Neuroprotective effects of donepezil through inhibition of GSK-3 activity in amyloid-β-induced neuronal cell death

Acetylcholinesterase inhibitors (AChE-inhibitors) are used for the treatment of Alzheimer's disease. Recently, the AChE-inhibitor donepezil was found to have neuroprotective effects. However, the protective mechanisms of donepezil have not yet been clearly identified. We investigated the neuroprotective effects of donepezil and other AChE-inhibitors against amyloid-β1–42 (Aβ42)-induced neurotoxicity in rat cortical neurons. To evaluate the neuroprotective effects of AChE-inhibitors, primary cultured cortical neurons were pre-treated with several concentrations of AChE-inhibitors for 24 h and then treated with 20 μM Aβ42 for 6 h. In addition to donepezil, other AChE-inhibitors (galantamine and huperizine A) also showed increased neuronal cell viability against Aβ42 toxicity in a concentration-dependent manner. However, we demonstrated that donepezil has a more potent effect in inhibiting glycogen synthase kinase-3 (GSK-3) activity compared with other AChE-inhibitors. The neuroprotective effects of donepezil were blocked by LY294002 (10 μM), a phosphoinositide 3 kinase inhibitor, but only partially by mecamylamine (10 μM), a blocker of nicotinic acetylcholine receptors. Additionally, donepezil's neuroprotective mechanism was related to the enhanced phosphorylation of Akt and GSK-3β and reduced phosphorylation of tau and glycogen synthase. These results suggest that donepezil prevents Aβ42-induced neurotoxicity through the activation of phosphoinositide 3 kinase/Akt and inhibition of GSK-3, as well as through the activation of nicotinic acetylcholine receptors.     

Increased Na+/Ca2+ Exchanger Activity Promotes Resistance to Excitotoxicity in Cortical Neurons of the Ground Squirrel (a Hibernator)

Ground squirrel, a hibernating mammalian species, is more resistant to ischemic brain stress than rat. Gaining insight into the adaptive mechanisms of ground squirrels may help us design treatment strategies to reduce brain damage in patients suffering ischemic stroke. To understand the anti-stress mechanisms in ground squirrel neurons, we studied glutamate toxicity in primary cultured neurons of the Daurian ground squirrel (Spermophilus dauricus). At the neuronal level, for the first time, we found that ground squirrel was more resistant to glutamate excitotoxicity than rat. Mechanistically, ground squirrel neurons displayed a similar calcium influx to the rat neurons in response to glutamate or N-methyl-D-aspartate (NMDA) perfusion. However, the rate of calcium removal in ground squirrel neurons was markedly faster than in rat neurons. This allows ground squirrel neurons to maintain lower level of intracellular calcium concentration ([Ca²⁺]_i) upon glutamate insult. Moreover, we found that Na⁺/Ca²⁺ exchanger (NCX) activity was higher in ground squirrel neurons than in rat neurons. We also proved that overexpression of ground squirrel NCX2, rather than NCX1 or NCX3, in rat neurons promoted neuron survival against glutamate toxicity. Taken together, our results indicate that ground squirrel neurons are better at maintaining calcium homeostasis than rat neurons and this is likely achieved through the activity of ground squirrel NCX2. Our findings not only reveal an adaptive mechanism of mammalian hibernators at the cellular level, but also suggest that NCX2 of ground squirrel may have therapeutic value for suppressing brain ischemic damage.     

Protective Effects of Vitamin E against Oxidative Damage Induced by Aβ1-40Cu（Ⅱ） Complexes

β-amyloid peptide (Aβ) is considered to be responsible for the formation of senile plaques,which is the hallmark of Alzheimer's disease (AD).Oxidative stress,manifested by protein oxidation andlipid peroxidation,among other alterations,is a characteristic of AD brain.A growing body of evidence hasbeen presented in support of Aβ_(1-40) forming an oligomeric complex that binds copper at a CuZn superoxidedismutase-like binding site. Aβ_(1-40)Cu(Ⅱ) complexes generate neurotoxic hydrogen peroxide (H_2O_2) from O_2via Cue reduction,though the precise reaction mechanism is unclear.The toxicity of Aβ_(1-40) or the Aβ_(1-40)Cu(Ⅱ)complexes to cultured primary cortical neurons was partially attenuated when ( )-α-tocopherol (vitamin E)as free radical antioxidant was added at a concentration of 100 μM.The data derived from lactate dehydro-genase (LDH) release and the formation of H_2O_2 confirmed the results from the MTT assay.These findingsindicate that copper binding to Aβ_(1-40) can give rise to greater production of H_2O_2, which leads to a break-down in the integrity of the plasma membrane and subsequent neuronal death.Groups treated with vitaminE exhibited much slighter damage,suggesting that vitamin E plays a key role in protecting neuronal cellsfrom dysfunction or death.     

Pharmacological induction of ischemic tolerance in hippocampal slices by sarcosine preconditioning

Brain ischemic tolerance is a protective mechanism induced by a preconditioning stimulus, which prepare the tissue against harmful insults. Preconditioning with N-methyl-d-aspartate (NMDA) agonists induces brain tolerance and protects it against glutamate excitotoxicity. Recently, the glycine transporters type 1 (GlyT-1) have been shown to potentiate glutamate neurotransmission through NMDA receptors suggesting an alternative strategy to protect against glutamate excitotoxicity. Here, we evaluated the preconditioning effect of sarcosine pre-treatment, a GlyT-1 inhibitor, in rat hippocampal slices exposed to ischemic insult. Sarcosine (300mg/kg per day, i.p.) was administered during seven consecutive days before induction of ischemia in hippocampus by oxygen/glucose deprivation (OGD). To access the damage caused by an ischemic insult, we evaluated cells viability, glutamate release, nitric oxide (NO) production, lactate dehydrogenase (LDH) levels, production of reactive oxygen species (ROS), and antioxidant enzymes as well as the impact of oxidative stress in the tissue. We observed that sarcosine reduced cell death in hippocampus submitted to OGD, which was confirmed by reduction on LDH levels in the supernatant. Cell death, glutamate release, LDH levels and NO production were reduced in sarcosine hippocampal slices submitted to OGD when compared to OGD controls (without sarcosine). ROS production was reduced in sarcosine hippocampal slices exposed to OGD, although no changes were found in antioxidant enzymes activities. This study demonstrates that preconditioning with sarcosine induces ischemic tolerance in rat hippocampal slices submitted to OGD.     

Effects of Aβ1–42 on the Subunits of KATP Expression in Cultured Primary Rat Basal Forebrain Neurons

ATP-sensitive potassium channels (KATP) play a crucial role in coupling metabolic energy to the membrane potential of cells, thereby functioning as cellular "metabolic sensors." Recent evidence has showed a connection between the amyloid neurotoxic cascade and metabolic impairment. With regard to their neuroprotection in other neuronal preparations, KATP channels may mediate a potential neuroprotective role in Alzheimer's disease (AD). To investigate the effects of Abeta1-42 on the subunits of KATP expression in cultured primary rat basal forebrain cholinergic neurons, primary rat basal forebrain neurons were cultured and evaluated. The subunits of KATP: Kir6.1, Kir6.2, SUR1 and SUR2 expressing changes were observed by double immunofluorescence and immunoblotting when the neurons were exposed to Abeta1-42(2 microM) for different time (0, 24, 72 h). We found a significant increase in the expression of Kir6.1 and SUR2 in the cultured neurons being exposed to Abeta1-42 for 24 h, while Kir6.2 and SUR1 showed no significant change. However, after being treated with Abeta1-42 for 72 h, the expression of the four subunits was all increased significantly compared with the control. These findings suggest that being exposed to Abeta1-42 for different time (24 and 72 h) induces differential regulations of KATP subunits expression in cultured primary rat basal forebrain cholinergic neurons. The change in composition of KATP may contribute to resist the toxicity of Abeta1-42.     

Potencies and selectivities of inhibitors of acetylcholinesterase and its molecular forms in normal and Alzheimer's disease brain

Eight inhibitors of acetylcholinesterase (AChE), tacrine, bis-tacrine, donepezil, rivastigmine, galantamine, heptyl-physostigmine, TAK-147 and metrifonate, were compared with regard to their effects on AChE and butyrylcholinesterase (BuChE) in normal human brain cortex. Additionally, the IC50 values of different molecular forms of AChE (monomeric, G1, and tetrameric, G4) were determined in the cerebral cortex in both normal and Alzheimer's human brains. The most selective AChE inhibitors, in decreasing sequence, were in order: TAK-147, donepezil and galantamine. For BuChE, the most specific was rivastigmine. However, none of these inhibitors was absolutely specific for AChE or BuChE. Among these inhibitors, tacrine, bis-tacrine, TAK-147, metrifonate and galantamine inhibited both the G1 and G4 AChE forms equally well. Interestingly, the AChE molecular forms in Alzheimer samples were more sensitive to some of the inhibitors as compared with the normal samples. Only one inhibitor, rivastigmine, displayed preferential inhibition for the G1 form of AChE. We conclude that a molecular form-specific inhibitor may have therapeutic applications in inhibiting the G1 form, which is relatively unchanged in Alzheimer's brain.     

TRPC6 inhibited NMDA receptor activities and protected neurons from ischemic excitotoxicity

Excitotoxicity induced by NMDA receptor‐mediated intracellular Ca²⁺ ([Ca²⁺]_i) overload is a major cause of delayed neuronal death in cerebral ischemia. Transient receptor potential canonical (TRPC) 6 protects neurons from ischemic brain damage. However, the mechanisms by which TRPC6 protects neurons are largely unknown. Here, we reported that TRPC6 suppressed the [Ca²⁺]_i elevation induced by NMDA and protected neurons from excitotoxicity. Over‐expressing or down‐regulating TRPC6 suppressed or aggravated Ca²⁺ overload under excitotoxicity, respectively. TRPC6 protected cultured neurons from damage caused by NMDA toxicity or oxygen glucose deprivation (OGD). Moreover, the infarct volume in TRPC6 transgenic (Tg) mice was smaller than that in wild‐type (WT) littermates. The TRPC6 Tg mice had better behavior performance and lower mortality than their WT littermates. Thus, TRPC6 inhibited NMDA receptor‐triggered neurotoxicity and protected neurons from ischemic brain damage. Increase in TRPC6 activity could be a potential strategy for stroke prevention and therapy.     

The Acetylcholinesterase Inhibitors Competitively Inhibited an Acetyl l-Carnitine Transport Through the Blood–Brain Barrier

We investigated the interaction of acetylcholinesterase (AChE) inhibitors with acetyl-L-carnitine (ALCAR) transporter at the blood-brain barrier (BBB). ALCAR uptake by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB cells), as an in vitro model of BBB, were characterized by cellular uptake study using [(3)H]ALCAR. In vivo brain uptake of [(3)H]ALCAR was determined by brain uptake index after carotid artery injection in rats. In results, the transport properties for [(3)H]ALCAR by TR-BBB cell were consistent with those of ALCAR transport by the organic cation/carnitine transporter 2 (OCTN2). Also, OCTN2 was confirmed to be expressed in the cells. The uptake of [(3)H]ALCAR by TR-BBB cells was inhibited by AChE inhibitors such as donepezil, tacrine, galantamine and rivastigmine, which IC(50) values are 45.3, 74.0, 459 and 800 μM, respectively. Especially, donepezil and galantamine inhibited the uptake of [(3)H]ALCAR competitively, but tacrine and rivastigmine inhibited noncompetitively. Furthermore, [(3)H]ALCAR uptake by the rat brain was found to be significantly decreased by quinidine, donepezil and galantamine. Our results suggest that transport of AChE inhibitors such as donepezil and galantamine through the BBB is at least partly mediated by OCTN2 which is involved in transport of ALCAR.     

Chronic Ethanol Exposure Potentiates NMDA Excitotoxicity in Cerebral Cortical Neurons

Abstract: The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined. Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA. Addition of NMDA (25–100 μ M ) to the culture dishes for 25 min in Mg²⁺-free buffer resulted in a dose-dependent increase in LDH accumulation. Phase-contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes. Chronic exposure of neuronal cultures to ethanol (100 m M ) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA-stimulated LDH release by 36 and 22% in response to 25 μ M and 50 μ M NMDA, respectively. Neither basal LDH release nor that in response to maximal NMDA (100 μ M ) stimulation was altered by chronic alcohol exposure. In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 m M ) only during the NMDA exposure period significantly reduced LDH release by ∼ 50% in both control and chronically treated dishes. This reduction by acute ethanol was also observed under phase-contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes. These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.     

Reduction of calcium release from the endoplasmic reticulum could only provide partial neuroprotection against beta-amyloid peptide toxicity

Beta-amyloid (Abeta) peptide has been suggested to play important roles in the pathogenesis of Alzheimer's disease (AD). Abeta peptide neurotoxicity was shown to induce disturbance of cellular calcium homeostasis. However, whether modulation of calcium release from the endoplasmic reticulum (ER) can protect neurons from Abeta toxicity is not clearly defined. In the present study, Abeta peptide-triggered ER calcium release in primary cortical neurons in culture is modulated by Xestospongin C, 2-aminoethoxydiphenyl borate or FK506. Our results showed that reduction of ER calcium release can partially attenuate Abeta peptide neurotoxicity evaluated by LDH release, caspase-3 activity and quantification of apoptotic cells. While stress signals associated with perturbations of ER functions such as up-regulation of GRP78 was significantly attenuated, other signaling machinery such as activation of caspase-7 transmitting death signals from ER to other organelles could not be altered. We further provide evidence that molecular signaling in mitochondria play also a significant role in determining neuronal apoptosis because Abeta peptide-triggered activation of caspase-9 was not significantly reduced by attenuating ER calcium release. Our results suggest that neuroprotective strategies aiming at reducing Abeta toxicity should include molecular targets linked to ER perturbations associated with ER calcium release as well as mitochondrial stress.     

Secreted Abeta does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein

Antibodies against APP, a precursor of Abeta deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Abeta mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Abeta40 and Abeta42 in the cultured media were undetectable by an assay capable of detecting 100 nM Abeta peptides. However, exogenously treated Abeta1-42 or Abeta1-43 required >3 microM to exert neurotoxicity, and 25 microM Abeta1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Abeta1-42, whereas serum attenuated toxicity by Abeta1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His(657)-Lys(676), but not the Abeta1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Abeta.     

Overexpression of glutathione peroxidase increases the resistance of neuronal cells to Abeta-mediated neurotoxicity

Senile plaques are neuropathological manifestations in Alzheimer's disease (AD) and are composed mainly of extracellular deposits of amyloid beta-peptide (Abeta). Various data suggest that the accumulation of Abeta may contribute to neuronal degeneration and that Abeta neurotoxicity could be mediated by oxygen free radicals. Removal of free radicals by antioxidant scavengers or enzymes was found to protect neuronal cells in culture from Abeta toxicity. However, the nature of the free radicals involved is still unclear. In this study, we investigated whether the neuronal overexpression of glutathione peroxidase (GPx), the major hydrogen peroxide (H2O2)-de-grading enzyme in neurons, could increase their survival in a cellular model of Abeta-induced neurotoxicity. We infected pheochromocytoma (PC12) cells and rat embryonic cultured cortical neurons with an adenoviral vector encoding GPx (Ad-GPx) prior to exposure to toxic concentrations of Abeta(25-35) or (1-40). Both PC12 and cortical Ad-GPx-infected cells were significantly more resistant to Abeta-induced injury. These data strengthen the hypothesis of a role of H2O2 in the mechanism of Abeta toxicity and highlight the potential of Ad-GPx to reduce Abeta-induced damage to neurons. These findings may have applications in gene therapy for AD.     

Effect of Agmatine Against Cell Death Induced by NMDA and Glutamate in Neurons and PC12 Cells

1. Aims: Agmatine is an endogenous guanido amine and has been shown to be neuroprotective in vitro and in vivo. The aims of this study are to investigate whether agmatine is protective against cell death induced by different agents in cultured neurons and PC12 cells.2. Methods: Cell death in neurons, cultured from neonatal rat cortex, was induced by incubating with (a) NMDA (100 M) for 10 min, (b) staurosporine (protein kinase inhibitor, 100 nM) for 24 h, and (c) calcimycin (calcium ionophore, 100 nM) for 24 h in the presence and absence of agmatine (1 M to 1 mM). Cell death in PC12 cells was induced by exposure to glutamate (10 mM), staurosporine (100 nM), and calcimycin (100 nM). The activity of lactate dehydrogenase (LDH) in the medium was measured as the marker of cell death and normalized to cellular LDH activity.3. Results: Agmatine significantly reduced the medium LDH in NMDA-treated neurons but failed to reduce the release of LDH induced by staurosporin or calcimycin. In PC12 cells, agmatine significantly reduced LDH release induced by glutamate exposure, but not by staurosporine or calcimycin. Agmatine itself neither increased LDH release nor directly inhibited the enzyme activity.4. Conclusion: We conclude that agmatine protects against NMDA excitotoxicity in neurons and PC12 cells but not the cell death induced by protein kinase blockade or increase in cellular calcium.     

Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-1-nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-beta (Abeta) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Abeta25-35 in the presence or absence of Cu or Fe. Abeta25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Abeta to perturb neuronal GSH homeostasis. Abeta25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.     

银杏内酯B对谷氨酸诱导海马神经元损伤的影响

目的：观察银杏内酯B（ginkgolide B,GB）在不同给药模式下对谷氨酸所致海马神经元损伤的影响。方法：采用Co2超临界萃取的方法制备GB,建立新生Wistar大鼠原代培养的海马神经元谷氨酸毒性模型,采用台盼蓝染色、程序性细胞死亡检测技术及乳酸脱氢酶测定的方法,观察预处理与急救两种给药模型下不同剂量GB的神经保护作用,并与MK-801急性给药相比较。结果：GB在两种给药模式下均能不同程度地提高细胞存活率,降低凋亡率,减少LDH漏出量,且在一定范围内保护作用呈剂量依赖的方式。其中预处理的效果明显优于急救给药处理。但均弱于MK-801组。结论：GB对谷氨酸细胞毒性损伤有保护作用,预防性用药效果更佳。GB可能不仅仅通过拮抗血小板活化因子（PAF）受体等下游事件实现其神经保护作用。如果我们重视其预处理给药的显著效果。将其用于高危人群的预防干预可能有更大价值。     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.     

Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia

Glutamate excitotoxicity to a large extent is mediated through activation of the N-methyl-D-aspartate (NMDA)-gated ion channels in several neurodegenerative diseases and ischemic stroke. Minocycline, a tetracycline derivative with antiinflammatory effects, inhibits IL-1beta-converting enzyme and inducible nitric oxide synthase up-regulation in animal models of ischemic stroke and Huntington's disease and is therapeutic in these disease animal models. Here we report that nanomolar concentrations of minocycline protect neurons in mixed spinal cord cultures against NMDA excitotoxicity. NMDA treatment alone induced microglial proliferation, which preceded neuronal death, and administration of extra microglial cells on top of these cultures enhanced the NMDA neurotoxicity. Minocycline inhibited all these responses to NMDA. Minocycline also prevented the NMDA-induced proliferation of microglial cells and the increased release of IL-1beta and nitric oxide in pure microglia cultures. Finally, minocycline inhibited the NMDA-induced activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells, and a specific p38 MAPK inhibitor, but not a p44/42 MAPK inhibitor, reduced the NMDA toxicity. Together, these results suggest that microglial activation contributes to NMDA excitotoxicity and that minocycline, a tetracycline derivative, represents a potential therapeutic agent for brain diseases.     

Tissue plasminogen activator requires plasminogen to modulate amyloid-beta neurotoxicity and deposition

Tissue plasminogen (plgn) activator (tPA) modulates neuronal death in models of stroke, excitotoxicity, and oxidative stress. Amyloid-beta (Abeta) appears central to Alzheimer's disease and is neurotoxic to neurons in vitro. Here, we evaluate tPA effects on Abeta toxicity. We report that tPA alone had no effect on Abeta toxicity. However, in combination with plgn, tPA reduced Abeta toxicity in a robust fashion. Moreover, the combined tPA and plgn treatment markedly inhibited Abeta accumulation. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to a sample of tPA, plgn, and Abeta resulted in a marked reduction of Abeta degradation. We interpret the actions of tPA and plgn within the context of the ability of plasmin to degrade Abeta.     

Beta-amyloid increases somatostatin expression in cultured cortical neurons

In beta-amyloid (Abeta)-induced neurotoxicity, activation of the NMDA receptor, increased Ca2+ and oxidative stress are intimately associated with neuronal cell death as normally seen in NMDA-induced neurotoxicity. We have recently shown selective sparing of somatostatin (SST)-positive neurons and increased SST expression in NMDA agonist-induced neurotoxicity. Accordingly, the present study was undertaken to determine the effect of Abeta25-35-induced neurotoxicity on the expression of SST in cultured cortical neurons. Cultured cortical cells were exposed to Abeta25-35 and processed to determine the cellular content and release of SST into medium by radioimmunoassay and SST mRNA by RT-PCR. Abeta25-35 induces neuronal cell death in a concentration- and time-dependent fashion, increases SST mRNA synthesis and induces an augmentation in the cellular content of SST. No significant changes were seen on SST release at any concentration of Abeta25-35 after 24 h of treatment. However, Abeta25-35 induces a significant increase of SST release into medium only after 12 h in comparison with other time points. Most significantly, SST-positive neurons are selectively spared in the presence of a lower concentration of Abeta25-35, whereas, in the presence of higher concentrations of Abeta25-35 for extended time periods, SST-positive neurons decrease gradually. Furthermore, Abeta25-35 induces apoptosis at lower concentrations (5 and 10 micromol/L) and necrosis at higher concentrations (20 and 40 micromol/L). Consistent with the increased accumulation of SST, these data suggest that Abeta25-35 impairs cell membrane permeability. Selective sparing of SST-positive neurons at lower concentrations of Abeta25-35 at early time points directly correlates with the pathophysiology of Alzheimer's disease.     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.