EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

Pregnenolone Protects Mouse Hippocampal (HT-22) Cells against Glutamate and Amyloid Beta Protein Toxicity

In the present work we have examined whether the neurosteroid pregnenolone has any neuroprotective effects against glutamate and amyloid beta protein neurotoxicity using immortalized clonal mouse hippocampal cell line (HT-22). The neurosteroid pregnenolone protects HT-22 cells against both 5 mM glutamate and 2 M amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was attained at 500 nM pregnenolone, against both 5 mM glutamate as well as 2 M amyloid beta protein induced HT-22 cell death. Furthermore, using confocal immunoflourescence microscopy we observed that 20 hours of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptor (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 500 nM pregnenolone treatment for 24 hours, followed by 20 hours treatment with 5 mM glutamate resulted in dramatic reduction in GR nuclear localization. These results show that (i) pregnenolone has neuroprotective effects against both glutamate and amyloid beta protein neuropathology and (ii) prevention of glucocorticoid receptor (GR) localization to the nucleus may be involved in the observed neuroprotective effects of pregnenolone against glutamate neurotoxicity.     

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.     

Calpain inhibitory flavonoids isolated from Orostachys japonicus

The n-butanol (n-BuOH) fraction of Orostachys japonicus A. Berger (Crassulaceae) significantly inhibited calpain activity. Through the activity-guided isolation from the n-BuOH fraction, herbacetin 8-O-α-D-ribopyranoside (1), kaempferol (2), quercetin (3), afzelin (4), astragalin (5), isoquercetin (6) and quercitrin (7) were obtained. Their structures were determined by spectroscopic techniques. Among them, compound 3 and 5 had significant calpain inhibitory activities.     

A single intramuscular injection of rAAV‐mediated mutant erythropoietin protects against MPTP‐induced parkinsonism

Erythropoietin (Epo) is neuroprotective in a number of preparations, but can lead to unacceptably high and even lethal hematocrit levels. Recent reports show that modified Epo variants confer neuroprotection in models of glaucoma and retinal degeneration without raising hematocrit. In this study, neuroprotective effects of two Epo variants (EpoR76E and EpoS71E) were assessed in a model of Parkinson's disease. The constructs were packaged in recombinant adeno‐associated viral (rAAV) vectors and injected intramuscularly. After 3 weeks, mice received five daily injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and were killed 5 weeks later. The MPTP‐lesioned mice pretreated with rAAV.eGFP (negative control) exhibited a 7‐ to 9‐Hz tremor and slower latencies to move on a grid test (akinesia). Both of these symptomatic features were absent in mice pretreated with either modified Epo construct. The rAAV.eGFP‐treated mice lesioned with MPTP exhibited a 41% reduction in tyrosine hydroxylase (TH)‐positive neurons in the substantia nigra. The rAAV.EpoS71E construct did not protect nigral neurons, but neuronal loss in mice pretreated with rAAV.EpoR76E was only half that of rAAV.eGFP controls. Although dopamine levels were normal in all groups, 3,4‐dihydroxyphenylacetic acid (DOPAC) was significantly reduced only in MPTP‐lesioned mice pretreated with rAAV.eGFP, indicating reduced dopamine turnover. Analysis of TH‐positive fibers in the striatum showed normalized density in MPTP‐lesioned mice pretreated with rAAV.EpoS71E, suggesting that enhanced sprouting induced by EpoS71E may have been responsible for normal behavior and dopaminergic tone in these mice. These results show that systemically administered rAAV‐generated non‐erythropoietic Epo may protect against MPTP‐induced parkinsonism by a combination of neuroprotection and enhanced axonal sprouting.     

Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line‐derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up‐regulated in primary RMG after glial cell line‐derived neurotrophic factor stimulation, provides neuroprotective and pro‐survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b^rd1 retinas. To identify stimulated pathways in the retina, we treated Pde6b^rd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation‐site‐specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro‐survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.

     

Thermosensitive heparin‐poloxamer hydrogel encapsulated bFGF and NGF to treat spinal cord injury

The application of growth factors (GFs) for treating chronic spinal cord injury (SCI) has been shown to promote axonal regeneration and functional recovery. However, direct administration of GFs is limited by their rapid degradation and dilution at the injured sites. Moreover, SCI recovery is a multifactorial process that requires multiple GFs to participate in tissue regeneration. Based on these facts, controlled delivery of multiple growth factors (GFs) to lesion areas is becoming an attractive strategy for repairing SCI. Presently, we developed a GFs‐based delivery system (called GFs‐HP) that consisted of basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and heparin‐poloxamer (HP) hydrogel through self‐assembly mode. This GFs‐HP was a kind of thermosensitive hydrogel that was suitable for orthotopic administration in vivo. Meanwhile, a 3D porous structure of this hydrogel is commonly used to load large amounts of GFs. After single injection of GFs‐HP into the lesioned spinal cord, the sustained release of NGF and bFGF from HP could significantly improve neuronal survival, axon regeneration, reactive astrogliosis suppression and locomotor recovery, when compared with the treatment of free GFs or HP. Moreover, we also revealed that these neuroprotective and neuroregenerative effects of GFs‐HP were likely through activating the phosphatidylinositol 3 kinase and protein kinase B (PI3K/Akt) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signalling pathways. Overall, our work will provide an effective therapeutic strategy for SCI repair.     

Neuroprotection in acute ischemic stroke – current status

With the growing understanding of the mechanism of cell death in ischemia, new approaches for treatment such as neuroprotection have emerged. The basic aim of this strategy is to interfere with the events of the ischemic cascade, blocking the pathological processes and preventing the death of nerve cells in the ischemic penumebra. This concept involves inhibition of the pathological molecular events which eventually leads to the influx of calcium, activation of free radicals and neuronal death. Despite encouraging data from experimental animal models, all clinical trials of neuroprotective therapies have to date been unsuccessful. This article reviews some of the reasons for the failure of neuroprotection in the clinical trials so far. Despite all the negative reports, we believe it would be wrong to give up at this point, since there is still reasonable hope of finding an effective neuroprotection for stroke.