyelination in the Corpus Callosum and Hippocampal Progenitors in Young Adult and Aged Mice.

Cuprizone is commonly used to induce neuronal demyelination in mice. In the present study, we compared the cuprizone-induced demyelination in the corpus callosum and investigated the effects of cuprizone on proliferating cells and neuroblasts in the dentate gyrus of young adult and aged mice. 5-week- and 23-month-old mice were fed a normal diet or a 0.2% cuprizone-enriched diet for 5 weeks. Mice fed a cuprizone-supplemented diet showed a significant reduction in myelin basic protein-positive structures in the corpus callosum, with the reduction in myelinated fibers being confirmed by electron microscopic analysis. In addition, we observed a marked increase in Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts in young adult mice in response to cuprizone treatment, although not in aged mice, as the basal levels of these cells were significantly lower in these older mice. Furthermore, Ser133-phosphorylated cAMP response element-binding protein (pCREB)-positive nuclei and brain-derived neurotrophic factor (BDNF) protein levels were significantly reduced in young adult mice following cuprizone treatment in young adult, although again not in the aged mice. However, in both young adult and aged mice, there were no significant reductions in hippocampal mature neurons in response to cuprizone treatment. These observations indicate that in the mice of both age groups a cuprizone-supplemented diet contributes to an increase in demyelination in the corpus callosum and neural progenitor cells in the dentate gyrus, although the damage is more pronounced in young adult mice. This demyelination and reduction in neural progenitor cells may be associated with changes in the levels of BDNF and pCREB in the dentate gyrus.

Tat-p27 Ameliorates Neuronal Damage Reducing α-Synuclein and Inflammatory Responses in Motor Neurons After Spinal Cord Ischemia.

p27Kip1 (p27) regulates the cell cycle by inhibiting G1 progression in cells. Several studies have shown conflicting results on the effects of p27 against cell death in various insults. In the present study, we examined the neuroprotective effects of p27 against H2O2-induced oxidative stress in NSC34 cells and against spinal cord ischemia-induced neuronal damage in rabbits. To promote delivery into NSC34 cells and motor neurons in the spinal cord, Tat-p27 fusion protein and its control protein (Control-p27) were synthesized with or without Tat peptide, respectively. Tat-p27, but not Control-27, was efficiently introduced into NSC34 cells in a concentration- and time-dependent manner, and the protein was detected in the cytoplasm. Tat-p27 showed neuroprotective effects against oxidative stress induced by H2O2 treatment and reduced the formation of reactive oxygen species, DNA fragmentation, and lipid peroxidation in NSC34 cells. Tat-p27, but not Control-p27, ameliorated ischemia-induced neurological deficits and cell damage in the rabbit spinal cord. In addition, Tat-p27 treatment reduced the expression of α-synuclein, activation of microglia, and release of pro-inflammatory cytokines such as interleukin-1ß and tumor necrosis factor-α in the spinal cord. Taken together, these results suggest that Tat-p27 inhibits neuronal damage by decreasing oxidative stress, α-synuclein expression, and inflammatory responses after ischemia.

Physical Stress Induced Reduction of Proliferating Cells and Differentiated Neuroblasts Is Ameliorated by Fermented Laminaria japonica Extract Treatment.

Laminaria japonica is widely cultivated in East Asia, including South Korea. Fucoidan, a main component of L. japonica, protects neurons from neurological disorders such as ischemia and traumatic brain injury. In the present study, we examined the effects of extract from fermented L. japonica on the reduction of proliferating cells and neuroblasts in mice that were physically (with electric food shock) or psychologically (with visual, auditory and olfactory sensation) stressed with the help of a communication box. Vehicle (distilled water) or fermented L. japonica extract (50 mg/kg) were orally administered to the mice once a day for 21 days. On the 19th day of the treatment, physical and psychological stress was induced by foot shock using a communication box and thereafter for three days. Plasma corticosterone levels were significantly increased after exposure to physical stress and decreased Ki67 positive proliferating cells and doublecortin immunoreactive neuroblasts. In addition, western blot analysis demonstrated that physical stress as well as psychological stress decreased the expression levels of brain-derived neurotrophic factor (BDNF) and the number of phosphorylated cAMP response element binding protein (pCREB) positive nuclei in the dentate gyrus. Fermentation of L. japonica extract significantly increased the contents of reduced sugar and phenolic compounds. Supplementation with fermented L. japonica extract significantly ameliorated the increases of plasma corticosterone revels and decline in the proliferating cells, neuroblasts, and expression of BDNF and pCREB in the physically stressed mice. These results indicate that fermented L. japonica extract has positive effects in ameliorating the physical stress induced reduction in neurogenesis by modulating BDNF and pCREB expression in the dentate gyrus.

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

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.

Phosphoglycerate Mutase 1 Prevents Neuronal Death from Ischemic Damage by Reducing Neuroinflammation in the Rabbit Spinal Cord.

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that increases glycolytic flux in the brain. In the present study, we examined the effects of PGAM1 in conditions of oxidative stress and ischemic damage in motor neuron-like (NSC34) cells and the rabbit spinal cord. A Tat-PGAM1 fusion protein was prepared to allow easy crossing of the blood-brain barrier, and Control-PGAM1 was synthesized without the Tat peptide protein transduction domain. Intracellular delivery of Tat-PGAM1, not Control-PGAM1, was achieved in a time- and concentration-dependent manner. Immunofluorescent staining confirmed the intracellular expression of Tat-PGAM1 in NSC34 cells. Tat-PGAM1, but not Control-PGAM1, significantly alleviated H2O2-induced oxidative stress, neuronal death, mitogen-activated protein kinase, and apoptosis-inducing factor expression in NSC34 cells. After ischemia induction in the spinal cord, Tat-PGAM1 treatment significantly improved ischemia-induced neurological impairments and ameliorated neuronal cell death in the ventral horn of the spinal cord 72 h after ischemia. Tat-PGAM1 treatment significantly mitigated the ischemia-induced increase in malondialdehyde and 8-iso-prostaglandin F2α production in the spinal cord. In addition, Tat-PGAM1, but not Control-PGAM1, significantly decreased microglial activation and secretion of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α induced by ischemia in the ventral horn of the spinal cord. These results suggest that Tat-PGAM1 can be used as a therapeutic agent to reduce spinal cord ischemia-induced neuronal damage by lowering the oxidative stress, microglial activation, and secretion of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α.

Pyridoxine Deficiency Exacerbates Neuronal Damage after Ischemia by Increasing Oxidative Stress and Reduces Proliferating Cells and Neuroblasts in the Gerbil Hippocampus.

We investigated the effects of pyridoxine deficiency on ischemic neuronal death in the hippocampus of gerbil (n = 5 per group). Serum pyridoxal 5'-phosphate levels were significantly decreased in Pyridoxine-deficient diet (PDD)-fed gerbils, while homocysteine levels were significantly increased in sham- and ischemia-operated gerbils. PDD-fed gerbil showed a reduction in neuronal nuclei (NeuN)-immunoreactive neurons in the medial part of the hippocampal CA1 region three days after. Reactive astrocytosis and microgliosis were found in PDD-fed gerbils, and transient ischemia caused the aggregation of activated microglia in the stratum pyramidale three days after ischemia. Lipid peroxidation was prominently increased in the hippocampus and was significantly higher in PDD-fed gerbils than in Control diet (CD)-fed gerbils after ischemia. In contrast, pyridoxine deficiency decreased the proliferating cells and neuroblasts in the dentate gyrus in sham- and ischemia-operated gerbils. Nuclear factor erythroid-2-related factor 2 (Nrf2) and brain-derived neurotrophic factor (BDNF) levels also significantly decreased in PDD-fed gerbils sham 24 h after ischemia. These results suggest that pyridoxine deficiency accelerates neuronal death by increasing serum homocysteine levels and lipid peroxidation, and by decreasing Nrf2 levels in the hippocampus. Additionally, it reduces the regenerated potentials in hippocampus by decreasing BDNF levels. Collectively, pyridoxine is an essential element in modulating cell death and hippocampal neurogenesis after ischemia.

Phosphoglycerate Mutase 1 Promotes Cell Proliferation and Neuroblast Differentiation in the Dentate Gyrus by Facilitating the Phosphorylation of cAMP Response Element-Binding Protein.

In a previous study, we observed a significant increase in phosphoglycerate mutase 1 (PGAM1) levels after pyridoxine treatment. In the present study, we investigated the effects of PGAM1 on novel object recognition, cell proliferation, and neuroblast differentiation in the dentate gyrus. We generated a Tat-PGAM1 fusion protein to cross the blood-brain barrier and neuronal plasma membrane. We administered the Tat peptide, control-PGAM1, or Tat-PGAM1 fusion protein to 8-week-old mice once a day for 3 weeks and tested novel object recognition memory. The mice were then euthanized to conduct western blot analysis for polyhistidine expression and immunohistochemical analysis for Ki67, doublecortin, and phosphorylated cAMP response element-binding protein. Mice treated with Tat peptide showed similar exploration times for familiar and new objects and the discrimination index was significantly lower in this group than in the control group. Tat-PGAM1 moderately increased the exploration time of new objects when compared to familiar objects, while the discrimination index was significantly higher in the Tat-PGAM1-treated group, but not in the control-PGAM1-treated group, when compared with the control group. Higher PGAM1 protein expression was observed in the hippocampus of Tat-PGAM1-treated mice when compared with the hippocampi of control, Tat peptide-, and control-PGAM1-treated mice, using western blot analysis. In addition, the numbers of proliferating cells and differentiated neuroblasts were significantly lower in the Tat peptide-treated group than in the control group. In contrast, the numbers of proliferating cells and differentiated neuroblasts in the dentate gyrus were higher in the Tat-PGAM1-treated group than in the control group. Administration of Tat-PGAM1 significantly facilitated the phosphorylation of cAMP response element-binding protein in the dentate gyrus. Administration of control-PGAM1 did not show any significant effects on novel object recognition, cell proliferation, and neuroblast differentiation in the dentate gyrus. These results suggest that PGAM1 plays a role in cell proliferation and neuroblast differentiation in the dentate gyrus via the phosphorylation of cAMP response element-binding protein in the hippocampus.

Leaf extracts from Dendropanax morbifera Léveille mitigate mercury-induced reduction of spatial memory, as well as cell proliferation, and neuroblast differentiation in rat dentate gyrus.

BACKGROUND: The brain is susceptible to methylmercury toxicity, which causes irreversible damage to neurons and glia and the leaf extract Dendropanax morbifera Léveille (DML) has various biological functions in the nervous system. In this study, we examined the effects of DML on mercury-induced proliferating cells and differentiated neuroblasts. METHODS: Dimethylmercury (5 µg/kg) and galantamine (5 mg/kg) was administered intraperitoneally and/or DML (100 mg/kg) was orally to 7-week-old rats every day for 36 days. One hour after the treatment, novel object recognition test was examined. In addition, spatial probe tests were conducted on the 6th day after 5 days of continuous training in the Morris swim maze. Thereafter, the rats were euthanized for immunohistochemical staining analysis with Ki67 and doublecortin and measurement for acetylcholinesterase (AChE) activity. RESULTS: Dimethylmercury-treated rats showed reduced discrimination index in novel object recognition test and took longer to find the platform than did control group. Compared with dimethylmercury treatment alone, supplementation with DML or galatamine significantly ameliorated the reduction of discrimination index and reduced the time spent to find the platform. In addition, the number of platform crossings was lower in the dimethylmercury-treated group than in controls, while the administration of DML or galantamine significantly increased the number of crossings than did dimethylmercury treatment alone. Proliferating cells and differentiated neuroblasts, assessed by Ki67 and doublecortin immunohistochemical staining was significantly decreased in the dimethylmercury treated group versus controls. Supplementation with DML or galantamine significantly increased the number of proliferating cells and differentiated neuroblasts in the dentate gyrus. In addition, treatment with dimethylmercury significantly increased AChE activity in hippocampal homogenates, while treatment with dimethylmercury+DML or dimethylmercury+galantamine significantly ameliorated this increase. CONCLUSIONS: These results suggest that DML may be a functional food that improves dimethylmercury-induced memory impairment and ameliorates dimethylmercury-induced reduction in proliferating cells and differentiated neuroblasts, and demonstrates corresponding activation of AChE activity in the dentate gyrus.

PFKFB3 ameliorates ischemia-induced neuronal damage by reducing reactive oxygen species and inhibiting nuclear translocation of Cdk5.

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) plays an essential role in glycolysis and in the antioxidant pathway associated with glutathione. Therefore, we investigated the effects of PFKFB3 on oxidative and ischemic damage. We synthesized a fusion protein of transactivator of transcription (Tat)-PFKFB3 to facilitate its passage into the intracellular space and examine its effects against oxidative stress induced by hydrogen peroxide (H2O2) treatment and ischemic damage caused by occlusion of the common carotid arteries for 5 min in gerbils. The Tat-PFKFB3 protein was efficiently delivered into HT22 cells in a concentration- and time-dependent manner, with higher levels observed 18 h after treatment. Furthermore, treatment with 6 µM Tat-PFKFB3 demonstrated intracellular delivery into HT22 cells, as analyzed through immunocytochemical staining. Moreover, it significantly ameliorated the reduction of cell viability induced by 200 µM H2O2 treatment. Tat-PFKFB3 treatment also alleviated H2O2-induced DNA fragmentation and reactive oxygen species formation in HT22 cells. In gerbils, the intraperitoneal administration of 2 mg/kg Tat-PFKFB3 efficiently delivered the substance to all hippocampal areas, including the hippocampal CA1 region. This administration significantly mitigated ischemia-induced hyperlocomotion, long-term memory deficits, and ischemic neuronal death in the hippocampal CA1 region after ischemia. Additionally, treatment with 2 mg/kg Tat-PFKFB3 significantly ameliorated the translocation of Cdk5 from the cytosol to the nucleus in the hippocampal CA1 region 24 h after ischemia, but not in other regions. The treatment also significantly reduced reactive oxygen species formation in the CA1 region. These findings suggest that Tat-PFKFB3 reduces neuronal damage in the hippocampal CA1 region after ischemia through the reduction of Cdk5 signaling and reactive oxygen species formation. Therefore, Tat-PFKFB3 may have potential applications in reducing ischemic damage.

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

Malate dehydrogenase (MDH) plays an important role in the conversion of malate to oxaloacetate during the tricarboxylic acid cycle. In this study, we examined the role of cytoplasmic MDH (MDH1) in hydrogen peroxide (H2O2)-induced oxidative stress in HT22 cells and ischemia-induced neuronal damage in the gerbil hippocampus. The Tat-MDH1 fusion protein was constructed to enable the delivery of MDH1 into the intracellular space and penetration of the blood-brain barrier. Tat-MDH1, but not MDH1 control protein, showed significant cellular delivery in HT22 cells in a concentration- and time-dependent manner and gradual intracellular degradation in HT22 cells. Treatment with 4 µM Tat-MDH1 significantly ameliorated 200 µM H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation in HT22 cells. Transient increases in MDH1 immunoreactivity were detected in the hippocampal CA1 region 6-12 h after ischemia, but MDH1 activity significantly decreased 2 days after ischemia. Supplementation of Tat-MDH1 immediately after ischemia alleviated ischemia-induced hyperlocomotion and neuronal damage 1 and 4 days after ischemia. In addition, treatment with Tat-MDH1 significantly ameliorated the increases in hydroperoxides, lipid peroxidation, and reactive oxygen species 2 days after ischemia. Tat-MDH1 treatment maintained the redox status of the glutathione system in the hippocampus 2 days after ischemia. These results suggest that Tat-MDH1 exerts neuroprotective effects by reducing oxidative stress and maintaining glutathione redox system in the hippocampus.

Tat-heat shock protein 10 ameliorates age-related phenotypes by facilitating neuronal plasticity and reducing age-related genes in the hippocampus.

We investigated the effects of heat shock protein 10 (HSP10) protein on memory function, hippocampal neurogenesis, and other related genes/proteins in adult and aged mice. To translocate the HSP10 protein into the hippocampus, the Tat-HSP10 fusion protein was synthesized, and Tat-HSP10, not HSP10, was successfully delivered into the hippocampus based on immunohistochemistry and western blotting. Tat-HSP10 (0.5 or 2.0 mg/kg) or HSP10 (control protein, 2.0 mg/kg) was administered daily to 3- and 21-month-old mice for 3 months, and observed the senescence maker P16 was significantly increased in aged mice and the treatment with Tat-HSP10 significantly decreased P16 expression in the hippocampus of aged mice. In novel object recognition and Morris water maze tests, aged mice demonstrated decreases in exploratory preferences, exploration time, distance moved, number of object contacts, and escape latency compared to adult mice. Treatment with Tat-HSP10 significantly improved exploratory preferences, the number of object contacts, and the time spent swimming in the target quadrant in aged mice but not adults. Administration of Tat-HSP10 increased the number of proliferating cells and differentiated neuroblasts in the dentate gyrus of adult and aged mice compared to controls, as determined by immunohistochemical staining for Ki67 and doublecortin, respectively. Additionally, Tat-HSP10 treatment significantly mitigated the reduction in sirtuin 1 mRNA level, N-methyl-D-aspartate receptor 1, and postsynaptic density 95 protein levels in the hippocampus of aged mice. In contrast, Tat-HSP10 treatment significantly increased sirtuin 3 protein levels in both adult and aged mouse hippocampus. These suggest that Tat-HSP10 can potentially reduce hippocampus-related aging phenotypes.

Purpurin ameliorates D-galactose-induced aging phenotypes in mouse hippocampus by reducing inflammatory responses.

Purpurin, an anthraquinone, has potent anti-oxidant and anti-inflammatory effects in various types of brain damage. In a previous study, we showed that purpurin exerts neuroprotective effects against oxidative and ischemic damage by reducing pro-inflammatory cytokines. In the present study, we investigated the effects of purpurin against D-galactose-induced aging phenotypes in mice. Exposure to 100 mM D-galactose significantly decreased cell viability in HT22 cells, and purpurin treatment significantly ameliorated the reduction of cell viability, formation of reactive oxygen species, and lipid peroxidation in a concentration-dependent manner. Treatment with 6 mg/kg purpurin significantly improved D-galactose-induced memory impairment in the Morris water maze test in C57BL/6 mice and alleviated the reduction of proliferating cells and neuroblasts in the subgranular zone of the dentate gyrus. In addition, purpurin treatment significantly mitigated D-galactose-induced changes of microglial morphology in the mouse hippocampus and the release of pro-inflammatory cytokines such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In addition, purpurin treatment significantly ameliorated D-galactose-induced phosphorylation of c-Jun N-terminal kinase and cleavage of caspase-3 in HT22 cells. These results suggest that purpurin can delay aging by reducing the inflammatory cascade and phosphorylation of the c-Jun N-terminal in the hippocampus.

CHIP ameliorates neuronal damage in H2O2-induced oxidative stress in HT22 cells and gerbil ischemia.

Carboxyl terminus of Hsc70-interacting protein (CHIP) is highly conserved and is linked to the connection between molecular chaperones and proteasomes to degrade chaperone-bound proteins. In this study, we synthesized the transactivator of transcription (Tat)-CHIP fusion protein for effective delivery into the brain and examined the effects of CHIP against oxidative stress in HT22 cells induced by hydrogen peroxide (H2O2) treatment and ischemic damage in gerbils by 5 min of occlusion of both common carotid arteries, to elucidate the possibility of using Tat-CHIP as a therapeutic agent against ischemic damage. Tat-CHIP was effectively delivered to HT22 hippocampal cells in a concentration- and time-dependent manner, and protein degradation was confirmed in HT22 cells. In addition, Tat-CHIP significantly ameliorated the oxidative damage induced by 200 µM H2O2 and decreased DNA fragmentation and reactive oxygen species formation. In addition, Tat-CHIP showed neuroprotective effects against ischemic damage in a dose-dependent manner and significant ameliorative effects against ischemia-induced glial activation, oxidative stress (hydroperoxide and malondialdehyde), pro-inflammatory cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) release, and glutathione and its redox enzymes (glutathione peroxidase and glutathione reductase) in the hippocampus. These results suggest that Tat-CHIP could be a therapeutic agent that can protect neurons from ischemic damage.

Phosphoglycerate kinase 1 protects against ischemic damage in the gerbil hippocampus.

Phosphoglycerate kinase 1 (PGK1) is a metabolic enzyme that converts 1,3-diphosphoglycerate to 3-phosphoglycerate. In the current study, we synthesized a PEP-1-PGK1 fusion protein that can cross the blood-brain barrier and cell membrane, and the effects of PEP-1-PGK1 against oxidative stress were investigated HT22 cells and ischemic gerbil brain. The PEP-1-PGK1 protein and its control protein (Con-PGK1) were treated and permeability was evaluated HT22 cells. The PEP-1-PGK1 was introduced into HT22 cells depending on its concentration and incubation time and was gradually degraded over 36 h after treatment. PEP-1-PGK1, but not Con-PGK1, significantly ameliorated H2O2-induced cell damage and reactive oxygen species formation in HT22 cells. Additionally, PEP-1-PGK1, but not Con-PGK1, mitigated ischemia-induced hyperlocomotion 1 d after ischemia and 4 d after ischemia of neuronic cell death. PEP-1-PGK1 treatment significantly alleviated the raised lactate and succinate dehydrogenase activities in the early (15 min to 6 h) and late (4 and 7 d) stages of ischemia, respectively. In addition, PEP-1-PGK1 treatment ameliorated the decrease in ATP and pH levels in the late stage (2-7 d) of ischemia. Nuclear factor erythroid-2-related factor 2 (Nrf2) levels accelerated the ischemia-induced increase in the hippocampus 1 d after ischemia after PEP-1-PGK1 treatment. Neuroprotective and ameliorative effects were prominent at a low concentration (0.1 mg/kg), but not at a high concentration (1 mg/kg), of PEP-1-PGK1. Collectively, low concentrations of PEP-1-PGK1 prevented neuronal stress by increasing energy production.

Neuroprotective Effects of Purpurin Against Ischemic Damage via MAPKs, Bax, and Oxidative Stress Cascades in the Gerbil Hippocampus.

Purpurin has various effects, including anti-inflammatory effects, and can efficiently cross the blood-brain barrier. In the present study, we investigated the effects of purpurin on oxidative stress in HT22 cells and mild brain damage in the gerbil hippocampal CA1 region induced by transient forebrain ischemia. Oxidative stress induced by H2O2 was significantly ameliorated by treatment with purpurin, based on changes in cell death, DNA fragmentation, formation of reactive oxygen species, and pro-apoptotic (Bax)/anti-apoptotic (Bcl-2) protein levels. In addition, treatment with purpurin significantly reduced the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK), and p38 signaling in HT22 cells. Transient forebrain ischemia in gerbils led to a significant increase in locomotor activity 1 day after ischemia and significant decrease in number of surviving cells in the CA1 region 4 days after ischemia. Administration of purpurin reduced the travel distance 1 day after ischemia and abrogates the neuronal death in the hippocampal CA1 region 4 days after ischemia based on immunohistochemical and histochemical staining for NeuN and Fluoro-Jade C, respectively. Purpurin treatment significantly decreased the activation of microglia and astrocytes as well as the increases of nuclear factor kappa-light-chain-enhancer of activated B cells p65 in the hippocampal CA1 region 4 days after ischemia and ameliorated the ischemia-induced transient increases of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the hippocampus 6 h after ischemia. In addition, purpurin significantly alleviated the ischemia-induced phosphorylation of JNK, ERK, and p38 in the hippocampus 1 day after ischemia. Furthermore, purpurin treatment significantly mitigated the increases of Bax in the hippocampus 1 day after ischemia and the lipid peroxidation based on malondialdehyde and hydroperoxides levels 2 days after ischemia. These results suggest that purpurin can be one of the potential candidates to reduce neuronal damage and inflammatory responses after oxidative stress in HT22 cells or ischemic damage in gerbils.

The neuroprotective effects of phosphoglycerate mutase 5 are mediated by decreasing oxidative stress in HT22 hippocampal cells and gerbil hippocampus.

Phosphoglycerate mutase 5 (PGAM5), a glycolytic enzyme, plays an important role in cell death and regulation of mitochondrial dynamics. In this study, we investigated the effects of PGAM5 on oxidative stress in HT22 hippocampal cells and ischemic damage in the gerbil hippocampus to elucidate the role of PGAM5 in oxidative and ischemic stress. Constructs were designed with a PEP-1 expression vector to facilitate the intracellular delivery of PGAM5 proteins. We observed time- and concentration-dependent increases in the intracellular delivery of the PEP-1-PGAM5 protein, but not its control protein (PGAM5), in HT22 cells, and morphologically demonstrated the localization of the transduced protein, which was stably expressed in the cytoplasm after 12 h of PEP-1-PGAM5 treatment. PEP-1-PGAM5 treatment significantly ameliorated cell death, reactive oxygen species formation, DNA fragmentation, and the reduction of cell proliferation induced by H2O2 treatment in HT22 cells. In addition, PEP-1-PGAM5 was effectively delivered to the gerbil hippocampus 8 h after treatment, and ischemia-induced hyperlocomotion and neuronal death in the hippocampal CA1 region were significantly alleviated 1 and 4 days after ischemia, respectively. Ischemia-induced microglial activation was also mitigated by treatment with 1.0 mg/kg PEP-1-PGAM5. At 3 h after ischemia, PEP-1-PGAM5 treatment significantly ameliorated the increase in lipid peroxidation, as assessed by malondialdehyde and hydroperoxide levels, and decreased glutathione levels (increases in glutathione disulfide, the oxidized form of glutathione) in the hippocampus. Two days after ischemia, treatment with PEP-1-PGAM5 significantly alleviated the ischemia-induced reduction in glutathione peroxidase activity and further increased superoxide dismutase activity in the hippocampus. The neuroprotective effects of PEP-1-PGAM5 are partially mediated by a reduction in oxidative stress, such as the formation of reactive oxygen species, and increases in the activity of antioxidants such as glutathione peroxidase and superoxide dismutase.

Gynura procumbens Root Extract Ameliorates Ischemia-Induced Neuronal Damage in the Hippocampal CA1 Region by Reducing Neuroinflammation.

Gynura procumbens has been used in Southeast Asia for the treatment of hypertension, hyperglycemia, and skin problems induced by ultraviolet irradiation. Although considerable studies have reported the biological properties of Gynura procumbens root extract (GPE-R), there are no studies on the effects of GPE-R in brain damages, for example following brain ischemia. In the present study, we screened the neuroprotective effects of GPE-R against ischemic damage and neuroinflammation in the hippocampus based on behavioral, morphological, and biological approaches. Gerbils received oral administration of GPE-R (30 and 300 mg/kg) every day for three weeks and 2 h after the last administration, ischemic surgery was done by occlusion of both common carotid arteries for 5 min. Administration of 300 mg/kg GPE-R significantly reduced ischemia-induced locomotor hyperactivity 1 day after ischemia. Significantly more NeuN-positive neurons were observed in the hippocampal CA1 regions of 300 mg/kg GPE-R-treated animals compared to those in the vehicle-treated group 4 days after ischemia. Administration of GPE-R significantly reduced levels of pro-inflammatory cytokines such as interleukin-1ß, -6, and tumor necrosis factor-α 6 h after ischemia/reperfusion. In addition, activated microglia were significantly decreased in the 300 mg/kg GPE-R-treated group four days after ischemia/reperfusion compared to the vehicle-treated group. These results suggest that GPE-R may be one of the possible agents to protect neurons from ischemic damage by reducing inflammatory responses.

Changes in the expression of the B subunit of vacuolar H+-ATPase, in the hippocampus, following transient forebrain ischemia in gerbils.

Objectives: Vacuolar H+-ATPase is a highly conserved enzyme that plays an important role in maintaining an acidic environment for lysosomal function and accumulating neurotransmitters in synaptic vesicles. In the present study, we investigated the time-dependent changes in the expression of vacuolar H+-ATPase V1B2 (ATP6V1B2), a major neuronal subtype of vacuolar H+-ATPase located in the hippocampus, after 5 min of transient forebrain ischemia in gerbils. We also examined the pH and lactate levels in the hippocampus after ischemia to elucidate the correlation between ATP6V1B2 expression and acidosis. Materials and Methods: Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 5 min and animals were sacrificed at various time points after ischemia for immunohistochemical staining of ATP6V1B2 and measurements of pH and lactate levels in the hippocampus. Results: ATP6V1B2 immunoreactivity was found to be transiently increased in the hippocampal CA1 region and dentate gyrus 12-24 hr after ischemia when the pH and lactate levels were decreased. In addition, ATP6V1B2 immunoreactivity significantly increased in the hippocampal CA3 and dentate gyrus, regions relatively resistant to ischemic damage, 4 days after ischemia, when the NeuN-positive, mature neuron numbers were significantly decreased in the hippocampal CA1 region. Conclusion: These results suggest that ATP6V1B2 expression is transiently increased in the hippocampus following ischemia, which may be intended to compensate for ischemia-related dysfunction of ATP6V1B2 in the hippocampus.