Mild hypothermia reduces apoptosis of mouse neurons in vitro early in the cascade.

Recent experimental work has shown that hypothermia with even small decreases in temperature is broadly neuroprotective, but the mechanism of this protection remains unclear. Although reduction of metabolism could explain protection by deep hypothermia, it does not explain the robust protection found with mild hypothermia. Several reports have suggested that ischemic apoptosis is reduced by hypothermia. The authors examined the effects of hypothermia on neuronal apoptosis using serum deprivation, a well-accepted model that induces neuronal apoptosis. Mild hypothermia (33 degrees C) significantly reduced the number of morphologically apoptotic neurons to less than half the number seen in normothermic culture temperatures (37 degrees C) after 48 hours. They examined the effect of hypothermia on several steps in the cascade. Caspase-3, -8, and -9 activity was significantly increased after 24 hours at 37 degrees C, and was significantly lower in cultures deprived of serum at 33 degrees C. Cytochrome c translocation was reduced by hypothermia. Western blot analysis failed to detect significant changes in Bax, bcl -2, or hsp -70 at early time points, whereas hypothermia significantly reduced cJun N-terminal kinase activation. The authors conclude that small decreases in temperature inhibit apoptosis very early, possibly at the level of the initiation of apoptosis, as suggested by reduced cJun N-terminal kinase activation and before the translocation of cytochrome c, with subsequent prevention of caspase activation.     

Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses

Synaptic dysfunction is increasingly viewed as an early manifestation of Alzheimer's disease (AD), but the cellular mechanism by which beta-amyloid (Abeta) may affect synapses remains unclear. Since cultured neurons derived from APP mutant transgenic mice secrete elevated levels of Abeta and parallel the subcellular Abeta accumulation seen in vivo, we asked whether alterations in synapses occur in this setting. We report that cultured Tg2576 APP mutant neurons have selective alterations in pre- and post-synaptic compartments compared to wild-type neurons. Post-synaptic compartments appear fewer in number and smaller, while active pre-synaptic compartments appear fewer in number and enlarged. Among the earliest changes in synaptic composition in APP mutant neurons were reductions in PSD-95, a protein involved in recruiting and anchoring glutamate receptor subunits to the post-synaptic density. In agreement, we observed early reductions in surface expression of glutamate receptor subunit GluR1 in APP mutant neurons. We provide evidence that Abeta is specifically involved in these alterations in synaptic biology, since alterations in PSD-95 and GluR1 are blocked by gamma-secretase inhibition, and since exogenous addition of synthetic Abeta to wild-type neurons parallels changes in synaptic PSD-95 and GluR1 observed in APP mutant neurons.     

Reactive oxidative species enhance amyloid toxicity in APP/PS1 mouse neurons

Objective To investigate whether intracellular amyloid β (iAβ) induces toxicity in wild type (WT) and APP/PS1 mice, a mouse model of Alzheimer’s disease. Methods Different forms of Aβ aggregates were microinjected into cultured WT or APP/PS1 mouse hippocampal neurons. TUNEL staining was performed to examine neuronal cell death. Reactive oxidative species (ROS) were measured by MitoSOXTM Red mitochondrial superoxide indicator. Results Crude, monomer and protofibrilAβ induced more toxicity inAPP/PS1 neurons than in WT neurons. ROS are involved in mediating the vulnerability of APP/PS1 neurons to iAβ toxicity. Conclusion Oxidative stress may mediate cell death induced by iAβ in neurons.     

Neuropeptide Y in noradrenergic neurons induces obesity in transgenic mouse models

Neuropeptide Y (NPY) in noradrenergic neurons plays an important role in modulating the release and effects of catecholamines in a prolonged stress response. Among other functions, it controls energy metabolism. Transgenic expression of Npy in noradrenergic neurons in mice allowed showing that it is critical for diet- and stress-induced gain in fat mass. When overexpressed, NPY in noradrenergic neurons increases adiposity in gene-dose-dependent fashion, and leads to metabolic disorders such as impaired glucose tolerance. However, the mechanisms of obesity seem to be different in mice heterozygous and homozygous for the Npy transgene. While in heterozygous mice the adipogenic effect of NPY is important, in homozygous mice inhibition of sympathetic tone leading to decreased lipolytic activity and impaired brown fat function, as well as increased endocannabinoid levels contribute to obesity. The mouse model provides novel insight to the mechanisms of human diseases with increased NPY due to chronic stress or gain-of-function gene variants, and a tool for development of novel therapeutics.     

Rifampicin reduces alpha-synuclein in a transgenic mouse model of multiple system atrophy

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by oligodendrocytic cytoplasmic inclusions containing abnormally aggregated alpha-synuclein. This aggregation has been linked to the neurodegeneration observed in MSA. Current MSA treatments are aimed at controlling symptoms rather than tackling the underlying cause of neurodegeneration. This study investigates the ability of the antibiotic rifampicin to reduce alpha-synuclein aggregation and the associated neurodegeneration in a transgenic mouse model of MSA. We report a reduction in monomeric and oligomeric alpha-synuclein and a reduction in phosphorylated alpha-synuclein (S129) upon rifampicin treatment. This reduction in alpha-synuclein aggregation was accompanied by reduced neurodegeneration. On the basis of its anti-aggregenic properties, we conclude that rifampicin may have therapeutic potential for MSA.     

Age-related impairments in operant DMTP performance in the PS2APP mouse, a transgenic mouse model of Alzheimer's disease

One of the earliest signs of Alzheimer's disease (AD) is loss of memory for recent events. This deficit in short term memory has been characterised in mild/moderate AD patients as a delay-dependent deficit in a delayed matching to sample (DMTS) task. PS2APP mice co-expressing hPS2mut and hAPPswe exhibit a spatial-temporal elevation in brain amyloid deposition and inflammation associated with temporal cognitive decline. The aim of the current study was to train PS2APP mice (C57BL/6JxDBA/2 mixed background) and appropriate control mice (B6D2F1 background) in a rodent delayed response task, the delayed matching to position (DMTP) task, prior to the onset of plaque formation and subsequently at 2-4 monthly intervals to investigate the effect of aging and increasing plaque load on DMTP performance. At 5 months of age (baseline) DMTP performance was equivalent with both PS2APP and control mice demonstrating a working memory curve across increasing delay intervals of 1-24s. A comparison of PS2APP and control mice across ages revealed a selective age-related, delay-dependent, impairment on choice accuracy in PS2APP mice, consistent with the cognitive decline and temporal amyloidosis previously described for this mouse model. These data are also relevant for other conditional transgenic mouse models which allow time-sensitive induction or inhibition of gene expression such that mice can be trained to perform the task prior to activation or inactivation of the gene and tested thereafter.     

A transgenic mouse model reveals fast nicotinic transmission in hippocampal pyramidal neurons

The relative contribution to brain cholinergic signaling by synaptic- and diffusion-based mechanisms remains to be elucidated. In this study, we examined the prevalence of fast nicotinic signaling in the hippocampus. We describe a mouse model where cholinergic axons are labeled with the tauGFP fusion protein driven by the choline acetyltransferase promoter. The model provides for the visualization of individual cholinergic axons at greater resolution than other available models and techniques, even in thick, live, slices. Combining calcium imaging and electrophysiology, we demonstrate that local stimulation of visualized cholinergic fibers results in rapid excitatory postsynaptic currents mediated by the activation of α7-subunit-containing nicotinic acetylcholine receptors (α7-nAChRs) on CA3 pyramidal neurons. These responses were blocked by the α7-nAChR antagonist methyllycaconitine and potentiated by the receptor-specific allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596). Our results suggest, for the first time, that synaptic nAChRs can modulate pyramidal cell plasticity and development. Fast nicotinic transmission might play a greater role in cholinergic signaling than previously assumed. We provide a model for the examination of synaptic properties of basal forebrain cholinergic innervation in the brain.     

APP17肽对APP转基因小鼠学习记忆能力和海马神经细胞凋亡的影响

目的观察APP(-βamyloid precursop protein,APP)17肽(APP695中319-335肽段)对APP转基因小鼠(APP695V717I)学习、记忆能力及海马神经细胞凋亡的影响。方法3月龄的APP695转基因小鼠随机分为模型组和APP17肽治疗组,正常对照组采用月龄和性别与之相匹配的C57BL/6J小鼠。APP17肽治疗组给予皮下注射APP17肽,每只每次0.34μg,每周3次;模型组和正常对照组给予等体积NS。应用水迷宫试验观察小鼠学习、记忆功能的变化,以流式细胞技术分析海马神经细胞凋亡率和线粒体膜电位的变化。结果(1)水迷宫试验结果显示,模型组小鼠存在明显学习和记忆功能障碍,其第3、4、5天游完全程的时间[(93.22±16.35)、(86.73±20.26)、(77.13±29.35)s]和错误反应次数[(6.63±2.16)、(5.81±2.13)、(5.33±1.41)次]均较正常对照组[分别为(70.89±20.19)、(61.25±21.88)、(54.63±16.92)s和(5.01±1.93)、(2.97±0.96)、(2.31±1.01)次]增多(P<0.05);APP17肽治疗组小鼠的行为学障碍明显轻于模型组(P<0.05),其上述水迷宫检测结果与正常对照组比较差异无统计学意义。(2)与正常对照组小鼠海马神经细胞凋亡发生率[(3.13±1.19)%]和线粒体膜电位[(176.39±13.88)mV]比较,模型组凋亡发生率[(8.06±2.31)%]显著增加(P<0.01)而线粒体膜电位[(97.51±15.73)mV]明显降低(P<0.01);APP17肽治疗组检测结果与正常对照组接近,海马神经细胞凋亡发生率[(4.38±1.26)%]低于模型组(P<0.01),线粒体膜电位[(168.35±19.29)mV]高于模型组(P<0.01)。结论APP695转基因小鼠存在学习和记忆功能障碍,且其海马神经细胞凋亡率增加,线粒体膜电位降低;APP17肽能够明显改善该转基因小鼠的学习和记忆能力,其作用机制可能是通过稳定线粒体膜电位及抑制凋亡发生而实现。     

Dynorphin reduces voltage-dependent calcium conductance of mouse dorsal root ganglion neurons

Dynorphin A (DYN) (1 microM) decreased somatic calcium-dependent action potential (CAP) duration of a portion of dorsal root ganglion (DRG) neurons in a naloxone reversible manner. Responses to DYN differed from responses to Leu-enkephalin in that only DYN decreases of somatic CAP duration were associated with decreased action potential after hyperpolarization and persisted after intracellular injection of the potassium channel blocker cesium. While Leu-enkephalin at 10 microM did not affect somatic CAP duration of DRG neurons impaled with cesium-filled micropipettes, dynorphin A (1-8), dynorphin B, and beta-neoendorphin were effective at 1 microM. During single electrode voltage clamp, DYN decreased inward current in a portion of DRG neurons under conditions that predominately isolated calcium current. Leak current was unaffected by dynorphin A. Therefore, we suggest that DYN decreases voltage-dependent calcium conductance. The action on calcium conductance appears specific for opioids with affinity for kappa-receptors.     

Time course of the development of Alzheimer-like pathology in the doubly transgenic PS1+APP mouse

Doubly transgenic mice expressing both a mutated amyloid precursor protein and a mutated presenilin-1 protein accumulate A(beta) deposits as they age. The early A(beta) deposits were found to be primarily composed of fibrillar A(beta) and resembled compact amyloid plaques. As the mice aged, nonfibrillar A(beta) deposits increased in number and spread to regions not typically associated with amyloid plaques in Alzheimer's disease. The fibrillar, amyloid-containing deposits remained restricted to cortical and hippocampal structures and did not increase substantially beyond the 12-month time point. Even at early time points, the fibrillar deposits were associated with dystrophic neurites and activated astrocytes expressing elevated levels of glial fibrillary acidic protein. Microglia similarly demonstrated increased staining for complement receptor-3 in the vicinity of A(beta) deposits at early time points. However, when MHC-II staining was used to assess the degree of microglial activation, full activation was not detected until mice were 12 months or older. Overall, the regional pattern of A(beta) staining resembles that found in Alzheimer disease; however, a progression from diffuse A(beta) to more compact amyloid deposits is not observed in the mouse model. It is noted that the activation of microglia at 12 months is coincident with the apparent stabilization of fibrillar A(beta) deposits, raising the possibility that activated microglia might clear fibrillar A(beta) deposits at a rate similar to their rate of formation, thereby establishing a relatively steady-state level of amyloid-containing deposits.     

Synaptic fatigue is more pronounced in the APP/PS1 transgenic mouse model of Alzheimer's disease

To search for potential mechanism that might alter synaptic transmission following Abeta increase we have examined the presynaptic component of transmitter release. As parameters of synaptic transmission that might underlie presynaptic mechanisms, we have used paired-pulse facilitation (PPF), post-tetanic potentiation (PTP), and synaptic fatigue (SF) at the connection between the hippocampal Schaffer-collateral pathway and CA1 pyramidal neurons in approximately 5 month old double transgenic mice overexpressing the mutated form of amyloid precursor protein (APPK670N, M671L) and presenilin 1 (PS1M146V). While the presynaptic mechanisms of PPF and PTP were not compromised in the APP/PS1 mice, SF was more pronounced in the double transgenic animals. The percentage of the 40th fEPSP slope over the first during the tetanus was 18 -/+ 3% in APP/PS1 vs. 26 -/+ 2% in WT. Thus, it is likely that presynaptic mechanisms underlying SF but not PPF and PTP, may account for synaptic dysfunction in APP/PS1 mice.     

A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease

Clinical, epidemiological, and laboratory studies suggest that cholesterol may play a role in the pathogenesis of Alzheimer's disease (AD). Transgenic mice exhibiting an Alzheimer's beta-amyloid phenotype were treated with the cholesterol-lowering drug BM15.766 and tested for modulation of beta-amyloid levels. BM15.766 treatment reduced plasma cholesterol, brain Abeta peptides, and beta-amyloid load by greater than twofold. A strong, positive correlation between the amount of plasma cholesterol and Abeta was observed. Furthermore, drug treatment reduced the amyloidogenic processing of the amyloid precursor protein, suggesting alterations in processing in response to cholesterol modulation. This study demonstrates that hypocholesterolemia is associated with reduced Abeta accumulation suggesting that lowering cholesterol by pharmacological means may be an effective approach for reducing the risk of developing AD.     

Characterization of dopaminergic midbrain neurons in a DBH:BDNF transgenic mouse.

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the survival and differentiation of central nervous system neurons, including dopaminergic cells in culture. To determine whether BDNF might play a role in the development of dopaminergic neurons in vivo, we used a previously characterized transgenic mouse (DBH:BDNF) that overexpresses BDNF in adrenergic and noradrenergic neurons as a result of fusion of the BDNF gene to the dopamine beta-hydroxylase (DBH) gene promoter. We quantified dopaminergic neuronal profiles at four midbrain coronal levels and compared DBH:BDNF transgenic animals with wild-type mice of the same genetic background. Analysis of sections immunostained with tyrosine hydroxylase (TH) showed that the mean number of dopaminergic neurons in the four selected midbrain sections was 52% greater (one-way analysis of variance, P < 0.0005) in transgenic mice (2,165 +/- 55 S. E.M., n = 4) than in control mice (1,428 +/- 71 S.E.M., n = 4). The increase in dopaminergic neuron profile count in DBH:BDNF transgenic animals was confirmed by analysis of the pars compacta of the substantia nigra on Nissl-stained sections. Surface area of the reference region of interest containing TH-immunoreactive neurons was similar in transgenic and control mice. Regional analysis of different midbrain areas containing dopaminergic neurons suggested that the increase in cell profile count occurs in a relatively homogeneous manner. Comparison of TH-immunoreactive cell size showed a tendency for smaller neurons in transgenic animals, but the difference was not statistically significant. We conclude that DBH:BDNF transgenic mice show increased number of TH-immunoreactive cells in the midbrain. We propose that BDNF rescues dopaminergic neurons from the perinatal period of developmental cell death as a consequence of increased anterograde transport of the neurotrophin via the coeruleonigral projection.     

APP/PS1双转基因AD传代小鼠的基因型鉴定及其行为学分析

目的 对APP/PS1双转基因AD传代小鼠进行基因型鉴定, 观察其学习记忆功能.方法 PCR鉴定APP/PS1双转基因AD传代小鼠的基因表型, 采用水迷宫实验检测记忆功能.结果 9只APP/PS1双转基因AD传代小鼠基因组DNA中5只小鼠基因组DNA扩增出约350 bp大小条带,认为成功转入APP和PS1基因.APP/PS1双转基因AD小鼠的水迷宫潜伏期比对照组小鼠明显延长.结论 利用PCR扩增APP基因可对APP/PS1双转基因AD传代小鼠进行基因型鉴定, APP/PS1双转基因AD小鼠能够较好地模拟AD患者的临床表现, 为AD的研究提供有效的实验动物模型.     

Sensorimotor gating and memory deficits in an APP/PS1 double transgenic mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder associated with cognitive deterioration and neuropsychiatric symptoms. Sensorimotor gating deficit has been identified in neuropsychiatric diseases. The aim of the present study was to evaluate the possible sensorimotor gating deficit and its correlation to memory impairment and cerebral β-amyloid (Aβ) plaque deposits in an amyloid precursor protein (APP)/presenilin-1 (PS1) double transgenic mouse model of AD. The sensorimotor gating in 3-, 7- and-22-month-old non-transgenic and transgenic mice was evaluated in a prepulse inhibition (PPI) task. Results revealed that the PPI was lower in the 7- and 22-month-old transgenic mice compared with the age-matched control, while the response to startle pulse-alone in the transgenic and non-transgenic mice was comparable. Congo red staining showed that Aβ neuropathology of transgenic mice aggravated with age, and the 3-month-old transgenic mice started to have minimum brain Aβ plaques, corresponding to the early stage of AD phenotype. Furthermore, memory impairment in the 7-month-old transgenic mice was detected in a water maze test. These results suggest that the sensorimotor gating is impaired with the progressing of AD phenotype, and its deficit may be correlated to cerebral Aβ neuropathology and memory impairment in the APP/PS1 transgenic mouse model of AD.     

Progressive and gender-dependent cognitive impairment in the APP(SW) transgenic mouse model for Alzheimer's disease.

To determine if early cognitive sensorimotor deficits exist in APP(SW) transgenic mice overexpressing human amyloid precursor protein (APP). Tg+ and Tg- animals at both 3 and 9 months of age (3M and 9M, respectively) were evaluated in a comprehensive battery of measures. The performance of all Tg+ mice at both ages was no different from all Tg- controls in Y-maze alternations, water maze acquisition, passive avoidance, and active avoidance testing. By contrast, results from other tasks revealed substantive cognitive deficits in Tg+ mice that were usually gender-dependent and sometimes progressive in nature. Between 3M and 9M, a progressive impairment was observed in circular platform performance by Tg+ males, as was a progressive deficit in visible platform testing for all Tg+ animals. Other transgenic effects included both impaired water maze retention and circular platform performance in 3M Tg+ females; this later effect was responsible for an overall (males + females) Tg+ deficit in circular platform performance at 3M. Sensorimotor testing revealed several Tg+ effects, most notably an increased activity of Tg+ males in both open field and Y-maze at 3M. Significant correlations between a number of behavioral measures were observed, although factor analysis suggests that each task measured components of sensorimotor/cognitive function not measured by other tasks. Finally, Tg+ mice had lower survivability than Tg- animals through 9M (85 vs. 96%). In summary, these results demonstrate the presence of gender-related and progressive cognitive deficits in APP(SW) transgenic mice at a relatively early age (i.e., prior to overt, beta-amyloid deposition in the brain), suggesting a pathophysiologic role for elevated levels of 'soluble' beta-amyloid in such impairments.     

Deposition of mouse amyloid beta in human APP/PS1 double and single AD model transgenic mice

The deposition of amyloid beta (Abeta) peptides and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). To investigate the relation between amyloid precursor protein (APP) production, amyloid beta deposition and the type of Abeta in deposits, i.e., human and/or mouse, we performed a histopathological analysis, using mouse and human specific antibodies, of the neocortex and hippocampus in 6, 12 and 19 months old APP/PS1 double and APP and PS1 single transgenic mice. There was a significant correlation between the human amyloid beta deposits and the intrinsic rodent amyloid beta deposits, that is, all plaques contained both human and mouse Abeta, and the diffuse amyloid beta deposits also colocalized human and mouse Abeta. Furthermore, some blood vessels (mainly leptomeningeal vessels) show labeling with human Abeta, and most of these vessels also label with mouse Abeta. Our findings demonstrate that the human amyloid deposits in APP/PS1 transgenic mice are closely associated with mouse Abeta, however, they do not precisely overlap. For instance, the core of plaques consists of primarily human Abeta, whereas the rim of the plaque contains both human and mouse amyloid beta, similarly, human and mouse Abeta are differentially localized in the blood vessel wall. Finally, as early as amyloid beta deposits can be detected, they show the presence of both human and mouse Abeta. Together, these data indicate that mouse Abeta is formed and deposited in significant amounts in the AD mouse brain and that it is deposited together with the human Abeta.     

Calpain inhibitors delay injury-induced apoptosis in adult mouse spinal cord motor neurons

Here, we investigated the effect of calpain inhibitors on apoptosis in organotypic adult spinal cord slices from mice. An increase in calpain I immunoreactivity was found in the nuclei of motor neurons from slices cultured for 30 min. After 4 h, the immunopositive motor neurons exhibited apoptotic changes including nuclear and chromatin condensation. Eight hours after excision, most motor neurons showed nuclear apoptotic features. Two calpain inhibitors, leupeptin and calpain inhibitor XI, inhibited apoptosis in the motor neurons while the caspase inhibitor Z-VAD.fmk had no effect. Leupeptin, but not calpain inhibitor XI and Z-VAD.fmk, also inhibited nucleosomal DNA fragmentation. These results suggest the involvement of calpain I in the induction of apoptosis in motor neurons of adult spinal cord and that apoptosis can be triggered independent of caspase activation.     

A novel approach for studying endogenous abeta processing using cultured primary neurons isolated from APP transgenic mice.

The central component of senile amyloid plaques in Alzheimer's disease (AD) is the beta-amyloid peptide (Abeta), derived from proteolytic processing of the amyloid precursor protein (APP). In this study, we developed an in vitro model to measure and identify soluble Abeta from primary cortical neurons. Neurons were isolated from mice transgenic for human APP695 containing the K670N, M671L double mutation. We characterized soluble Abeta using Western blot and ELISA assays. We found that the Abeta levels in conditioned media from these neurons were readily detectable and almost five times higher than in CSF. The majority of Abeta in the media was Abeta1-40; however, Abeta1-42 was also detectable. When the neurons were exposed to Phorbol 12-myristate 13-acetate (PMA), alpha1-antichymotrypsin, or alpha1-antitrypsin, the alterations of soluble Abeta levels were consistent with other models reported. Most importantly, the soluble Abeta in our model was remarkably stable, and aliquots were unchanged after prolonged incubations or repeated freeze/thaw cycles. The Abeta appeared to be monomeric by Western blot analysis. Soluble Abeta coimmunoprecipitated with endogenous mouse apolipoprotein E from the primary cultures. Taken together, our data demonstrated that using a Western blot assay to detect soluble Abeta from transgenic mouse overexpressing APP695 is sensitive, specific, and reliable and provides an accessible model for examining the neuronal metabolism of APP and Abeta.     

Fractalkine reduces N-methyl-d-aspartate-induced calcium flux and apoptosis in human neurons through extracellular signal-regulated kinase activation

Our purpose was to investigate in human neurons the neuroprotective pathways induced by Fractalkine (FKN) against glutamate receptor-induced excitotoxicity. CX(3)CR1 and FKN are expressed constitutively in the tested human embryonic primary neurons and SK-N-SH, a human neuroblastoma cell line. Microfluorometry assay demonstrated that CX(3)CR1 was functional in 44% of primary neurons and in 70% of SK-N-SH. Fractalkine induced ERK1/2 phosphorylation within 1 min and Akt phosphorylation after 10 min, and both phosphorylation decreased after 20 min. No p38 and SAPK/JNK activation was observed after FKN treatment. Application of FKN triggered a 53% reduction of the NMDA-induced neuronal calcium influx, which was insensitive to pertussis toxin and LY294002 an inhibitor of Akt pathway, but abolished by PD98059, an ERK1/2 pathway inhibitor. Moreover, FKN significantly reduced neuronal NMDA-induced apoptosis, which was pertussis toxin insensitive and abolished in presence of PD98059 and LY294002. In conclusion, FKN protected human neurons from NMDA-mediated excitotoxicity in at least two ways with different kinetics: (i) an early ERK1/2 activation which reduced NMDA-mediated calcium flux; and (ii), a late Akt activation associated with the previously induced ERK1/2 activation.