Song Hee Lee

Late treatment with choline alfoscerate (l-alpha glycerylphosphorylcholine, α-GPC) increases hippocampal neurogenesis and provides protection against seizure-induced neuronal death and cognitive impairment

Choline alfoscerate (α-GPC) is a common choline compound and acetylcholine precursor in the brain, which has been shown to be effective in the treatment of Alzheimers disease and dementia. α-GPC has been shown to enhance memory and cognitive function in stroke and Alzheimers patients but currently remains untested in patients suffering from epilepsy. This study aimed to evaluate whether α-GPC treatment after seizure can ameliorate seizure-induced cognitive impairment and neuronal injury. The potential therapeutic effects of α-GPC on seizure-induced cognitive impairment were tested in an animal model of pilocarpine-induced seizure. Seizures were induced by intraperitoneal injection of pilocarpine (25mg/kg) in male rats. α-GPC (250mg/kg) was injected into the intramuscular space once daily for one or three weeks from immediately after seizure, or from 3 weeks after the seizure onset for 3 weeks. Here we found that immediate 1-week treatment of α-GPC showed no neuroprotective effects and neurogenesis. Immediate 3-week treatment of α-GPC showed neuroprotective effect but no effect on neurogenesis. To evaluate the effect of late treatment of α-GPC on cognitive impairment following seizure, rats were injected α-GPC from 3 weeks after seizure for 3 weeks and subjected to a water maze test. In the present study, we found that administration of α-GPC starting at 3 weeks after seizure improved cognitive function through reduced neuronal death and BBB disruption, and increased neurogenesis. Therefore, α-GPC injection may serve as a beneficial treatment for improvement of cognitive function in epilepsy patients.

Zinc plus cyclo-(His-Pro) promotes hippocampal neurogenesis in rats

Zinc is a central actor in regulating stem cell proliferation and neurogenesis in the adult brain. High levels of vesicular zinc are found in the presynaptic terminals. It has been demonstrated that high levels of vesicular zinc are localized in the presynaptic terminals of the granule cells of the dentate gyrus (DG) and that neurogenesis occurs in the subgranular zone (SGZ). Furthermore, zinc chelation reduces hippocampal neurogenesis in pathological conditions such as hypoglycemia, epilepsy and traumatic brain injury. Here we test the effects of zinc plus cyclo-(His-Pro) (CHP) treatment on neurogenesis in the adult SGZ. In order to increase brain zinc, Sprague-Dawley (SD) rats, aged 5weeks, were given zinc plus CHP (ZC, 27mg/kg) orally available once per day for 2weeks. BrdU was intraperitoneally injected 2 times per day for 4 consecutive days starting 1week after initial ZC treatment. Neurogenesis was analyzed by BrdU, Ki67 and doublecortin (DCX) immunostaining. The number of progenitor cells and immature neurons were significantly increased in the DG following 2weeks of ZC treatment. Hippocampal vesicular zinc content was evaluated with TSQ staining. Vesicular TSQ fluorescent intensity was seen to increase in the mossy fiber area at 2weeks after ZC treatment. The present study demonstrates that zinc supplementation by ZC treatment increases hippocampal neurogenesis and levels of vesicular zinc. These findings provide evidence in support of the essential role of zinc in modulating hippocampal neurogenesis.

Zinc transporter 3 (ZnT3) gene deletion reduces spinal cord white matter damage and motor deficits in a murine MOG-induced multiple sclerosis model

The present study aimed to evaluate the role of zinc transporter 3 (ZnT3) on multiple sclerosis (MS) pathogenesis. Experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis, was induced by immunization with myelin oligodendrocyte glycoprotein (MOG35-55) in female mice. Three weeks after the initial immunization, demyelination, immune cell infiltration and blood brain barrier (BBB) disruption in the spinal cord were analyzed. Clinical signs of EAE first appeared on day 11 and reached a peak level on day 19 after the initial immunization. ZnT3 gene deletion profoundly reduced the daily clinical score of EAE. The ZnT3 gene deletion-mediated inhibition of the clinical course of EAE was accompanied by suppression of inflammation and demyelination in the spinal cord. The motor deficit accompanying neuropathological changes associated with EAE were mild in ZnT3 gene deletion mice. This reduction in motor deficit was accompanied by coincident reductions in demyelination and infiltration of encephalitogenic immune cells including CD4+ T cells, CD8+ T cells, CD20+ B cells and F4/80+ microglia in the spinal cord. These results demonstrate that ZnT3 gene deletion inhibits the clinical features and neuropathological changes associated with EAE. ZnT3 gene deletion also remarkably inhibited formation of EAE-associated aberrant synaptic zinc patches, matrix metalloproteinases-9 (MMP-9) activation and BBB disruption. Therefore, amelioration of EAE-induced clinical and neuropathological changes by ZnT3 gene deletion suggests that vesicular zinc may be involved in several steps of MS pathogenesis.

Melatonin Reduces Hypoglycemia-Induced Neuronal Death in Rats.

Melatonin, N-aceyl-5-methoxytryptamine, is the main secretory product of the pineal gland and has neuroprotective effects on several brain injuries, including ischemic stroke. In the present study, we hypothesized that exogenous melatonin may decrease hypoglycemia-induced neuronal death through the prevention of superoxide generation. To test our hypothesis, hypoglycemia was induced by injecting human insulin (10 U/kg, i.p.) in rats. Melatonin injection was started immediately after hypoglycemia (10 mg/kg, i.p.). The first melatonin injection was performed at the end of a 30-min isoelectric EEG period. The second and third injections were administered at 1 and 3 h after the first injection. Reactive oxygen species generation, as detected by dihydroethidium staining, was significantly reduced by melatonin treatment. Neuronal injury was reduced by the treatment of melatonin in the hippocampal CA1 and dentate granule cells. Microglia activation was robust in the hippocampus after hypoglycemia, which was almost completely prevented by melatonin treatment. Hypoglycemia-induced cognitive impairment was also significantly prevented by melatonin treatment. The present study suggests that melatonin has therapeutic potential to prevent hypoglycemia-induced brain injury.

Decreased cysteine uptake by EAAC1 gene deletion exacerbates neuronal oxidative stress and neuronal death after traumatic brain injury

Excitatory amino acid carrier type 1 (EAAC1), a high-affinity glutamate transporter, can expend energy to move glutamate into neurons. However, under normal physiological conditions, EAAC1 does not have a great effect on glutamate clearance but rather participates in the neuronal uptake of cysteine. This process is critical to maintaining neuronal antioxidant function by providing cysteine for glutathione synthesis. Previous study showed that mice lacking EAAC1 show increased neuronal oxidative stress following transient cerebral ischemia. In the present study, we sought to characterize the role of EAAC1 in neuronal resistance after traumatic brain injury (TBI). Young adult C57BL/6 wild-type or EAAC1−/− mice were subjected to a controlled cortical impact model for TBI. Neuronal death after TBI showed more than double the number of degenerating neurons in the hippocampus in EAAC1−/− mice compared with wild-type mice. Superoxide production, zinc translocation and microglia activation similarly showed a marked increase in the EAAC1−/− mice. Pretreatment with N-acetyl cysteine (NAC) reduced TBI-induced neuronal death, superoxide production and zinc translocation. These findings indicate that cysteine uptake by EAAC1 is important for neuronal antioxidant function and survival following TBI. This study also suggests that administration of NAC has therapeutic potential in preventing TBI-induced neuronal death.

The cancer chemotherapeutic agent paclitaxel (Taxol) reduces hippocampal neurogenesis via down-regulation of vesicular zinc

Chemotherapy-induced cognitive impairment (CICI) is increasingly recognized as a major unwanted side effect of an otherwise highly valuable life-saving technology. In part, this awareness is a result of increased cancer survival rates following chemotherapy. Altered hippocampal neurogenesis may play a role in mediating CICI. In particular, zinc could act as a key regulator of this process. To test this hypothesis, we administered paclitaxel (Px) to male C57BL/6 mice for set time periods and then evaluated the effects of Px treatment on hippocampal neurogenesis and vesicular zinc. We found that vesicular zinc levels and expression of zinc transporter 3 (ZnT3) were reduced in Px-treated mice, compared to vehicle-treated mice. Moreover, Px-treated mice demonstrated a significant decrease in the number of neuroblasts present. However, no difference in the number of progenitor cells were observed. In addition, zinc supplementation by treatment with ZnCl2 ameliorated the Px-induced decrease in hippocampal neurogenesis and cognitive impairment. These results suggest that via disruption of vesicular zinc stores in hippocampal mossy fiber terminals, chemotherapy may impinge upon one or more of the sequential stages involved in the maturation of new neurons derived via adult neurogenesis and thereby leads to the progressive cognitive decline associated with CICI.

Administration of Protocatechuic Acid Reduces Traumatic Brain Injury-Induced Neuronal Death

Protocatechuic acid (PCA) was first purified from green tea and has shown numerous biological activities, including anti-apoptotic, anti-inflammatory, and anti-atherosclerotic effects. The effect of PCA on traumatic brain injury (TBI)-induced neuronal death has not previously been evaluated. TBI is defined as damage to the brain resulting from external mechanical force, such as rapid acceleration or deceleration, impact, blast waves, or penetration by a projectile. TBI causes neuronal death in the hippocampus and cerebral cortex. The present study aimed to evaluate the therapeutic potential of PCA on TBI-induced neuronal death. Here, TBI was induced by a controlled cortical impact model using rats. PCA (30 mg/kg) was injected into the intraperitoneal (ip) space immediately after TBI. Neuronal death was evaluated with Fluoro Jade-B (FJB) staining at 24 h after TBI. Oxidative injury was detected by 4-hydroxy-2-nonenal (4HNE), glutathione (GSH) concentration was analyzed by glutathione adduct with N-ethylmaleimide (GS-NEM) staining at 24 h after TBI, and microglial activation in the hippocampus was detected by CD11b immunohistochemistry at one week after TBI. We found that the proportion of degenerating neurons, oxidative injury, GSH depletion, and microglia activation in the hippocampus and cortex were all reduced by PCA treatment following TBI. Therefore, our study suggests that PCA may have therapeutic potential in preventing TBI-induced neuronal death.

Prevention of hypoglycemia-induced hippocampal neuronal death by N-acetyl-L-cysteine (NAC).

Type 1 and type 2 diabetic patients who are treated with insulin or other blood glucose reducing agents for tight control of blood glucose levels are frequently at risk of experiencing severe hypoglycemia which can lead to seizures, loss of consciousness and death. Hypoglycemic neuronal cell death is not a simple result of low glucose supply to the brain, but, instead, results from a cell death signaling pathway that is started by the re-administration of glucose after glucose deprivation. Zinc is a biologically important element for physiological function of central nervous system. However, excessive zinc release from the presynaptic terminals and subsequent translocation into the postsynaptic neurons may contribute to neuronal death following hypoglycemia. N-acetyl-l-cysteine (NAC) acts as a zinc chelator that alleviates zinc-induced neuronal death processes. In addition, NAC restores levels of neuronal glutathione (GSH), a potent antioxidant, by providing a cell-permeable source of cysteine. Thus, we hypothesized that NAC treatment can reduce neuronal cell death, not only by increasing GSH concentration but also by zinc chelation. As a result, we found that NAC decreased the oxidative stress, zinc release and translocation, and improved the level of glutathione. Therefore, NAC administration alleviated hippocampal neuron death in hypoglycemia-induced rats.

Administration of Zinc plus Cyclo-(His-Pro) Increases Hippocampal Neurogenesis in Rats during the Early Phase of Streptozotocin-Induced Diabetes

The effects of zinc supplementation on hippocampal neurogenesis in diabetes mellitus have not been studied. Herein, we investigated the effects of zinc plus cyclo-(His-Pro) (ZC) on neurogenesis occurring in the subgranular zone of dentate gyrus after streptozotocin (STZ)-induced diabetes. ZC (27 mg/kg) was administered by gavage once daily for one or six weeks from the third day after the STZ injection, and histological evaluation was performed at 10 (early phase) or 45 (late phase) days after STZ injection. We found that the proliferation of progenitor cells in STZ-induced diabetic rats showed an increase in the early phase. Additionally, ZC treatment remarkably increased the number of neural progenitor cells (NPCs) and immature neurons in the early phase of STZ-induced diabetic rats. Furthermore, ZC treatment showed increased survival rate of newly generated cells but no difference in the level of neurogenesis in the late phase of STZ-induced diabetic rats. The present study demonstrates that zinc supplementation by ZC increases both NPCs proliferation and neuroblast production at the early phase of diabetes. Thus, this study suggests that zinc supplemented with a histidine/proline complex may have beneficial effects on neurogenesis in patients experiencing the early phase of Type 1 diabetes.

Acetylcholine precursor, citicoline (cytidine 5′-diphosphocholine), reduces hypoglycaemia-induced neuronal death in rats

Citicoline (cytidine 5′‐diphosphocholine) is an important precursor for the synthesis of neuronal plasma membrane phospholipids, mainly phosphatidylcholine. The administration of citicoline serves as a choline donor for the synthesis of acetylcholine. Citicoline has been shown to reduce the neuronal injury in animal models with cerebral ischaemia and in clinical trials of stroke patients. Citicoline is currently being investigated in a multicentre clinical trial. However, citicoline has not yet been examined the context of hypoglycaemia‐induced neuronal death. To clarify the therapeutic impact of citicoline in hypoglycaemia‐induced neuronal death, we used a rat model with insulin‐induced hypoglycaemia. Acute hypoglycaemia was induced by i.p. injection of regular insulin (10 U kg‐1) after overnight fasting, after which iso‐electricity was maintained for 30 minutes. Citicoline injections (500 mg/kg, i.p.) were started immediately after glucose reperfusion. We found that post‐treatment of citicoline resulted in significantly reduced neuronal death, oxidative injury and microglial activation in the hippocampus compared to vehicle‐treated control groups at 7 days after induced hypoglycaemia. Citicoline administration after hypoglycaemia decreased immunoglobulin leakage via blood‐brain barrier disruption in the hippocampus compared to the vehicle group. Citicoline increased choline acetyltransferase expression for phosphatidylcholine synthesis after hypoglycaemia. Altogether, the present findings suggest that neuronal membrane stabilisation by citicoline administration can save neurones from the degeneration process after hypoglycaemia, as seen in several studies of ischaemia. Therefore, the results suggest that citicoline may have therapeutic potential to reduce hypoglycaemia‐induced neuronal death.